WO 2008/127670 Al

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(43) International Publication Date PCT (10) International Publication Number 23 October 2008 (23.10.2008) WO 2008/127670 Al

(51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every AOlN 63/00 (2006.01) C12N 5/08 (2006.01) kind of national protection available): AE, AG, AL, AM, AOlN 65/00 (2006.01) AO, AT,AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, (21) International Application Number: EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, PCT/US2008/004738 IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, (22) International Filing Date: 11 April 2008 (11.04.2008) MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, (25) Filing Language: English SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (30) Priority Data: GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, 60/923,145 11 April 2007 (11.04.2007) US ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (AT,BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, (71) Applicants and FR, GB, GR, HR, HU, IE, IS, IT, LT,LU, LV,MC, MT, NL, (72) Inventors: HATHAWAY,Alecia [US/US]; P.O. Box NO, PL, PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, 100723, Forth Worth, TX 76185 (US). ORR, William, C. CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). [US/US]; 2075 So. University Blvd. #240, Denver, CO Published: 80210 (US). — with international search report — before the expiration of the time limit for amending the (74) Agents: BURTON, Carol, W. et al.; Hogan & Hartson claims and to be republished in the event of receipt of LLP, 1200 17th Street, Suite 1500, Denver, CO 80202 (US). amendments

(54) Title: AUTOLOGOUS/ ALLOGENEIC HUMAN DNA GRAFTING, ANTI-AND RESERVE AGING STEM CELL, AND AUTOLOGOUS/ALLOGENEIC HUMAN DNA GRAFTING, ANTI-AND REVERSE AGING STEM CELL, AND BONE MARROW COMPOSITIONS/METHODS

RELATED APPLICATION

The present application claims priority of U.S. Provisional Application No. 60/923,145 filed

April 11, 2007, which is incorporated herein in its entirety by this reference.

FIELD OF THE INVENTION

The present invention relates to anti-aging compositions.

BACK GROUND

Autologous stem cells have been found in many adult tissues such as brain, heart, and pancreas, in addition to bone marrow. The major limitation to the therapeutic use of these cells is their relatively quantities and their reduced potential for proliferation. To immortalize such cells, the use of cell feeders and/or growth factor genes to expand stem cell availability raises potential problems with contamination of cell feeders and tumorigenicity. Thus, there is an inherent limitation absent a means for proliferation.

To date, stem cells found in bone marrow, placenta and umbilical cord blood have been used extensively to repopulate the hematopoietic system. The use of cord blood has several unique advantages, including no risk to the donor, low risk of graft-versus-host disease, and rapid availability. However, the major disadvantage of placenta and cord blood transplantation, is the low number of hematopoietic progenitor cells (CD34.sup.+ cells) compared with bone marrow or mobilized peripheral blood. Unfortunately, the populations of pluripotent SCs are also limited.

A major limitation to stem cell-based therapy is the need to generate sufficient numbers of cells retaining their pluripotentiality. Cell number can be increased by introduction of growth stimulatory genes to produce sustained expansion. However, loss of differentiation potential and safety concerns has limited the usefulness of this approach. In addition, promoting cell growth in an undifferentiated state by co-incubation with feeder layers increases the risk for cross-transfer of pathogens. Therefore, the use purified recombinant growth factors has been widely applied to expand stem cell cultures. Thrombopoietin (TPO) has been shown to have multiple functions not only inducing differentiation to a platelet and megakaryocyte phenotype, but also stimulating the proliferation of hematopoietic cells (See B. Fishley, W. S. Alexander, Thrombopoietin signalling in physiology and disease, Growth Factors 22 (2004) 151-155; and D. J. Kuter, C. G. Begley, Recombinant human thrombopoietin: basic biology and evaluation of clinical studies, Blood 100 (2002) 3457-3469).

Blood stem cells (derived from bone marrow) may be able to generate both skeletal muscle and neurons. This facility of AS cells to generate specialized cell types of another type of tissue has been variously referred to as "plasticity," "unorthodox differentiation," or "transdifferentiation." Presently, there is evidence that AS cells can generate mature, fully functional cells, or that the cells have restored lost function in vivo. Collectively, studies on plasticity suggest that stem cell populations in adult mammals are not fixed entities, and that after exposure to a new environment, they may be able to populate other tissues and possibly differentiate into other cell types. However, as in other cases there are fundamental limitations on total SC potency.

Fortunately, several new methods have been devised for collecting, screening and proliferating undifferentiated pluripotent SCs from these and other embryonic, placental and umbilical cord sources, and others have been announced. Further, even other major break¬ throughs in pluripotent collection and proliferation procedures are on the horizon.

Unfortunately, one the greatest therapeutic opportunities for the use of these primordial SCs, when they do become plentiful, has yet to be discovered - e.g. an ability to effectively use them to retard or reverse aging.

Prior art research focuses primarily on utilizing human DNA that is obtained in stem cells harvested from donors other than the intended recipient (non-autologous, i.e. embryonic or fetal stem cells). Still other prior art explores autologous DNA obtained from stem cells collected from the donor in real-time or relatively current to the recipient's age and treatment period. Other art discloses both types involve "injecting" whole stem cells (whether autologous or not) into target tissue to promote repair, such as in spinal cord injury or damaged heart. United States Patent Application, 200401 51706, Shakhov, Alex;et al., August 5, 2004, for example provides a Method for treating a cytopathological disease or other medical condition in a mammal, including the steps of harvesting a biological specimen containing stem cells selected from peripheral blood and/or bone marrow from the body of a donor, then storing the harvested bone marrow for a predetermined waiting period before re-infusing it back into the same donor after the donor later contracts and is diagnosed with one or more of a cytopthalogical illness, a chronic fatigue syndrome, and/or damaged issue.

United States Patent Application 20040258673, Hirose, Thomas Gordon; et al., December 23, 2004, "Elective collection and banking of autologous peripheral blood stem cells (Hirose)," is invention using an individual's own peripheral blood stem cells for future healthcare uses. In Hirose an individual can elect to have his or her own stem cells collected, processed and preserved, while he or she is in healthy or "pre-disease" state, for future distribution for his or her healthcare needs. The Hirose process includes collection, processing, preservation and distribution of adult (including pediatric) peripheral blood stem cells during non-diseased state. Hirose stem cells collected will contain adequate dosage amounts, for one or more transplantations immediately when needed by the individual for future healthcare treatments. The Hirose collected adult or non-neonate child peripheral blood stem cells can be aliquoted into defined dosage fractions before cryopreservation so that cells can be withdrawn from storage without the necessity of thawing all of the collected cells. Hirose mentions in his claims at least 2 different collections at 2 different ages/weights

U.S. Patent Publication 20070053888, Hariri; Robert J., March 8, 2007, "Use of umbilical cord blood to treat individuals having a disease, disorder or condition (Hariri)," provides methods of using cord blood and cord blood-derived stem cells in high doses to treat various conditions, diseases and disorders. Hariri's high-dose cord blood and cord blood-derived stem cells have a multitude of uses and applications, including but not limited to, therapeutic uses for transplantation and treatment and prevention of disease, and diagnostic and research uses. In particular, Hariri's cord blood or cord blood-derived stem cells are delivered in high doses, e.g., at least 3 billion nucleated cells per treatment, where treatment may comprise a single or multiple infusions. The Hariri provides for the use of cord blood or cord blood- derived stem cells from multiple donors without the need for HLA typing. U.S. Patent Publication 200401973 10, Paul R.; et al. October 7, 2004, "Compositions and methods for using umbilical cord progenitor cells in the treatment of myocardial infarction (Sanberg)," provides compositions and methods for treating circulatory disorders, for treating myocardial infarctions, for producing cardiac muscle cells, and for treating injured tissue in an individual. More particularly, the present invention provides methods of treating circulatory disorders by administering an effective amount of a composition comprising an umbilical cord blood cell. In one Sanberg embodiment, the circulatory disorder is myocardial infarction.

U.S. Patent Publication 20060275271, Chow; Robert, December 7, 2006, "PLASMA- DEPLETED, NON-RED BLOOD CELL-DEPLETED CORD BLOOD COMPOSITIONS AND METHODS OF USE (Chow)," provides umbilical cord blood (UCB) compositions that possess the unique features of having plasma that is substantially depleted from the UCB unit and red blood cells (RBC) that are not depleted from the UCB unit. Such UCB units can be prepared by a process that combines plasma depletion with cryopreservation, selection, thawing, and/or transplantation of hematopoietic stem cells to provide superior clinical outcome by maximizing post-processing cell recovery and post-thaw infusion cell dose. Methods for treating a wide variety of malignant diseases and benign diseases associated with the hematopoietic system by administering the UCB compositions of the present invention are also provided.

The prior art (teaching stem cell, cord blood, bone marrow replacement) generally teaches prevention of disease and therapies and compositions are reactive —generally providing treatment after the onset of a disease. The prior art does do not teach proactive periodic primordial (preferably pluripotent) stem cell use with or with bone marrow infusion, much less at a frequency with dosages and conditions capable of affecting a global cellular renewal of all or essentially all cell tissue. The Hariri reference teaches progenitor cells, without suggesting a combination of stem cells with bone marrow in an ex-vivo composition, or a systematic/periodic infusion protocol.

Applicant can find no reference of an anti-aging method or composition employing proliferated primordial stem cells, preferably pluripotent. Nor does the Hirose autologous reference (representing an alternate healthcare insurance system) provide for periodic proactive infusion.

While the prior act incidentally recognizes 'renovating' or rejuvenating the tissues from stem cells, it does not distinguish on the requisite usage of biologically younger primordial stem cells for age reduction. Rather, the art principally deals with stem cells targeted at diseases and specific organs (the brain, the heart, the muscles, localities, end tissues, etc.).

SUMMARY OF INVENTION

The invention relates to an integration of 1) anti-aging compositions incorporating bone marrow, stem cells and combination, with methods of collecting, proliferating and manufacturing same, together with 2) the periodic systematic infusion of these compositions into a recipient, where recipient experiences the establishment of an earlier relative biological clock set-point within his body tissue, with respect to the number of cell generations- divisions. This is seen in the formation of a transient tissue chimera containing two distinct DNA chronological &/or biological aged tissues, where the younger tissue eventually out- replicates, transmogrifying the older tissue, maintained through the continuation/systematic series of swemic introductions (infusion) containing primordial stem cells (SC) and optionally bone marrow (BM) or both.

More specifically, the invention resides in the use of primordial stem cells (preferably pluripotent stem cells) and optionally for both) bone marrow together with a proactive systematic systemic means of introductions (infusion), wherein a global inchoate-transient tissue chimera is formed ever evolving toward a mature tissue chimera, characterized by markedly younger cellular tissues biologically relative to the initial chronological and biological age of the recipient. In other words, Applicants' invention provides for a global renewal of cellular structures with biologically younger tissue throughout recipient's entire body, whereby the natural chronological aging process of recipient is retarded or reversed.

Donor primordial totipotent, pluripotent and omnipotent stem cells, which are matched, do not require HLA typing, transmogrified and otherwise (non graft-versus-host rejecting-

NGvHR) will be employed in order to avoid autoimmune response. The art suggests that primordial SCs are largely absent host immune responses and thus should be largely void of this concern. However, in certain cases in the practice of this invention immune suppression is expressly contemplated. See, e.g., U.S. Publication Nos. 20050123525, 20060159666,

This invention incorporates both autologous and allogeneic methods, wherein the delivery of stem cells of each practice includes markedly younger DNA than the chronological age of recipient and a means to cause the formation of a transient tissue chimera.

An important element of the invention is an ex-vivo composition of compatible (non graft- versus-host rejecting- NGvHR) primordial SCs and bone marrow (BM), activated to enhance the formation of the transient tissue chimera.

Collection of stem cells from a living being (donor) is provided, preferably i) after donor's conception (absent damage to embryo), ii) umbilical cord and/or placenta collection, iii) and/or collection at birth through early life of a donor, are all contemplated. The collection of bone marrow is also an essential element. The preferred stem cell (and bone marrow) collection includes collecting as much as possible before donor's chronological age of 10. Autologous practice additionally includes stem cell and donor collection through out donor- recipient's life.

Augmentation and/or proliferation are important elements and are employed to expand stem cell and where possible bone marrow populations. Compositions and procedure for infusing same and compatible compositions thereof designed to avoid "graft versus host rejection" are essential in this practice and disclosed in the detailed description below.

Young primordial stem cell collection, preferably post conception and at birth, and the proliferation of these SC represent an especially essential element of this invention. Post conception collection of SC are made under the proviso that no damage is ever done to an embryo.

An essential element of the invention is a routine/systematic and periodic transportation (infusion) program for delivering of these anti-aging compositions into recipient such that a transient tissue chimera is formed and maintained. Infusions are contemplated throughout

recipient's entire life, probably not beginning much earlier than age 13 years of age.

This life long infusion regime will become a routine proactive regime consistent with recipient's age, weight, gender, and results desired. Infusion may be either orally, vascular and/or directly into recipient's bone marrow, or a combination. Infusion therapy contemplates single point entry or multiple point entry. Enhancement methods may also be employed, such as signally, stimulation, targeting and the like to increase bone marrow production of SCs after infusion and/or to target certain tissues.

The allogeneic practice of this invention resides in the collection of stem cells of a biologically younger donor, preferably embryonic SCs, at birth (cord or placental SC), and/or before age 10 SCs. These cells will be accumulated, separated, augmented and proliferated to achieve desired quantities and compositions necessary to achieve the objective of applicants' invention.

Stem cell proliferation, particularly contemplates growth of the preferred totipotent and pluripotent stem cells. In the autologous and allogeneic practices young stem cells and bone marrow samples will be stored and preserved, as required.

Thus, the essence of the invention resides in the collection of young DNA containing primordial stem cells (and bone marrow), the proliferation of said stem cells to provided the necessary quantities needed for a period infusion program, and the proactive periodic transfusions of these compatible stem cells, preferably together with bone marrow whereby said infusion program results in an adsorption of younger DNA containing stem cells throughout recipient's entire body, forming a transient tissue chimera —regenerating all cellular, tissue, bone, nerve, muscular and organic structure of recipient with said younger infused DNA.

The practice of this invention contemplates a wide range of mammals, preferably humans.

DETAILED DESCRIPTION OF INVENTION

Applicant's invention is predicated on 5 fundamental elements/steps:

1. Periodic collection of stem cell and/or bone material from a donor, whereby the mass of all collected samples has an average biological age ranging from after conception to 70 years +/-of age, with preferred biological age ranging from after conception to under 50, and a more preferred range from after conception to birth (umbilical cord) to under chronological age 10. Collection/harvesting optionally commence as soon as possible after conception as possible (absent damage to the embryo). 2. Concentration/sorting, augmentation and proliferation of stem cell (and/or bone marrow) such that resultant product is compatible with recipient (as provided below) and of sufficient quantities to be used routinely on a regular/periodic infusion schedule. Preservation and storage as needed. When providing for extended period of storage, storage must secure, appropriately logging, cataloging and controlling as required, does not cause or result in any appreciable aging or disability to samples, and that after storage said stem cell samples and/or bone marrow may be proliferated and augmented as necessary. Augmentation and/or proliferation are expressly contemplated after some period of storage, if any, prior to infusion.

3. Creation of infusion compositions comprising SCs and/or bone marrow.

4. Periodically on a proactive routine transfusion, infusion or grafting (infusion) said SC/BM compositions into the donor after recipient achieving a minimum age

normally not less than 13 years of age, which can benefit from said infusion, anticipated to be periodically over recipient's entire lifetime. The infusion compositions contemplates minimum concentrations of primordial totipotent or pluripotent stem cells (and optionally omnipotent) for minimum potency. Transformation of said SCs in the manufacture of Applicant's BM compositions is anticipated, wherein certain differentiated SC product many be generated. Such primordial SC differentiaton may be inhibited or enhanced.

5. The proactive periodic infusion regime will result in biologically younger transfused stem cells causing a global cellular integration of a body wide transient tissue chimera, which is characterized by the collective tissue composition of chronologically older DNA-containing cells being renewed and transmogrified by the biologically younger DNA-containing infused stem cells.

After each infusion or series of infusions expected biologically age differentials of the resulting cellular transient tissue chimera structure will contain younger tissue at least 3 months, 6 months, 1 year, 2, 3, 4, 5 to 10 years younger (or greater) than the original biological/chronological age of the recipient's cellular structure/tissue (prior to infusion) or the chronological age of recipient, comprising the balance of the chimera - until the tissue is substantially or essentially totally replaced. The tissue chimera will range from an inchoate to a mature tissue with two or more age different tissues. Each series of younger DNA infusion regimes (assuming each regime contains the same age DNA) will non-the-less result in variable ages within any given chimera. After a prolonged continuation of infusions of sufficient duration (and dosage) an inchoate-transient tissue chimera will advance into a more mature chimera, where the younger cells eventually transmogrify the older cells becoming the majority or preponderance of the chimera. Different tissues groups (e.g. skin, heart, nerves, bone, brain, etc) will experience difference rates of turn over under different time lines. Thus, it is expected that different tissue chimera (e.g. different organs) will achieve maturity (wherein a majority or preponderance of tissue is younger compared to the older/oldest tissue; or alternatively where a blend of younger tissue replaces the older tissue, and wherein this blend of younger tissue become a majority of the chimera). Naturally, with this being a life-long program, the chimeras will experience different combinations of younger tissue transmogrifying prior infused young chimera tissue (formerly representing the younger tissue, but now becoming the middle aged tissue, and eventually becoming the older aged tissue).

Naturally, there are many derivatives and variations of this process, which embody the aforementioned elements, and included in the appended claims.

As provided herein infusion and transfusion include injection, transplantation, grafting and/or implantation (infusion), and any other means of delivering SCs and/ BM into recipient. This includes a periodic initial regime and active routine of cellular/tissue maintenance after an initial regime. This routine of infusions is systematic preventative care and administered proactively prior to the onset of any disease, which may be contracted by recipient. It is one of a global regeneration of the body's entire cellular tissue to a early biological clock set point.

Basically, this invention embodies a elegant and unique delivery system to replace the body's older cell structures throughout the entire body with younger more vital DNA containing cells, resulting in a universal transformation of tissue structures and organs (on a global basis) into a younger version. Mammalian cells are pre-programmed, through their genome (DNA mechanism) so as to undergo a finite number of cellular replications - divisions. As the individual cells (which define the total tissue structure) progress along their continuum of number of cellular divisions, their rate of replication slows. Eventually, signs of aging become apparent, as damaged and worn out tissue cells are not timely replaced. Cells become worn, exhausted, damaged from environmental insults, and deprived of necessary co-factors and interactive influences from other failing bodily endocrine and physiologic functions.

Stem cells are cells with the ability to divide for indefinite periods in culture and to give rise to specialized cells. Stem cells are a self-renewing population of cells that can be found predominately within the bone marrow, but can be found in blood in other parts of the body (peripheral stem cells) in certain circumstances. Stems cells can also be found in a newborn's placenta and cord blood at time of delivery.

While research efforts are and have been underway to intervene in the aging process, none propose to simply replace the entire body's cells with younger, fresh cells through a self- adaptive (autologous) grafting process, employing the recipient's very body and DNA - in essence a younger self-cloning process of integrating the old until the new can take over. .

Mammalian stems cell can be collected, stored and re-infused at a later date into the bloodstream of the same being from which they were harvested at an earlier time in his/her life. They will disseminate throughout the entire body, lodging in all tissues. Interacting with factors and determinants specific to differentiated 'resident' tissue cells (already evolved to a specific and distinguishable type of tissue such as bone or muscle, etc.), the stem cells will be induced through interactions with the adopted tissues to 'differentiate' or evolved into the respective distinguishable tissues in which they find residence forming a graft and transient chimera of older and younger DNA-containing cells.

Possessing younger DNA, vital enzymes and co-factors, these younger stem cells will divide at faster rates than their adopted perspective resident mature host tissue cells and then mature into new and younger host cells, "replacing" the older resident cells, eventually resulting in an essentially homogeneous cellular composition with respect to the age of the cells' DNA (thus establishing a younger total body).

COLLECTION OF PRIMORDIAL STEM CELLS (SCs)

Collection and use of proliferated primordial SCs, particularly totipotent, pluripotent and omnipotent SCs is an express requirement/embodiment of this invention. Pluripotent SCs are the preferred SCs of this invention. Numerous methods are contemplated and are provided in the art. For example, United States Patent Application 20040120932, Zahner, Joseph Edward, June 24, 2004, discloses methods for deriving adultpluripotent stem cells from fully differentiated adult somatic cells by in vitro nuclear remodeling; US Patent publication 20020188963 provides for an isolated population of embryonic stem (ES) cells and methods of obtaining these ES cells. In one aspect, the target ES cells are obtained by co-culturing embryo cells from a target with non-target ES cells.

Applicants' invention includes Stem cell collection employing various means directly from an embryo, placenta, cord, cord blood, donor's bone marrow, donor's vascular blood stream, donor vascular department, donor tissue and organs, donor feces, from fetal, neonate and other sources. Donor's may be 3r party donors or autologous (donor-recipient). Recent breakthroughs permit collection of ES cells directly from live embryos absent damaging fetuses. Other means exist including that disclosed in US Patent Publication 20060084168 20030213008 Still others are disclosed below.

Placental stem cell collection provides an excellent resource for totipotent and pluripotent stem cell collection. It has been report that the placenta used as a bioreactor for endogenous cells, can provide various pluripotent and/or totipotent stem cells, by incubating the placenta for 48 hours with perfusate solution. See U.S. Patent 7,045,148, US Patent Publication 20030032179

The conventional placenta technique for SC collection is similar to that of cord blood. It is based on the use of a needle or cannula which is used with the aid of gravity to drain the cord blood from the placenta. Usually the needle or cannula is placed in the umbilical vein and the placenta is gently massaged to aid in draining the cord blood from the placenta. Thereafter the drained placenta has been considered to be of no use and has typically been discarded. A major limitation of stem cell procurement from cord blood has been the frequently inadequate volume of cord blood obtained resulting in insufficient cell numbers to reconstitute bone marrow after transplantation. The method requires access to freshly drained human placentas which have been subjected to a conventional cord blood recovery process by draining substantially all of the cord blood from the placenta. It is important that the placenta be properly stored and drained if it is to be a suitable source of embryonic stem cells. Generally, a placenta should be stored in an anticoagulant solution at a temperature of 5 to 25.degree. C. (centigrade) for no more than 48 hours prior to the collection of the cord blood. Suitable anticoagulant solutions are well known. An acceptable anticoagulant solution comprises a solution of heparin (1% w/w in 1:1000 solution). Generally, the drained placenta should be stored for no more than 36 hours before the embryonic-like stem cells are collected.

Acceptable embryonic-like stem cells (acceptable to Applicants' invention) may be extracted from placenta employing a method is based on the perfusion of the drained placenta with a suitable aqueous fluid such as an anticoagulant dissolved in any suitable aqueous isotonic fluid such as 0.9N sodium chloride solution. The anticoagulant for the perfusion liquid may comprise heparin or warfarin sodium at a concentration which is sufficient to prevent the formation of clots of any residual cord blood. Generally from 100 to 1000 units of heparin may be employed.

The extraction procedure is based on the passage of the perfusion liquid through either or both of the umbilical artery and umbilical vein using a gravity flow into the placenta which is suspended in such a manner that the umbilical artery and umbilical vein are at the highest point. It is preferred to connect the umbilical artery and the umbilical vein simultaneously to a pipette that is connected via a flexible connector to a reservoir of the perfusion liquid which is passed into the umbilical vein and artery and collected in a suitable open vessel from the surface of the placenta that was attached to the uterus of the mother during gestation.

The collection technique is based on the use of a sufficient amount of the perfusion liquid that will result in the collection of the cells left after the drainage of the cord blood. It has been observed that when the perfusion liquid is first collected, the liquid is colored with the residual red blood cells and tends to become clear as the perfusion liquid is passed through the placenta. Generally from 30 to 100 ml of perfusion liquid is adequate to collect the embryonic-like cells but more or less may be used depending on the observed results.

In one embodiment, the placenta may be used as a bioreactor for endogenous cells, including but not limited to lymphocytes and various kinds of pluripotent and/or totipotent stem cells, by incubating the placenta for 48 hours with perfusate solution.

Other methods of collecting placenta SCs are contemplated in Applicants invention, including those provided in US Patent 7,045,148, Hariri (May 16, 2006) and others are provided in US Patent Publications 20070043328, 20050272148 20020123141, 20050276792, 20050148034, 200501 18715, 20040161419, 20040028660 20030235909 20020160510

In the practice of Applicants' invention human stem cell compositions derived from bone marrow and blood are contemplated. Initially, bone marrow cells may be obtained from a source of bone marrow, e.g., iliac crests, tibiae, femora, spine, or other bone cavities. Other sources of human hematopoietic stem cells include embryonic yolk sac, fetal liver, fetal and adult spleen, blood, including adult peripheral blood, placenta and umbilical cord blood. For isolation of bone marrow from fetal bone or other bone source, an appropriate solution may be used to flush the bone, which solution will be a balanced salt solution, conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from about 5-25 mM. Convenient buffers include Hepes, phosphate buffers, lactate buffers, etc. Otherwise bone marrow may be aspirated from the bone in accordance with conventional ways.

Bone marrow contains three stem cell populations, hematopoietic stem cells, bone marrow stromal cells, and (possibly) endothelial progenitor cells. Bone marrow stromal cells are a mixed cell population of cells that generate bone, cartilage, fat, fibrous connective tissue, and the reticular network that supports blood cell formation (mesenchymal stem cells of the bone marrow also give rise to these tissues, and may constitute the same population of cells as the bone marrow stromal cells). Studies of hematopoietic stem cells from bone marrow demonstrate an ability to regenerate an entire tissue system, i.e., all cell types found in blood. Thus, bone marrow shows promise as a source for AS cells exhibiting plasticity, and further development of materials and techniques may allow the utilization of all three stem cell populations found in bone marrow.

Bone marrow compositions substantially free of cells dedicated to a particular lineage, cells carrying markers associated with lineage dedication, wherein the stem cells are able to regenerate and differentiate to populate the various hematopoietic and/or non-hematopietic lineages, are acceptable. A substantially homogenous composition may be obtained by selective isolation of cells free of markers associated with differentiated cells, while displaying epitopic characteristics associated with the stem cells, and by regeneration of the isolated stem cells in defined culture systems leading to different hematopoietic and/or non- hematopietic cell lineages.

The stem cells are characterized by both the presence of markers associated with specific epitopic sites identified by antibodies and the absence of certain markers as identified by the lack of binding of certain antibodies. It is not necessary that selection is achieved with a marker specific for stem cells. By using a combination of negative selection (removal of cells) and positive selection (isolation of cells), a substantially homogeneous stem cell composition can be achieved. A large proportion of differentiated cells to isolate primordial SCs may be removed by initially using a "relatively crude" separation. The source of the cells may be the bone marrow, fetal, neonate or adult or other hematopoietic cell source, e.g., fetal liver, peripheral blood, umbilical cord blood, and the like. For example, magnetic bead separations may be used initially to remove large numbers of lineage committed cells, namely major cell populations of the hematopoietic systems, including such lineages as T-cells, B-cells, (both pre-B and B-cells), myelomonocytic cells, or minor cell populations, such as megakaryocytes, mast cells, eosinophils and basophils. Desirably, at least about 70%, usually at least 80% of the total hematopoietic cells will be removed. It is not essential to remove every dedicated cell class, particularly the minor population members at the initial stage. Usually, the platelets and erythrocytes will be removed prior to sorting. Since there will be positive selection in the protocol, the dedicated cells lacking the positively selected marker will be left behind. However, it is preferable that there be negative selection for all of the dedicated cell lineages, so that in the final positive selection, the number of dedicated cells present is minimized. The stem cells are characterized by being for the most part CD34.sup.+, CD3.sup.-, CD7.sup.-, CD .sup.-, CDIO.sup.-, CDH.sup.-, CD15.suρ.-, CD19.sup.-, CD20.sup.-, CD33.sup.-, and Thy-l.sup.+. A highly stem cell concentrated cell composition is CD34.sup.+, CDIO.sup.-, CD19.sup.- and CD33.sup.-, more particularly in addition CD3.sup.- and CD .sup.-, preferably in addition Thy-l.sup.+. The CD3.sup.-, 8.sup.-, 10.sup.-, 19. sup.-, 20.sup.- and 33.sup.- . The CDlO/19/20 markers are associated with B-cells, CD3/4/8 markers are associated with T-cells, CD 14/ 15/33 cell markers are associated with myeloid cells. The Thy-1 marker is absent on human T-cells. Also, for human CD34.sup.+, rhodamine 123 can divide the cells into high and low subsets. See Spangrude, (1990) Proc. Natl. Acad. Sci. 87, 7433 for a description of the use of rhodamine 123 with mouse stem cells. Preferably these cells are rhodamine low. However, is non-the- less necessary to isolate the rare pluripotent human stem cell from the other cells contained in bone marrow and other hematopoietic sources.

Morphologic evaluation of the 34+Thy+Lin- cells indicates that the multipotent progenitors, "stem cells" are of medium size. Light scatter evaluation shows that "stem cells" have a blast cell profile with low side scatter. These observations indicate that the "stem cells" have a unique density profile. It has been found that the low density fractions from density fractionated human bone marrow are enriched for CD34+Thy+Lin- cells.

Various techniques may be employed to separate the cells by initially removing cells of dedicated lineage. Monoclonal antibodies are particularly useful for identifying markers (surface membrane proteins) associated with particular cell lineages and/or stages of differentiation. The antibodies may be attached to a solid support to allow for crude separation. The separation techniques employed should maximize the retention of viability of the fraction to be collected. For "relatively crude" separations, that is, separations where up to 10%, usually not more than about 5%, preferably not more than about 1%, of the total cells present having the marker, may remain with the cell population to be retained, various techniques of different efficacy may be employed. The particular technique employed will depend upon efficiency of separation, cytotoxicity of the methodology, ease and speed of performance, and necessity for sophisticated equipment and/or technical skill.

Procedures for separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, e.g., complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., plate, or other convenient technique. Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.

One procedure which may be used is in a first stage after incubating the cells from the bone marrow for a short period of time at reduced temperatures, generally about 4.degree. C , with saturating levels of antibodies specific for a particular cell type, e.g., CD3 and 8 for T-cell determinants, the cells may then be washed with a fetal calf serum (FCS) cushion. The cells may then be suspended in a buffer medium as described above and separated by means of the antibodies for the particular determinants, using various proteins specific for the antibodies or antibody-antigen complex.

Conveniently, the antibodies may be conjugated with markers, such as magnetic beads, which allow for direct separation, biotin, which can be removed with avidin or streptavidin bound to a support, fluorochromes, which can be used with a fluorescence activated cell sorter, or the like, to allow for ease of separation of the particular cell type. Any technique may be employed which is not unduly detrimental to the viability of the remaining cells.

Other procedures are found in the art for collecting the SCs of Applicants' invention from bone marrow sources. See generally for example, US Patents 5,914,108, 5,763,197, 5,750,397, 5,739,1 10, 5,716,827, 5,677,136, 5,658,761, 5,087,570, 5,061,620, 5,460,964, 5,436,151, 5,523,286, 5,643,741; US Patent publications 20060018889 200501 18144 20050164380 20030017588.

Cord and Cord Blood Stem Cell Collection is also contemplated and known in the art. For example see US Patent Publication 20060205071, 20040136967. There are a number of methods known in the art for the collection of SCs, which may be used in the practice of Applicants invention..

Generally the STEM CELLS implicated in Applicants' invention include (but are not limited to) embryonic stem cells, embryonic totipotent stem cell, embryonic pluripotent stem cells, embryonic omnipotent (multipotent) stem cells, embryonic progenitor stem cells, bone- derived stem cells, bone marrow/cord derived pluripotent stem cells, bone marrow/cord derived omnipotent stem cells, bone marrow/cord derived progenitors, hemeangioblast stem cells, angiohematopoietic stem cells, multipotent adult progenitor cells, tissue-derived pluripotent stem cell, muscle-derived stem cells, fat-derived stem cells, mesenchymal stem cells, neural stem cells, multipotent adult progenitor cells (MAPCs), vascular pericytes, blood or marrow derived CFU-GEMM, blood or marrow derived CD34.sup.+ stem cells or hematopoietic stem cells, CFU-blast, satellite cells (skeletal myoblast progenitors), angioblasts (endothelial cell progenitors), mesenchymal stem cells, embryonic stem cells. Some of these stem cells, e.g., neural stem cells, CD34+ cells or multipotent adult progenitor cells can differentiate into one or more of osteoblasts, chrondroblasts or chondrocytes, fibroblasts; adipocytes, skeletal myoblasts, smooth muscle cells, cardiac myocytes, endothelial cells, hepatocytes, neurons, glial cells, astrocytes or oligodendrocytes.The preferred practice however includes primordial stem cells, selected from totipotent, pluripotent and multipotent. The most preferred by totipotent and pluripotent . Bone marrow harvesting/collection is also an embodiment of Applicants' invention. Traditionally, bone marrow has been harvested using a bone marrow puncture needle, such as Thomas needle, to puncture ilium. The bone marrow puncture needle is constructed in such a manner as to insert and fit an inner needle into a tubular mantle while allowing the tip of the inner needle to project from the mantle. The bone marrow puncture needle, which is equipped with a handle, percutaneously punctures ilium, and once the tip of the needle reaches bone marrow, the inner needle alone is drawn out and the mantle is kept indwelling the bone marrow to be subsequently used. As a method for harvesting bone marrow using this bone marrow puncture needle, typically, an aspiration method is employed. The aspiration method is such that the mantle of the bone marrow puncture needle is connected to a syringe to collect bone marrow by virtue of the aspiration force. Other methods include a bone marrow perfusion method such that two bone marrow harvesting needles are kept indwelling respective ends of a long bone, such as humerus, with one of the needles connected to an injection syringe and the other connected to a centrifugal tube for harvesting bone marrow via a collection tube, so as to perfuse bone marrow with a medium such as sterilized and heparinized phosphate-buffered physical saline, and thereby a required quantity of bone marrow is harvested into a collection container at a time by injecting the medium slowly into the bone marrow in such a manner as to wash away the bone marrow. This method is desirable for long bone marrow harvesting, because the needle equipped with a drill is capable of providing a more powerful rotational power than a handle. Methods of harvesting and storing BM are provided in US Patents 7,008,394 and 5,456,267.

ENGINEERED BM is also contemplated within the practice of Applicants' invention. In certain instances the use of engineered bone marrow may be preferred. See for example: United States Patent Application, 20060134224, Flake; Alan W. ; et al., June 22, 2006, which provides for engineered bone marrow compositions comprising of bone marrow cells, pulverized bone, and type 1 collagen, which can be transplanted into the portal system of a patient. The engineered bone marrow provides a microenvironment, for engraftment of hematopoietic stem cells. It is anticipated that engineered BM may become a meaningful source of compatible BM for the practice of Applicants invention. Certainly, substitutes and artificial ingredients that accomplish Applicants object are contemplated. Engineered BM may have significant advantages in avoiding "grant v host rejections." SEPARATION will be required of SCs at some point after their collection (optionally after their storage). Collected stem cells will often be contained with other cells and other SCs, which are also collected. Separation must occur and fortunately separation technique also includes opportunities to improve both the quality of the SC material used in the invention, but also eases subsequent proliferation, manufacturing, handling costs and subsequent infusion costs. Separation and other technique anticipated are know in the art. See for example US Patent Application 20030199050 providing for cell separation using electric fields.

PLURIPOTENT STEM CELLS are stem cells that may be obtained from sources such as embryonic tissues, placenta, cord blood, bone marrow, and certain other sources. U.S. Pat. No. 6,200,806 describes pluripotent stem cells obtained from human embryonic tissue. These cells are preferred in the practice of Applicants invention because they are capable of proliferating in vitro without significant karyotype changes while maintaining a capacity to differentiate into endoderm, mesoderm, and ectoderm tissues. These cells are negative for the SSEA-I marker, positive for the SSEA-4 marker, and express alkaline phosphatase activity. These cells have euploid karyotypes and none of the chromosomes are obviously altered. U.S. Pat. No. 5,843,780 describes a purified preparation of primate embryonic stem cells that is capable of proliferation in an in vitro culture for and maintains a karyotype in which all the chromosomes characteristic of the primate species are present and not noticeably altered through prolonged culture. These cells maintain a potential to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues throughout the culture. These cells will typically not differentiate when cultured on a fibroblast feeder layer and they can differentiate to trophoblasts and produce chorionic gonadotropin when cultured at a high density.

Pluripotent cells have been obtained from preimplantation embryos of several animals, e.g., Evans, et al., Theriogenology 33(1): 125-128, 1990; Evans, et al., Theriogenology 33(1): 125- 128, 1990; Notarianni, et al., J. Reprod. Fertil. 41(Suppl.):51-56, 1990; Giles, et al., MoI. Reprod. Dev. 36:130-138, 1993; Graves, et al., MoI. Reprod. Dev. 36:424-433, 1993; Sukoyan, et al., MoI. Reprod. Dev. 33:418-431, 1992; Sukoyan, et al., MoI. Reprod. Dev. 36:148-158, 1993; lannaccone, et al., Dev. Biol. 163:288-292, 1994). Human embryonic carcinoma cells, which are pluripotent cells obtained from teratocarcinomas resemble human embryonic stem cells (Andrews, et al., Lab. Invest. 50(2): 147- 162, 1984; Andrews, et al., in: Robertson E., ed. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Oxford: IRL press, pages 207-246, 1987). Embryonic carcinoma cells can be induced to differentiate in culture, which is characterized by the loss of specific cell surface markers (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) and the appearance of new markers. As provided above Pluripotent SCs can be found in BM, placenta, cord, cord blood, certain tissue and other sources. See 20060216821. Applicants' invention expressly contemplates protocols and technique for the isolation and collection of pluripotent SCs.

An optimal Pluripotent SC source includes EMBRYONIC stem (ES) cells, which can proliferate indefinitely in an undifferentiated state. Furthermore, ES cells are readily easy to separate into primordial totipotent or pluripotent cells. These primordial SCs can generate into all of the cells present in the body (bone, muscle, brain cells, etc.). ES cells have been isolated from the inner cell mass of the developing murine blastocyst (Evans et al., Nature 292:154-156, 1981; Martin et al., Proc. Natl. Acad. Sci. U.S.A. 78:7634-7636, 1981; Robertson et al., Nature 323:445-448, 1986; Doetschman et al., Nature 330:576-578, 1987; and Thomas et al., Cell 5 1:503-512, 1987; U.S. Pat. No. 5,670,372). Additionally, human cells with ES properties have recently been isolated from the inner blastocyst cell mass (Thomson et al., Science 282:1 145-1 147, 1998) and developing germ cells (Shamblott et al., Proc. Natl. Acad. Sci. U.S.A. 95:13726-13731, 1998) (see also U.S. Pat. No. 6,090,622, WO 00/70021 and WO 00/27995).

REVERSE GENETIC TRANSITION/ENGINEERING of committed (omnipotent or progenitor) SCs back into pluripotent SCs form is also contemplated [cites ]

PROLIFERATION OF PRIMORDIAL SC is an essential embodiment.

Applicant's invention is predicated upon one or more methods of proliferating collected primordial SCs, especially pluripotent SCs (while all SCs may be included). Existing proliferation methods are now available, which appear able to achieve Applicants' quantity needs to practice this invention, and more processes are expected. See for example, United

States Patent Application 2007001001 1, Parsons; Xuejun Huang ; et al., January 11, 2007, which provides for cultivation in serum-free, feeder-free, and conditioned-medium-free - the long-term growth of undifferentiated pluripotent stems. Also, see 20060030040, Yang; Mei- Ju ; et al. February 9, 2006, which discloses a method of culturing embryonic stem cells that remain substantially undifferentiated while maintaining their pluripotency to differentiate into subsequent germ layer cells. Others provide for pluripotent SC proliferation from bone marrow culture, see 20070077201, 20060134784. Still others from placenta, cord and cord blood, see 20070059824, 20060134784. 20060078993. Also, see US Patent Applications 20060216821, 20060127370, 20050221480, 20040087016, 20030212024, 20030186439. In 20060127370, Niwa; Hitoshi ; et al., June 15, 2006, provides for the proliferation of undifferentiated pluripotent stem cells that retain their differentiation potency by culturing the pluripotent stem cells in a medium free of a feeder cell, or a serum. This is attained by using a culture medium for pluripotent stem cells, which is supplemented with an inhibitor of an adenylate cyclase activity. In United States Patent Application, 20060030041, Furcht; Leo T., et al., February 9, 2006, provides for isolated stem cells of non-embryonic origin in their undifferentiated or differentiated state to form cells of multiple tissue types.

Thus, proliferation of placenta, cord, cord blood, bone marrow and vascular department derived pluripotent SCs is expressly contemplated in the invention. See U.S. Patent 5,914,108, Publications 20060134784 and 20070059824, with the later disclosing methods of providing for umbilical cord blood-derived pluripotent fibroblast-like-macrophages.

The AUGMENTATION step - like the proliferation step, may be exercised at any stage of the invention, including after collection, prior to storage, after storage, in the manufacture of the sera contain SCs, or prior to infusion.

Numerous forms of augmentation and augmentation methods are anticipated. Unlimited examples include augmentation to increase SC potency, reduce weaker SC product, increase population of stem cells, reduce or reverse differentiation or differentiation potential, specialized differentiation/tissue genetic targeting, avoiding NGvHR or immune response desensitizing, initiation of primordial SC proliferation, initiation of SC division, signaling, targeting, and the like. An example of a hematopoietic augmenting method includes United States Patent Application 20030054549, Takebe, Minoru ; et al., March 20, 2003, which provides a stem cell-augmenting material capable of augmenting hematopoietic stem cells of bone marrow, and embryonic stem cells- using isoflavone aglycone (which has estrogen-like activity) that does not block the enzyme activity of cell proliferation factor. Also see U.S. Patent Publication Nos. 20060233840 20060034767 20030022249 20020183297 20060068414

STORAGE AND STEM CELL BANKING, logging, security and other measures insuring preservation and storage (long term and short term) are contemplated. Numerous art references incorporate the elements of the requisite system(s), which must be employed in this invention. It is known that Stem cells are routinely cryopreserved (see e.g., Boyse et al., U.S. Pat. No. 5,004,681, "Preservation of Fetal and Neonatal Hematopoietic Stem and Progenitor Cells of the Blood", Boyse et al.,) See U.S. Pat. No. 5,192,553 and 20050106554, with the later providing for cryopreservation of pluripotent stem cells. Other art provides for extended SC preservation at ambient temperature. Also see US Patent Publications 20060167401 20050276792, 20040258673 20060233768 20060095319 20060063141 20060060494 20040258673 20030220244 20030054331 20020094550

STEM CELL CULTURJNG, MAINTENANCE, GROWTH AND PRESERVATION METHODS ~ The culturing, maintenance, preservation, enhancement, growth and storage of collected BM and SC material, cultures, and combination BM/SC compositions (including Applicant's "Activated" BM/SC composition) are an important element of this invention. The art provides numerous methods of variously achieving of these objectives, which are incorporated in full by reference. Incorporated referenced US Patents include 6,673,904, 6,667,391, 5,914,108 and incorporated referenced US Patent Publications (not intended to be limiting) include: 200700781 13,20070073186, 20070072830, 20070072293, 20070059826. 20070054258 20030166894 20020132224 20030180784 20070053890, 20070050218, 20070048726, 20070048350,20070042341 20070042337 20070026042 20070026036 20070025961 20070015830 20060293724 20060292689 20060280727 20060275886 20060270038 20060269907 20060258003 20060246043 20060223050 20060210544 20060204482 20060200043 20060188867 20060167401 20060148017 20060142818 20060134070 20060134069 20060129225 20060127375 20060122373 200601 16603 20060094693 20060093999 20060083720 20060078872 20060063738 20060063719 20060057137 20060040894 20060034941 20060024657 20060024386 20060019234

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20050164380

The PREPARATION of Applicants infusion SC and optionally B M compositions may also

employ various differentiation inhibition methods/agents- in order to prevent differentiation

and in certain cases proliferation, as may be required. Thus, inhibiting methods and agents may be required in the preparation of the ex-vivo primordial SC proliferation cultures and in the formulation of Applicants' SC/BM composition(s), including Applicants' activated composition. Inhibiting agents are well known in the art. See 6. Qi, X. et al. BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways. Proc Natl Acad Sci USA 101, 6027-6032 (2004); Smith, A. G. et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336, 688- 690 (1988); Williams, R. L. et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336, 684-687 (1988); Ying, Q. L., Nichols, J., Chambers, I. and Smith, A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115, 282-292 (2003). Also see for example United States Patent Application 20050153941, Miyabayashi, Tomoyuki ; et al., July 14, 2005 which provides for a differentiation inhibiting agent which allows an embryonic stem cell culture in an undifferentiated state without use of any feeder cell, prepared by culturing using a differentiation inhibiting agent of a tetrahydroisoquinoline derivative. Other references disclosing differentiation/proliferation inhibiting agents/methods, include US Patent 7,1 15,267, 6,432,917, 6,022,848, 5,861,483 ; and, US Patent Publications 200501 18713, 20040229350, 20030082803, 20050153941, 20050106725 20050266553 20050079607

Likewise, inducement of proliferation and/or differentiation may also be required in the practice of the invention. The art provides for a number of inducing means. See for example US Patent 7,1 15,267, 5,650,299 and US Patent publications 20050239201 "Methods of inducing differentiation of stem cells into a specific cell lineage." Also see 20050227353, 20050191744.

Bone Marrow Activation and Stimulation

The bone marrow is presumed to be more replete with SCs than any other body site, and is represented by all lines-types in proportion to their level of differentiation. Recipient's in- vivo BM will serve as the germination centers or factories of SCs generation in the practice of this invention. BM contains other critical constituents that serve in the process of SC differentiation and specific tissue commitment (i.e. production of progenitor SCs and the like). Applicants' invention utilizes recipient's BM itself to promote and produce, through its natural processes, the needed younger SCs destined to assume residence within specific tissue location.

Direct infusion of Applicant's SCs compositions, including said activated composition inspires recipient's own BM to commence the eventual manufacturing of the desired SC, progenitors and others that will be delivered to tissue sites throughout recipient's body. Thus, toward this goal, the BM is appropriately stimulated and/or activated by one of a variety of disclosed means under this invention to accelerate natural production and deployment of younger DNA aged containing SCs of varying differentiation - SCs from the newly formed seeded BM centers, which have formed as a consequence of the systematic systemic introductions/infusions of the biologically younger DNA containing SCs (of applicants' invention) into the recipient sera/BM.

In the practice of this invention, recipients' BM will ultimately generate sufficient younger DNA containing SCs to populate recipients' entire body. These elevated levels of SCs must be for a reasonable to a reasonably prolong period to effectuate Applicants' tissue chimeras. Elevated SC activity can generally be measured as a function of the elevated stem cell count in recipient's blood stream (a provided herein). Thus, elevated SC count in recipient's blood stream is normally a function of new steam cell generation from recipient's BM -in turn resulting from the practice of Applicants' invention.

Applicants' expect that there may be a gestation period for recipient's own bone marrow to become, itself sufficiently regenerated with the introduction of young DNA containing SCs, before it will in turn generate the increase in necessary SCs for the rest of the body. Thus, the need for a systematic infusion program to commence this indigenous BM activity.

Naturally, immediately after a vascular infusion/injection in the blood stream, one would expect the SC count to be elevated.

However, the indigenous generation of SCs within the bone marrow is a preferred practice of this invention, where said self-generated younger DNA age containing SCs ultimately incorporate into the recipient's end organs and tissues in which the younger DNA containing and, thus eventually differentiated cells (also through promotion and stimulation processes) assume a statistical preponderance defining the recipient's body. AVOIDANCE OF GRAFT-VERSUS-HOST OR AUTOIMMUNE REACTION is an important element of this invention. Graft-versus-host reaction is an inflammatory response that is unique to allogeneic transplantation. It is an attack of the "new" SCs or bone marrow's immune cells against the recipient's tissues. This can occur even if the donor and recipient are HLA-identical because the immune system can still recognize other differences between their tissues. It is aptly named graft-versus-host reaction because bone marrow transplantation is the only transplant procedure in which the transplanted cells must accept the body rather than the body accepting the new cells.

Acute reaction typically occurs in the first 3 months after an infusion or transplantation and may involve the skin, intestine, or the liver. Chronic graft-versus-host reaction may also develop after allogeneic transplant and is the major source of late complications. In addition to inflammation, chronic graft-versus-host reaction may lead to the development of fibrosis, or scar tissue, similar to scleroderma or other autoimmune reactions and may cause functional disability, and the need for prolonged immunosuppressive therapy. Graft-versus- host reaction may be mediated by T cells when they react to foreign peptides presented on the MHC of the host. Removal of these T cells before donation can lessen the risk of this reaction. As provided herein Applicant's invention is to be practiced in a way to avoid any graft-versus-host reaction.

The use of embryo, placenta and cord SCs substantially mitigates these concerns, as these are normally non-HLA typing.

However, in certain practice an immune reaction is expressly contemplated and methods of suppression will be administered. Various means are contemplated and many are within the skill of the art. See for example, United States Patent Application 20060147428, Sachs; David H., July 6, 2006, which provides methods for restoring or inducing immunocompetence, including the step of introducing donor thymic tissue into the recipient. Autoimmune suppression is thus a contemplated practice and is know in the art. Also see US Patent Publications 20040156834, 20070077201, 200601 11316, 20040198762, 20060153819 Other methods include adaptation in-vitro, wherein graft v host mitigation takes place. For example in donor SC and/or BM compositions, where said compositions include recipient-host DNA and adaptive means, prior to injections. A beneficial aspect of the Graft-versus-Host phenomenon is the "GRAFT VERSUS TUMOR EFFECT" or "graft versus leukemia" effect. For example, leukemia patients with chronic graft-versus-host reaction after an allogeneic transplant have a lower risk of leukemia relapse. This is due to a therapeutic immune reaction of the grafted donor lymphocytes, more specifically, the Natural Killer cells, against the reacted bone marrow of the recipient. This lower rate of relapse accounts for the increased success rate of allogeneic transplants compared to transplants from identical twins, and indicates that allogeneic HSCT is a form of immunotherapy. Graft vs tumor is also the major mechanism of benefit of non-myeloablative transplants which do not employ high dose chemotherapy or radiation. As provided herein Applicant's invention may be practiced in order to enhance any graft-versus-tumor effect and therefore will employ appropriate compositions and method that elicit an immune response.

Formation of a dual aged global TRANSIENT TISSUE CHIMERA is one of the most essential elements and objects (if not the most important) of Applicants' invention. It is one of the most distinguishing structures of Applicants invention over the prior art.

Applicants' Transient Tissue Chimera is a distinct in-vivo tissue composition/structure comprised of two genetically distinct types/DNA tissue cell ages. In Applicants' invention a transmogrification of sorts occurs wherein there is a combination of younger aged DNA tissue in a tissue structure together with older DNA containing tissue. The evolvement of this transient tissue chimera eventually produces a replacement of the older tissue with the younger tissue. This transmogrification is a cumulative process and requires systematic infusions (multiple infusions/reinfusions) of Applicants' SCs (and/or SC/BM) compositions, in accordance with the practice of this invention. .

Human chimeras were first discovered with the advent of blood typing when it was found that some people had more than one blood type. Most of them proved to be "blood chimeras" ~ non-identical twins who shared a blood supply in the uterus. Those who were not twins are thought to have blood cells from a twin that died early in gestation. Twin embryos often share a blood supply in the placenta, allowing blood stem cells to pass from one and settle in the bone marrow of the other. About 8% of non-identical twin pairs are chimeras. Many more people are microchimeras and carry smaller numbers of foreign blood cells that may have passed from mother across the placenta, or persist from a blood transfusion. In vitro fertilization (IVF) is also contributing to the number of human chimeras. To improve success rates, two or more embryos are placed in the uterus so women who have IVF have more twin pregnancies than usual. More twins mean more chimeras. In Greek mythology, the Chimera was an awesome fire-breathing monster with the head of a lion, the body of a goat, and the tail of a serpent. The Chimera was killed by the hero Bellerophon mounted, in most versions of the tale, on Pegasus, the winged horse.

In Applicant's invention a "transient tissue chimera" is not an organic original structure, but rather a new structure being introduced in-vivo. This new tissue structure commences as a two part distinctively intertwined differing DNA aged tissue ~ having two distinct ages and likely two distinct sets of genomes and/or DNA/RNA. The blended/interspersed or dual DNA aged tissue is heterogeneously interwoven within a given tissue structure (muscle, nerve, brain, bone, etc.). The age differential within the chimera eventually diminishes with/after the continuation of periodic (regular/sustained) infusion of the same younger DNA- with the younger DNA tissue of the chimera ultimately dominating, replacing the older DNA tissue. While the entire biological process is still yet known, a global renewal of tissue of all (or virtually all) tissue groups is contemplated.

1. EXAMPLE —An in-vivo transient tissue structure comprised of [at least] two distinct DNA ages, optionally being at least 10 years in age difference.

2. EXAMPLE ~A transient tissue structure comprised of [at least] two distinct DNA aged genomes.

3. EXAMPLE —A transient tissue structure comprised of [at least] two distinct DNA aged tissues whereby youngest DNA tissue transmogrifies and replaces the older DNA tissue within said chimera.

4. EXAMPLE —A transient tissue structure comprised of [at least] two distinct DNA ages, whereby said structure incorporates one or more vital organs and the age difference between DNA is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more, years.

5. EXAMPLE —A transient tissue structure comprised of [at least] two distinct DNA ages, whereby said structure incorporates the majority of the body's tissues, and the age difference between the DNA is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 years.

6. EXAMPLE —A transient tissue structure comprised of two distinct DNA ages, whereby the younger DNA tissue comprises a plurality of at least 10%, 20%, 30%, 40%, or a majority of 50%, 60%, 70%, 80%, 90%, 95%, or more, of the chimera.

7. EXAMPLE ~A transient tissue structure comprised of two distinct DNA ages, whereby the younger DNA tissue transmogrifies the older DNA containing tissue and become 95%, or more [100%], of the chimera.

8. EXAMPLE —A transient tissue structure of the above Examples, wherein after a single, or series of continuing, infusions containing stems cells of said younger DNA, said transient tissue structure of said chimera becomes predominately the age of the younger DNA.

9. EXAMPLE—The Examples above, wherein said chimera is produced after multiple periodic infusion of composition containing a plurality of SCs, and optionally bone marrow, optionally derived from a donor and/or recipient-host (an autologous donor).

10. EXAMPLE —The Example above, wherein after multiple periodic infusion recipient's blood stream enjoys a minimum of a 500% increase in stem cell population (optionally, as measured by use an appropriate marker(s) provided herein or that is otherwise provided in the art).

INJECTION or INTRODUCTION (INFUSION) of Applicants SC and/or BM compositions, as provided herein includes transfusion, infusion, injection, ingestion, transportation, engraftment, inoculation, adsorption, absorption, intercalation, nano-continuous/nano -based or supported transport, physical transport, and all other transport means, whereby Applicants' compositions can be delivered/provided into recipient's body, body tissue, rather orally, via bone marrow, skeletal system, blood stream, vascular department, or any into or by any other system . As provided herein and in Applicants' appended claims term "introduction," "transportation/transport" and "infusion" are interchangeable terms.

PERIODIC INFUSION REGIMES - As provided herein, an essential element of Applicant's invention is its periodic frequency and active/systematic regimes of infusions, which are required to form the transient tissue chimera. Single and/or haphazard infusions of SCs are not acceptable and do not accomplish Applicants' object of creating a body wide transient tissue chimera, where younger DNA containing tissue eventually replaces older DNA containing tissue.

11. EXAMPLE - A periodic infusion of SC and optionally BM made on an average frequency 1 to 12 times/month over a period ranging from 1 to 12 months/year [ideally continuously for a period of at 2-4 months] with at least two infusions in this

12 month period, and then follow-on infusions of a minimum one infusion per year for a continuous period of time thereafter, or on another frequency sufficient to create a transient tissue chimera characterized as having two distinct DNA tissue ages [optionally, wherein said age difference is at least 10 year, and wherein at least 10%, 25%, or 50% of the chimera tissue is said younger DNA aged tissue].

12. EXAMPLE - A periodic infusion of SC and optionally BM to a recipient under chronological age 20 with said infusion made on an average frequency of 1 to 12 times/month over a cycle period ranging from at least 1 to 3 consecutive months with 1or more cycles in any 12 to 12 month period, or on another frequency sufficient to create a transient tissue chimera characterized as having tissue of two distinct DNA ages [optionally, wherein said age difference is at least 10 year, wherein at least 10%, 25%, or 50% of the chimera tissue is said younger aged DNA].

13. EXAMPLE - A periodic infusion of SC and optionally BM to a recipient over chronological age 20 with said infusion made on an average frequency of 1 to 12 times/month over a cycle period ranging from at least 1 to 3 consecutive months with

1, or on another frequency sufficient to create a transient tissue chimera characterized as having tissue of [at least] two distinct DNA ages [optionally, wherein said age difference between youngest and oldest is at least 10 years, wherein at least 10%, 25%, 30%, 40%, 50% or more of the transient tissue chimera is said youngest aged DNA].

14. EXAMPLE - A periodic infusion of SC and optionally BM into a recipient at least 30 years of age, wherein said periodic introductions/infusions are made on an average frequency of 1 to 12 times/month over a cycle period ranging for at least 2 consecutive months [preferably 3 or more] within a 12 month period, and on an annual cycle of at least one infusion thereafter for in a period of 1- 100 years, or on another frequency sufficient to create a transient tissue chimera characterized as having tissue of [at least] two distinct DNA ages [optionally, wherein said age difference between youngest and oldest is at least 10 years, wherein at least 10%, 25%, 30%, 40%, 50% or more of the transient tissue chimera is said youngest aged DNA].

15. EXAMPLE - A periodic infusion of SC and optionally BM into a recipient on an average frequency of at least once in a single month over a period of 1 to 6 months period or on another frequency sufficient to create a transient tissue chimera characterized as having tissue of [at least] two distinct DNA ages [optionally, wherein said age difference between youngest and oldest is at least 10 years, wherein at least 10%, 25%, 30%, 40%, 50% or more of the transient tissue chimera is said youngest aged DNA].

16. EXAMPLE —A periodic infusion of SC and optionally BM into a recipient over chronological age 20, wherein said introduction/infusion is made on an average frequency of 1 to 12 times/month over a cycle period ranging from at least 1 to 3 consecutive months with 1 or more cycles in any given annual period, or on another frequency sufficient to create a transient tissue chimera characterized as having tissue of [at least] two distinct DNA ages [optionally, wherein said age difference between youngest and oldest is at least 10 years, wherein at least 10%, 25%, 30%, 40%, 50% or more of the transient tissue chimera is said youngest aged DNA].

17. EXAMPLE —A periodic infusion of SC and optionally BM on an average frequency of 1 to 4 times/month for a least one month over a 6 month period, or on another frequency sufficient to create a transient tissue chimera characterized as having tissue of [at least] two distinct DNA ages [optionally, wherein said age difference between youngest and oldest is at least 10 years, wherein at least 10%, 25%, 30%, 40%, 50% or more of the transient tissue chimera is said youngest aged DNA]. 18. EXAMPLE - A periodic infusion of SC and optionally BM on an average of once to four times a month for a period of at least two to nine consecutive months in any single calendar year, or on another frequency sufficient to create a transient tissue chimera characterized as having tissue of [at least] two distinct DNA ages [optionally, wherein said age difference between youngest and oldest is at least 10 years, wherein at least 10%, 25%, 30%, 40%, 50% or more of the transient tissue chimera is said youngest aged DNA].

19. EXAMPLE - A periodic infusion of SCs and optionally BM, including the EXAMPLES above, wherein said infusion is made at least once a month in a cycle ranging from 2 to 6 (or more) consecutive months of any given annual period.

20. EXAMPLE —A periodic infusion of SCs and optionally BM, including the EXAMPLES above, wherein said infusion is made at least once a month in a cycle ranging from 2 to 12 consecutive months of any annual period.

2 1. .EXAMPLE —A periodic infusion of SCs and optionally BM on a frequency sufficient to create a transient tissue chimera characterized as having tissue of [at least] two distinct DNA ages [optionally, wherein said age difference between youngest and oldest is at least 10 years, wherein at least 10%, 25%, 30%, 40%, 50% or more of the transient tissue chimera is said youngest aged DNA].

22. EXAMPLE - A periodic infusion regime of SCs and optionally BM wherein a transient tissue chimera is formed containing at least two distinct DNA ages, and wherein after a continuation of infusions containing SCs and optionally BM, said transient tissue chimera enjoys a majority population of tissue being the youngest DNA age.

The art suggests stem cells comprise approximately 0.1-1.0% of the total nucleated cells as measured by the surrogate CD34+ cells. See 20040258673. The formation of a dual aged transient tissue chimera is predicated upon a periodic infusion regime with sufficient dosage that in turn operates to elevate recipients' bone marrow and blood stream stem cell count for a prolonged period of time. While minimal and upper levels and durations are yet unknown, it is expected that there will be some upper limit where the body's spleen and other organs will disoose of excess quantities However minimum bone marrow and blood stream quantities of 0.3 to 3.0% of the total nucleated cells are expected minimums. Minimums of 1% to 4%, with about 3% of the total nucleated cells in the bone marrow or blood stream being SCs over a prolonged period of at least 1 to 52/weeks within a year is preferred.

23. EXAMPLE - A periodic infusion regime of SCs and optionally BM, wherein after infusion for a period of at least 3 days to 52 weeks in any given year, recipients average blood stream SC volume is at least .0.2 to 5.0% of total nucleated cells.

24. EXAMPLE - A periodic infusion regime of SCs and optionally BM, wherein after infusion for a period of at least 2 weeks to 52 weeks in any given year, recipients average blood stream SC volume is at least .0.3 to 5.0% of total nucleated cells.

25. EXAMPLE - A periodic infusion regime of SCs and optionally BM, wherein after infusion for a period of at least 4 weeks to 52 weeks in any given year, recipients average blood stream SC volume is at least .0.3 to 5.0% of total nucleated cells.

26. EXAMPLE - A periodic infusion regime of SCs and optionally BM, wherein after infusion for a period of at least 8 weeks to 52 weeks in any given year, recipients average blood stream SC volume is at least .0.3 to 5.0% of total nucleated cells.

27. EXAMPLE - A periodic infusion regime of SCs and optionally BM, wherein after infusion for a period of at least 12 weeks to 52 weeks in any given year, recipients average blood stream SC volume is at least .0.3 to 5.0% of total nucleated cells.

28. EXAMPLE —A periodic infusion regime of SCs and optionally BM, wherein after infusion for a period of at least 8 weeks to 52 weeks in any given year, recipients average blood stream SC volume is at least .1.5 to 5.0%. of total nucleated cells

29. EXAMPLE —A periodic infusion regime of SCs and optionally BM, wherein after infusion for a period of at least 12 weeks to 52 weeks in any given year, recipients average blood stream SC volume is at least 1.5 to 5.0% of total nucleated cells.

30. EXAMPLE - A periodic infusion regime of SCs and optionally BM, wherein after infusion for a period of at least 8 weeks to 52 weeks in any given year, recipients average blood stream SC volume is about 2.0%, or greater, of total nucleated cells.

Appropriate markers may be used to measure these volumes. Additional volumes/amounts H t l t d j id d i th S l t l S if ti A principal embodiment of applicants' invention is predicated upon stimulating the inherent production of these younger DNA containing SCs within the body's own bone marrow. By producing a continuous volume of these younger DNA containing SCs itself, the body will naturally generate the means for creating Applicants' global transient tissue chimera. Thus, Applicants' dosages and treatment regime of periodic infusions, which produce the dual age transient tissue chimera, are a critical element of this invention.

IN-VIVO STEM CELL EXPANSION primarily via bone marrow activity, which is accomplished via Applicants' dosages, SC and/or BM compositions, periodic infusions is contemplated. It is the enhanced activity of the bone marrow, in turn producing younger aged DNA containing SCs that fill the recipient's body for a continued period of time, in turn creating the dual age transient tissue chimera that represents the essence of Applicants' invention. Thus, Applicants' invention contemplates various methods of improving SC expansion and proliferation in-vivo after infusion. Applicant will also employ existing method in achieving this goal. See for example United States Patent Application, 20070077201

Employing MARXERS may be appropriate to determine if minimal SC volumes are achieved. Applicants' contemplated practice and appended claims provide for various increases of blood stream populations of stem cells. Different compositions and methods are likely to product different results, different stem cell types, which will populate the blood stream. It is contemplated that various markers will be established as appropriate to determine that these limitations (results) are secured. In the case of hematopoietic progenitor cells, it is known that CD34.sup.+ cells may be a measuring determinate. A highly stem cell concentrated cell composition could see a CD34.sup.+, CD 10.sup.-, CDl 9.sup.- and CD33.sup.-, more particularly in addition CD3.sup.- and CD8.sup.-, preferably in addition Thy-l.sup.+. The CD3.sup.-, 8.sup.-, 10.sup.-, 19.sup.-, 20.sup.- and 33.sup.- .The CDl 0/1 9/20 markers are associated with B-cells, CD3/4/8 markers are associated with T- cells, CD14/15/33 cell markers are associated with myeloid cells. The Thy-1 marker is absent on human T-cells. Also, for human CD34.sup.+, rhodamine 123 can divide the cells into high and low subsets. See Spangrude, (1990) Proc. Natl. Acad. Sci. 87, 7433 for a description of the use of rhodamine 123 with mouse stem cells. Preferably the cells are rhodamine low. However, the blood circulation of embryo totipotent, pluripotent and multipotent SCs (or proliferated derivations) may implicate different markers.

ALLOGENEIC PRACTICE~The allogeneic embodiment of this invention is similar to the autologous practice, except that the original source of the biologically younger stem cell is from a party other than recipient. In this practice an essential element is the conversion or use of a stem cell that is DNA compliant or modifiable to the DNA of recipient. Research suggests a species of pluripotent stem cells from a 3rd party donor may be sufficiently absent DNA characterization to become DNA compliant to that of recipient. See (cite).

DNA compliance under this method, as in the case of the autologous practice, is achieved when the characterization of the transfused stem cell is fully recognized as being biologically younger DNA capable of causing the global integration of stem cells in recipient whereby these biologically younger DNA containing stem cells are integrated into recipient's tissues throughout his/her entire body. This integration forms a transient chimera, which is characterized by the collective tissue composition of the recipient whose chronologically older DNA-containing cells are renewed by the biologically younger DNA-containing infused stem cells, whereby the biologically age differential of resulting cellular structure comprising the resulting transient tissue chimera is 5 years younger or more than the original biological age of the recipient's cellular structure/tissue prior to infusion.

Combination of recipient's DNA with biologically younger -transfusion non-HLA typing donor stem cells —in either an in-vitro or in-vivo process and combination process, whereby a younger identical DNA, DNA compliant or otherwise sufficiently similar stem cell to recipient results, such that it is sufficient to effect a global renewal/integration of recipient's cellular tissue with biologically younger DNA as provided under Applicants' infusion means.

The stem cells of this practice include the entire continuum of undifferentiated to differentiated or tissue committed stem cells. In the autologous practice of this invention the collection of a full range of undifferentiated and more differentiated or tissue committed stem cells from the donor for preservation until later transfusion is contemplated. Cell proliferation (augmentation) without new or further differentiating of such is expressly contemplated where the population of stem cells, in the ratio collected, can be preserved. Laboratory proliferation of differentiated cells—e.g. heart muscle cells, blood cells, or nerve cells, is controlled by the chemical composition of the culture medium, alteration of the surface of the culture dish, or by cellar modify by insertion of specific genes. See Chapters 5-9 and Appendices B and C of the NIH report Stem Cells: Scientific Progress and Future Research Directions.

The essence of this invention is the periodic transfusion of sufficient plurality of younger DNA stem cells compared to the biological age of the recipient, whereby said cells are absorbed into the entire body so as to retard the biological aging process.

Thus, the method contains various means and combination to cause the integration of these biologically younger stem cells to be absorbed and included in the tissues throughout recipient's body, whereby recipient's natural biological aging process is retarded. This is an essential element of the invention.

The dosage levels of the transfused stem cells are expected alone to be sufficient to achieve this result by natural absorption. Thus, the effective concentrations and quantities and frequency of transfused stem cells into the recipient are an essential element of this invention. Furthermore, the collection of bone marrow and its later infusion into recipient, independently of and/or simultaneously with stem cells is expected to also achieve this result. Thus, the infusion of young bone marrow directly or indirectly into recipient's bone marrow is contemplated means. Another would be the infusion of stem cells directly or indirectly into the bone marrow (simultaneously with/or independently of bone marrow infusion). Stimulation of bone marrow activity prior to, during, and after stem cell transfusion is an express embodiment.

Applicants' believe that enhanced bone marrow activity with corresponding production of stem cells and related structure is a critical element in achieving the result of global tissue renewal under this invention. Independent and simultaneous use of signalers, cellular receptors, physiological techniques, drugs, meditation, psychological techniques, electronic stimuli, audio inducers/stimuli (music), and other means are contemplated. Signaling means to encourage specialized adsorption and usage of transfused stem cells.

This means of insuring that said infusion achieves a global integration of biologically younger stem cells within the chronologically older DNA containing cells contemplates facilitation or separate mechanisms, which will yield such results. Contemplated techniques, include:

a. Bone marrow grafting/infusion

i. Independently or in conjunction with stem cell infusion

b. Systematic infusion technique

c. Periodicy of infusion

d. Frequency/duration infusion therapy

i. multiple infusions over a relatively short peri od

ii. Prolonged infusion

1. Continuous multi-hour or multi-day infusion technique.

e. Saturated or massive dosage infusion technique

i. Dosage sufficient to achieve saturation level for minimal dosage amount and/or infusion period

f. Multi-point transfusion technique

g. Stem cell modification

h. Stem cell compositional modification/tailoring

i. Resonance Therapy

j . Magnetic therapy

k. Electronic

1. Electro-Evaporation. See United States Patent, 6,972,013,

m. Gene therapy modification. See for example United States Patent, 7,1 15,417,

n. Grafting technique

o. Hormone therapy

i. Growth hormones. See 20060247170, 20060094655

p. Electrical induction or stimulation q. Audio, music micro-vibrations or ultrasound based induction or stimulation. See for example: 20040191906

r. Visual based induction or stimulation

s. Light therapy, including photothermal, photochemical and photomodulatory therapy. See for example: 6,936,044

t. stem cell trafficking, see 6,814,961

u. Intracellular signaling molecules/technique, see 200601 15813

v. Diet

i. Antigenic sensitive diet (reducing immune sensitivities)

w. Protein therapy

x. Anti-body therapy

y. Protein therapy/injection

z. Stem cell tailoring technique

aa. Stem cell blend proportion technique

i. Transfusion samples containing certain markers, agents or progenitor stem cells

bb. Tunable Matrixes, see 20070026518

The biological age differentiation between the transfused DNA and bone marrow that recipient receives is also a critical element of this invention. The invention's effectiveness is predicated upon the DNA age of the transfused stem cells and/or bone marrow being younger than the biological age of recipient. The younger the DNA age compared to the biological age of the recipient, the better anticipated tissue renewal is expected.

One embodiment of this invention contemplates a mathematical tailoring of anti-aging effect. Under this embodiment huge differences between the biological age (average biological age) of the transfused DNA and recipient are preferred. However, another embodiment expressly contemplates, especially if significant quantities of stem cell material are available, employing the youngest DNA stem cell material possible for as long as possible. 31. EXAMPLE —An autologous anti-aging method comprising: Periodic collection of whole stem cells and/or bone marrow from donor-recipient from shortly after his/her conception to chronological age 500 years, whereby said collection of stem cells provides a plurality of embryonic and/or adult cells selected from the group consisting of totipotent stem cells, pluripotent stem cells, multipotent stem cells, progenitor stem cells and combination thereof; Providing for long term storage of said donor-recipient stem cells and/or bone marrow in sterile conditions in non-breachable containers; Thawing a portion of said stored stem cells and/or bone marrow after a period of storage; Periodically auto-transfusing or infusing said stem cells and/or bone marrow back into donor-recipient, whereby said infusion results in an integration of biologically younger DNA containing stem cells and/or bone marrow into donor-recipient' s bone marrow & tissues throughout his/her body.

32. EXAMPLE - The autologous example above wherein said collections are on average a minimum of once every year, once every two years, once every three years, once every four years for the first 30 years of donor-recipient's life.

33. EXAMPLE ~ The autologous example above wherein said infusions/transfusions are on average a minimum of once every year, once every two years, once every three years, once every four years, once every five years after chronological age 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or later of the donor-recipient's life.

34. EXAMPLE ~ An autologous anti-aging method comprising: Periodic collection of stem cells and/or bone marrow from a donor from after conception; Providing for the long term storage of said donor-recipient stem cells and/or bone marrow in sterile conditions in non-breachable containers; Thawing a portion of said stored stem cells and/or bone marrow after a period of storage; Periodic and regular infusions or infusion of said stem cells and/or bone marrow into the donor starting after age 10 years, whereby said periodic infusions result in an average biological age of the new body comprised of replaced tissues from the donor DNA at least 5 years younger than the actual chronological age of said recipient.

35. EXAMPLE —An anti-aging method comprising: Periodic collection of stem cells and/or bone marrow from DNA related donor from shortly after his/her conception to chronological age 500 years, whereby said collection provides a plurality of embryonic and/or adult cells selected from the group consisting of totipotent stem cells, pluripotent stem cells, multipotent stem cells, progenitor stem cells and combination thereof; Optionally, providing for the long term storage of said donor- recipient stem cells and/or bone marrow in sterile conditions in non-breachable containers; Optionally, thawing a portion of said stored stem cells and/or bone marrow after a period of storage; Periodically transfusing or infusing said stem cells and/or bone marrow into DNA related or compatible recipient, whereby said infusion results in a global integration of biologically younger stem cells into recipient's tissues throughout his/her body.

36. EXAMPLE ~ An anti-aging method comprising: A periodic collection of stem cells and/or bone marrow from DNA related donor from shortly after his/her conception to chronological age 500 years, whereby said collection provides a plurality of embryonic and/or adult cells selected from the group consisting of totipotent stem cells, pluripotent stem cells, multipotent stem cells, progenitor stem cells and combination thereof; Optionally, providing for the long term storage of said donor- recipient stem cells and/or bone marrow in sterile conditions in non-breachable containers; Optionally, thawing a portion of said stored stem cells and/or bone marrow after a period of storage; Periodically transfusing or infusing said stem cells and/or bone marrow into DNA related or compatible recipient, whereby said infusion results in a global integration of biologically younger stem cells into recipient's tissues throughout his/her body.

37. EXAMPLE —An anti-aging method comprising: The collection of stem cells and/or bone marrow from a biologically suitable donor from after conception providing embryonic and/or adult cells selected from the group consisting of totipotent stem cells, pluripotent stem cells, multipotent stem cells, progenitor stem cells; Optionally, providing for the long term storage of said donor-recipient stem cells and/or bone marrow in sterile conditions in non-breachable containers; Optionally, thawing a portion of said stored stem cells after a period of storage; The adaptation of said stem cells to comport with the DNA structure of recipient; Periodic transfusing of infusing a quantity of said stem cells into recipient, on the proviso that said transfused stem cells are biologically younger than recipient, whereby said infusion results in an integration of said stem cells into recipient's tissues throughout his/her body

38. EXAMPLE —A composition containing DNA in a stem cell carrier and optionally bone marrow, whereby said DNA is delivered in sufficient useable quantity to an autologous donor/recipient to regenerate existing cell structure on a global body basis; wherein said regeneration provides a 5 year differential between the donor/recipient's chronological age and his biological age.

39. EXAMPLE —The anti-aging methods and compositions above, wherein said infusion results in biologically younger DNA containing stem cells being integrated into recipient's tissues throughout his/her entire body causing formation of a transient chimera, characterized by the collective tissue composition of the recipient current chronologically older DNA-containing cells with biologically younger DNA- containing infused stem cells, where the biologically age of the resulting transient tissue chimera is at least 5 years younger than the chronological age of recipient.

40. EXAMPLE —An Allogeneic Anti-Aging method that: a) collects stem cells from cloned embroyonic steam cells, whereby a plurality of said cells are from the group consisting of Totipotent stem cells, Pluripotent stem cells, and Multipotent stem cells; b) increasing the pool of stem cells by cell proliferation means (see United States Patent Application, 20030232430, Cibelli, Jose; et al. December 18, 2003, Methods for making and using reprogrammed human somatic cell nuclei and autologous and isogenic human stem cells); c) providing long term storage of donor-recipient stem cells in sterile and cell protective conditions in non-breachable containers or other acceptable storage or cryogenic conditions; d) retrieving a portion of said stored stem cells after a period of time; e) periodically transfusing said stem cells back into the recipient after such time as said donor's chronological age exceeds the chronological or biological age of the DNA contained in said transfused stem cells by at least 10, 16, 18, 20, 22, 26, 28, 30 years, whereby said infusion results in an integration of biologically younger DNA containing stem cells through-out recipient's entire body; optionally infusing younger DNA containing bone marrow prior to or simultaneously with said infusion of embroyonic stem cells. Applicants' invention contemplates EMBODIMENTS including those expressed herein and those that are not. For example, the use of a Telomerase as concurrent therapy and/or as co- additive in Applicants' compositions will be considered. Telomerase is known to be an anti- aging enzyme that is important in cellular proliferation, which may be beneficial in combination with other elements of Applicants' invention. Various methods of producing and using telomerase in anti-aging therapy are known. See 20070042962 20060094043 20040253701 20040086518 20030213008 20030099660 20030077758 20030077757 20030059787 20030044953 20030032075 20030009019 20020187471 20020164786 20020155100 20010048917

It is an embodiment of this invention to combine donor stems cells with host/recipient stem cells. It is contemplated that a pre-initiation conferring the host cellular & DNA attributes onto the donor cells IN VITRO or EX-VIVO may be desirable, while at the same time of augmenting the quantities (growing) the collective stem cell mixture .

However, it is important in the practice of proliferation to realize the concept of cellular senescence that cells have a genetically programmed limitation on the number of cellular divisions - whether in vivo, in vitro or ex-vivo. This most likely due to the lack of other dynamic chemical communications/signals/triggers/suppressors that would be available in the body (in vivo), as these cells are in isolation from the total body. Prior research suggests that unaltered cells could only be 'passed' (freshly replated for new growth) @ 50 times or more in culture and would progressively reduce in growth or slow in division time, and not being replaced with new progeny - would wither and die (just like the aging process, but in- vitro on an accelerated basis). If harvested freshly cells are added to an already existing cell culture having undergone several generations of divisions - in-vitro cultures tend to last much longer than those that were not 'freshened' despite the continuous process of replating (taking a sample and introducing a fresh media). It has been observed that when cells divide and begin to crowd each other - division slows or ceases - so 'passing' to new plates (new replication media) to keep the line going becomes critical. It is important to keep cell healthy as cells 'altered' by a virus or other defect no longer seemed to have the senescence factor..

In the practice of this invention, Applicants' do not want to 'alter' cells or DNA into losing programmed senescence - or risk creating cancer (this is the fundamental process of tumor cells - when they lose their dynamic give and take communicating ability and divide with abandon). Applicants' do however contemplate technique during formation of the transient tissue chimera to 'freshen' the replicating cellular DNA with younger DNA that still contains the dynamic feedback and response mechanisms including senescence.

Thus, one embodiment of the augmentation and proliferation process includes 'fueling' by new fresh stem cells - either from cord donor source or from saved younger host stem cells whether in vitro, ex-vivo or in vivo. .

To sum: younger stems are pooled together - stored and/or grown to some predetermined number of divisions to achieve minimal dosage, and then infused into the recipient.

Another embodiment is the pre-mingling (commingling) of the donor cord and/or other source stem cells, and recipients stem cells (preferably young stem cells) permits transmogrification of the donor stem cells (DNA) to the recipientt's. However, this is not a requisite step - but possibly and enhancing one. Donor (cord) stem cells and younger recipient stem cells can be commingled and replicated (optionally stored) for later plating or direct infusion into the recipient.

Another embodiment includes commingling donor stem cells and younger or concurrent aged recipient DNA stem cells, which are pooled and grown in culture to augment, proliferate, and to preconfer host/recipient attributes through several divisions - but not so many as to approach cellular senescence. This would permit direct infustion. This would permit bypassing the storage step and/or permit storing the mixture for later use. Thus, embodiment would relying upon the donor's stem cell DNA for revitalization of the recipient/host's body - but the recipient's cell contribution would be 'priming' the donor-cord DNA - thus enhancing or transmogrifying it to the host/recipient's attributes prior to infusion - promoting greater acceptance and uptake. Applicants' preferred practice of employing pluripotent stem cells, which are typically non-HLA typing, reduces or eliminates immune sensitivities.

The prior embodiment contemplates donor-cord stems/DNA admixing these with any type, age host tissues - but would be directing a specificity of cellular differentiation ~ which really is the mainstay of the research going on today in stem cell and tissue repair research. I was trying to stay away from getting this specific on tissues ~ as our initial goal was to renew the 'entire' body. However, ovaries contain a preset # of ova, which are in a miotic stasis (frozen in the DNA division state and are haploids - 23 chromosomes). I don't what affect infusions of young revitalizing DNA would have on this. The outer covering of the ovary (containing full complement DNA - 46 chromosomes) is dynamic and dividing and would be subject to renewal. In order for tissues to renew - they have to actively divide. Thus, perhaps there would be no affect on the ova-follicules - which would be a problem for aging women - in that these are central to the stimulation and production of critical hormones - the estrogens etc. which influence the thyroid and others. So, on one hand you could have some tissue renewal but incomplete rejuvenation because we cannot create new ova.

While it may be less of a consideration in men —same with spermatogenesis. However, these are NOT preset in numbers in embryogenesis like ova and are an ongoing production factory. This process is essential for the production of testosterone and other factors for male youthfulness.

While 'renewal' of global tissues in women occurs (with the exception of the ovaries estrogen factory), it would be likely that a supply exogenous estrogen to complement the rejuvenating process would be required.

In the case of a non-autologous or allogeneic practice, the use of human embryonic stem cells are a preferred practice. They are easily accessible for controlled and specific genetic manipulation. When this facility is combined with their rapid growth, remarkable stability, and ability to mature in vitro into multiple cell types of the body, human embryonic stem cells are an attractive tools. Pathways for human embryonic stem cells in non-autologous practice include genetically manipulated embroyonic cells to introduce appropriate genetic information. This genetic information may either be active or awaiting later activation, once the modified embryonic stem cell has differentiated into the desired cell type.

Embryonic stem cells can be additionally beneficial since these cells can be differentiated in vitro into many cell types, including presumably tissue-specific stem cells, they may provide a constant in vitro source of cellular material. Thus, "adult" stem cells derived from embryonic stem cells may be utilized to optimize protocols for propagation and genetic manipulation techniques for purposes of acquiring optimal cellular material. The practice of this invention contemplates genetic manipulation of stem cells. This practice is contemplated in the non-autologous portion of the invention. For this purpose genes may be introduced into cells by transfection or transduction. It is contemplated that transfection utilizing chemical or physical methods to introduce new genes into cells would be employed. Usually, small molecules, such as liposomes, as well as other cationic-lipid based particles are employed to facilitate the entry of DNA encoding the gene of interest into the cells. Brief electric shocks are additionally used to facilitate DNA entry into living cells. All of these techniques may be applied to various stem cells of this invention, including human embryonic stem cells. Recipient DNA may disappear after days or weeks, and in rare cases, integrates randomly into host chromosomal DNA. In vitro drug selection strategies allow the isolation and expansion of cells that are stably transfected, as long as they significantly express the newly introduced recipient's gene. Transduction utilizes viral vectors for DNA transfer. Viruses, by nature, introduce DNA or RNA into cells very efficiently. Engineered viruses can be used to introduce almost any genetic information into cells. However, there are usually limitations in the size of the introduced gene. Additionally, some viruses (particularly retroviruses) only infect dividing cells effectively, whereas others (lentiviruses) do not require actively dividing cells. In most cases, the genetic information carried by the viral vector is stably integrated into the host cell genome (the total complement of chromosomes in the cell). In this practice an important parameter that must be carefully monitored is the random integration into the host genome, since this process can induce mutations that lead to malignant transformation or serious gene dysfunction. However, several copies of the recipient's gene may also be integrated into the genome, helping to bypass positional effects and gene silencing. Positional effects are caused by certain areas within the genome and directly influence the activity of the introduced gene. Gene silencing refers to the phenomenon whereby over time, most artificially introduced active genes are turned off by the host cell, a mechanism that is not currently well understood. In these cases, integration of several copies may help to achieve stable gene expression, since a subset of the introduced genes may integrate into favorable sites. In the past, gene silencing and positional effects were a particular problem in mouse hematopoietic stem cells. These problems led to the optimization of retroviral and lentiviral vector systems by the addition of genetic control elements (referred to as chromatin domain insulators and scaffold/matrix attachment regions) into the vectors, resulting in more robust expression in differentiating cell systems, including human embryonic stem cells. See USE OF GENETICALLY MODIFIED STEM CELLS IN EXPERIMENTAL GENE THERAPIES, by Thomas P. Zwaka Center for Cell and Gene Therapy & Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, "Regenerative Medicine 2006," http://stemcells.nih.gov/info/scireport/2006report.htm. Thus, this or other means are contemplated in the genetic adoption of stem cells in the practice of this invention.

The invention contemplates means for the proliferation/enhancement of stem cell generation within the donor, himself, prior to collection [CITE Existing Art References]

Another embodiment is the proliferation of stem cells, including embroyonic stem cells from the donor. Cell proliferation without differentiating methods/techniques are contemplated. Applicants' have found the presence of a protein called Oct-4, which help characterize undifferentiated cells typically make is an important marker that can be used in the practice of the invention to confirm that cell differentiation is not occurring.

Thus, Applicants' invention expressly contemplates stem cell proliferation, whereby a starting population of stem cells after several months in the laboratory can yield millions of cells. If the resulting cells are unspecialized, like a preferred parent stem cell, said cells are capable of long-term self-renewal. This is an express object of this invention regarding stem cell growth after collection under this invention.

Said method contemplates growing cells in the laboratory known as cell culture. Human embryonic and other stem cells are isolated by transferring the inner cell mass into a plastic laboratory culture dish that contains a nutrient broth known as culture medium. The cells divide and spread over the surface of the dish. The inner surface of the culture dish is typically coated with embryonic skin cells that have been treated so they will not divide. This coating layer of cells a feeder layer give cells a sticky surface to which they can attach. Also, the feeder cells release nutrients into the culture medium. Recently, scientists have devised alternative ways of growing embryonic stem cells. Over the course of several days, the cells of the inner cell mass proliferate and begin to crowd the culture dish. When this occurs, they are removed gently and plated into several fresh culture dishes. The process of replating the cells is repeated many times and for many months, and is called subculturing. After six months or more, an original population of 30 cells of the inner cell mass can yield millions of embryonic stem cells. Embryonic stem cells that have proliferated in cell culture for six or more months do so under this embodiment without differentiating, and are genetically normal to the originally collected embryonic stem cells. (Cite). Other methods exist for progenitor cell proliferation including United States Patent Application, 200601 10368, Prosper Cardoso; Felipe; et al., May 25, 2006, "Medium for culturing autologous human progenitor stem cells and applications thereof."

These contemplated techniques can incorporate means of preserving the ratio of collected stem cell types/species or one to increasing non-differentiated portion of stem cell types, for example to include a higher population of undifferentiated stem cell types. Examples of such techniques would include: [CITE Existing Art References].

These proliferation methods may be employed immediately after collection or at any point in the process, up to immediately to transfusion into the recipient. Applicants' preferred practice is immediately after collection, assuming techniques are adequately developed.

Applicants' practice contemplates embryonic stem cell collection of the donor-recipient or of a live DNA related donor, for example including_[CITE Existing Art References]

"Regenerative Medicine 2006," is a report/reference describing advances made since 2001 and outlines the expectations for future developments in stem cell research, including stem cell biology, using cells from embryos, fetal tissue, and adult tissues. See http://stemcells.nih.gov/info/scireport/2006report.htm. This reference is incorporated herein in its entirety by reference.

Harvesting of stem cells in adults is usually obtained from the peripheral circulation, however a surgical bone marrow method can be used. The donor may receive a stem cell inducing agent (such as Neupogen - G-CSF) a few days before collecting the stem cells. These inducing agents force the stem cells from the bone marrow into the general circulation. Typically it takes 1 day to collect the necessary number of stem cells from a healthy donor. However, in early infancy or youth this period may be extended.

The practice of this invention anticipates means to keep stem cells within their unspecialized mode for purposes of their self renewal, including for a period of years; and identifying (and improving upon) the signals that upon or after transfusion will permit their optimal conversion into specialized cells.

The use of these and signal means induced by certain physiologic or other conditions can induce transfused unspecialized stem cells to become specialized cells with special functions of recipient, such as becoming the beating cells of the heart muscle or the insulin-producing cells of a pancreas.

Thus, it is contemplated that signaling means will be employed to encourage generalized adsorption and usage of transfused stem cells throughout recipient after transfusion. In certain circumstances this will be used to encourage specialized adsorption and usage of transfused stem cells.

The invention contemplates a number of different storage means. These are generally known in the art [CITE Existing Art References, included by reference]. The long term preservation of the healthiest stem cells and bond marrow cultures/samples possible with appropriate identification and tagging are essential in the practice of this invention.

The invention also contemplates the usage of immune suppression, as may be required. Immune suppression in transfusion and grafting of blood and bone marrow is generally known in the prior art and existing art methods are contemplated in the practice of this invention. See United States Patent Application, 20050191309, Kakkis, Emil D, et al.; September 1, 2005," Induction of antigen specific immunologic tolerance."

As contemplated in this invention, limitation, structure and enablement of autologous anti- aging and allogeneic anti-aging methods shall be free transferable. For example the infusion frequency, proliferation of the autologous method shall be applicable to the allogeneic methods.

EXAMPLE

A process or method employing: a) periodicity of infusions offset by the deliberate and regular periodic collections of a person's stem cells, under a protocol that would follow a staggered or stair-stepped ascending schedule - the earliest collected stem cells are utilized first; b) where those collected in the early years (such as in the first decade) would be used for the first infusions in the 1st, 2" , 3r , 4th and later decades (depending on supply quantities), and where stem cells collected in the 1st, 2n , 3r , 4 and later decades would be used in the 5th and 6th decades, and so on - with obvious adjustment as time, supply and need would dictate.

EXAMPLE

then making periodic auto-transfusions of the stored stem cells, which would be thawed for use, back into same donor (recipient) starting after a responsible age (likely to be after donor's chronological age 10 to 30 years); f) conducting this protocol in such a manner that the infusion of stem cell would result in an integration of biologically younger stem cells into recipient's tissues throughout his/her entire body

It is worth bearing in mind that each successive decade of life will actually be characterized by a younger specimen, resulting from prior infusions of younger stem cells that have consequently replaced the older tissue cells. New supplies of younger stem cells will be produced in the younger person to be used and stored for later use despite their chronological age.

In actuality, the use of Applicants' invention may be elected at any point in a person's life - realizing that optimal benefit of extending youth is achieved using the youngest possible DNA-containing stem cells - (with at least an age differential of 10 years and ideally obtained in the first decade of life). This is the biggest challenge, however - obtaining adequate quantities of stem cells during one's 1st decade to survive years of storage for use later in life. While focus on harvesting stem cells after conception is contemplated in the practice of this invention, harvesting in later stages is also expressly contemplated. Later stages of embryonic development, prior to and after child birth, during very early childhood through infancy are emphasized. Regular collections should continue, throughout the span of a person's life, in order to assure supplies later on, together with continuous periodic infusions to enjoy optimal anti-aging benefit.

Controlled-rate freezing with temperature curve monitoring is required. Until required for infusion, stem cells products may be stored in the vapor phase of liquid nitrogen. Storage period is weeks to years, stem cell products must be adequately preserved for extended periods after cryopreservation.

After thawing, stem cell activity is checked for viability. Because granulocytes do not survive cryopreservation, loss of this cell fraction from the collection is expected. To allow survival during freezing and thawing, cells may be optionally placed in a medium containing 7.5-10% dimethyl sulfoxide (or a better medium as may be discerned). Because cells lose viability over a short period in a dimethly sulfoxide medium, infusing the cells immediately after thawing is important embodiment of this invention.

Periodic and regular collection makes it possible to store the necessary minimum amounts of autologous whole stem cell (the autologous DNA carrier) to practice this invention, and is an essential element.

As contemplated herein periodic collection may begin after conception, in utero or at birth (umbilical cord) and continue until prior to the death of the individual. It is anticipated that this invention will be practiced over a recipient's normal life span, which therein will range from birth to age 50, 100, 150, 200, 300, 400, 500, or more years. The ultimate success of the invention has yet to be determined. It is conceivable human life could be extended to age 1000 years.

Periodic stem cell collection will depend upon the specific program results desired, age at which donor starts the regime and other factors. However, expected collections will start at birth and last a life time. The contemplated collection of stem cells is by harvesting stem cells, through sterile mass blood collection and/or cellular dialysis or continuous circulating cell screening techniques, or others contemplated.

It is fully expected that processes/devices and/or methods, including protector, promoter, enhancing, inducing, suppressing, and/or inhibiting, and/or other agents, known and/or which will be developed specific for: 1) harvesting of targeted stem cells from the vascular compartment, bone marrow, and the like, 2) screening and concentration, and the like, and 3) the stem cells subsequent placement back into donor/recipient's tissues at later date, will be employed.

It is contemplated that these processes/devices and/or methods may or may not be concurrently used during collection and/or infusion, and may be used at some time before and/or after said collection and/or infusion.

These processes/devices and/or methods are incorporated into the claims of this invention.

The continuous collection of stem cells is of paramount importance. It is desirable under this invention to obtain sufficient quantities with a goal of no less than 50 to 500 grams per year, more preferably 100 to 2000 grams. Lesser amounts of 1 to 20 grams per year would also be acceptable. Ideally, collection will need to target as much as possible in the earliest years.

A preferred collection period would include collecting 5 grams to 1000 grams of packed stem cells on a frequently of 1 to every 6 months. Another desirable periodic collection frequency includes at least once every 12 months. Other frequencies include once every 1 to 5 years. Yet another example would be collections of no less than once a year for a donor's age 1 & 2, twice a year for ages 3 to 5, three times a year ages 6 through 15, four times a year 16 years or older. Other periods/frequencies are contemplated. Even so, periodic collection, as contemplated in the claims hereto, include periods that are irregular in length and random in nature.

Typically it takes 3 to 5 days to collect the necessary number of stem cells - collection targets of about 5 million expressing cells - per kilogram of the donor's weight - is the goal, but as few as 2 million cells would be acceptable. .Collection quantities less than this number may be expanded or augmented through replication or culture techniques.

Thus, a principal embodiment of this invention is dedicated periodic collection, so that there exists a major amount of youthful stem cells stored by an early age, sufficient to accomplish the life time regime of this invention. An objective of this invention is to have collected at least 0.01 to 3 kilograms of concentrated stem cell material by a donor's chronological age of 20 to 30 years. Acceptable amounts would be 0.02 to 0.5 kilograms. Similar amounts at chronological age of 30 to 40 years would be acceptable. Concentrated stem cell material of at least 0.2 to 1.0 kilograms would however be more desirable. Concentrations ranging from about 1 kilogram to 6 kilograms and from 1.5 kilograms to 5 kilograms by age 30 would be more desirable. Concentration ranges outside these ranges are expressly contemplated in the practice of this invention.

The invention further distinguishes in that it requires a life-long commitment of the recipient and is for the sole use of the donor-recipient.

The current art employs the technique of auto-transfusions as on an as needed basis for completely differentiated whole blood, red blood cells, immune cells, blood clotting factors & clotting cells for transfusions, in response to emergency, life threatening incidents or anticipated surgeries for patients with rare blood types, such as AB (-). Collection, storage and intended use of blood is sporadic and not conducted over a person's lifetime. The intended product of collection are red blood cells predominately with a smaller percentage of white cells and blood clotting components, as is the case industry wide in blood donation.

Applicants' invention is distinguished, because its autologous blood components are concentrated to reflect a plurality of stem cells for an infusion of younger DNA into the human body for global uptake throughout the body's tissues and/or incorporation into bone marrow stem cell production centers. Thus, it is a completely different product than disclosed in the prior art.

EXAMPLE

A composition containing DNA in a stem cell carrier, where the DNA can be delivered in sufficient useable quantity to a recipient to regenerate/replace his entire existing cellular tissues on a global body basis - by means of the stem cells differentiating into respective specific tissues and these newly differentiated tissues replicating rates out-pacing the older tissues' replicating rates.

EXAMPLE A composition containing DNA in a stem cell carrier, where the DNA is supplied by a mixture of stem cells wherein 5% to 98% are embryonic undifferentiated stem cells and the balance are a combination of adult pluripotent, multipotent, progenitor stem cells and combination, where the DNA is delivered in sufficient useable quantity and means to a recipient to regenerate/replace (over time) his existing cellular tissues/structure on a global body basis.

The above composition is collected according to a predetermined life-time regular regimen with an emphasis on the person's 1st and 2nd decades, with stem cell auto-transfusions performed on a predetermined periodicity throughout a person's life, distinguished from being transfused on a stand-by basis, for example, in response to an emergency, anticipated surgery, or specific healthcare desire.

It is further distinguished because it does not target site specific tissue repairs or regeneration. Instead it is an anti-aging mechanism that performs by replacing older tissue cells throughout the "entire" body with cells containing younger DNA.

Applicant's invention employs a technique whereby 'whole' stem cells are globally 'infused' via the blood stream, disseminating throughout the whole of the body, rather than site specific injections. The invention, thus, capitalizes on the body's natural design and physiologic mechanisms, which permit the receiving and lodging of the newly introduced stem cells into the tissue; integration, feedback and stimulation that results in differentiation and maturation of the stem cells into the adopted resident tissues. It distinguishes in that no other interventions, chemically or mechanically are performed or induced to affect this result, unlike the current art. And the current art exploring anti-aging modalities and technology employs no similar or obvious modal.

The prior art also utilizes stem cells or their components for the purpose of growing or generating tissues in a laboratory setting for later use in repairing or regenerating specific target tissues within the body. Other prior art focuses on specific techniques exploring sub¬ components of stem cells and their genetic material in an attempt to control the determinants that regulate growth and cellular behavior.

Instant invention intervenes with the aging process and operates to replace damaged tissue globally through the use of stem cells and/or their components It simply recruits the body's own inherent and complex processes to reset its biological clock, through saving its own natural resources (younger stem cells) without complex interference to natural body processes or regulation mechanisms; and re-introduces these said resources to the whole body, through a simply infusion into the blood stream. Claimed invention globally disseminates stem cells that carry younger autologous DNA throughout the entire body. Infused younger stems will migrate directly to specific tissues and will also populate bone marrow stem cell production centers, replacing (out-replicating) older resident stem cells and eventually "replace" the body's existing older tissue cells .

The body does not regress, but is transformed to a younger more vital stage. Acquired attributes would be transferred by the body's inherent redundancies in biological systems. For example, we know that memory and other 'information' is transferred to new cells from older ones. Thus, the claimed invention extends youth.

There will be eventual genetic fade. That is, each successive collection point will yield stem cells a little bit older than the proceeding. Thus, a person will still age, but theoretically much more slowly. However, the person's biological age and chronological age will widen. The biological age will advance, but at a slower rate than the person's actual chronological age. It will take much longer to reach the end of the reverse moving platform. But, it will eventually be reached.

Extending youth is to be distinguished from extending longevity - as this is occurring, through enhancements in medical services and technology; public health and preventive medicine via vaccines, medications, early detection and intervention of diseases, enhanced nutrition, and reduced exposure to environmental insults etc., healthful living choices and habits; and through improved environmental engineering.

Applicant's invention posses no risk to the individual from rejection reaction, nor requires use of medications to prevent rejection reactions, delicate or risky invasive procedures or toxic effects, which is a distinguishing feature.

The following is excluded from risk exception, such as idiosyncratic reactions or anaphylaxis to any carrier solution or residue, equipment or its materials used to collect or re-infuse the stem cells or to unforeseen events due to poor technique, sanitation, inventory error or other errors - as its potential occurrence is not unique The economics of longer life-spans would be offset by cost-shifting of expensive sick care consumptive costs and the long-term financial and social commitment of sustaining lives suffering with chronic illnesses, as people would live longer - healthier!

Essential elements/structure of claims/examples within this description are intended to interchangeable to between various claims/examples. For example, critical elements contained in a composition's claim/example that would constitute or represent a logical critical limitation of a different example or composition (or method), where would be included if such inclusion be logically. The disclosure of this invention intends this interchangeability of critical feature.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention are contemplated in addition to those described herein, after further consideration of the references, and/or which will become apparent to those skilled in the art from the foregoing description. The skill of the art is changing rapidly and this invention anticipates certain improvements in the collection, proliferation, differentiation, genetic engineering, preservation of SCs, injection methods, and all other aspects of this invention— wherein for example, when improved methods of collection, proliferation and other techniques of employing pluripotent SCs are developed. New improved signaling, genetic engineering, and all other improved methods/compositions in the practice of this invention are herein expressly contemplated in this invention. Such improvements and means, if it not now expressly provided, are intended to fall within the scope of the appended claims.

All references cited herein (including cited patents, published patent applications, publications, references below, AND the references contained in these references) are FULLY incorporated herein by reference ~ in their entirety and for all purposes to the same extent as if each individual publication, patent or patent application, reference and references within the cited reference were specifically and individually indicated to be incorporated by reference in its entirety for all purposes in this specification. Said incorporated references are intended to provide background, state of the art, direction, and practice suggestions, and are to read in conjunction with this disclosure/specification in an entirety. Thus, this disclosure and appended claims may be modified as required to reflect the provided references. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

Applicant's autologous practice is predicated upon:

• Periodic collection of stem cell material from a donor, whereby the mass of all collected samples has an average biological age ranging from after conception, at birth and to 70 years +/-of age, with preferred biological age ranging from birth to under 50, and a more preferred range from after conception, birth and to under age 30.

• Concentration/sorting this stem cell material such that it can be effectively stored for very extended times, absent appreciable aging, and then later reused as a live organic medium. Sorting into primordial SC components and then proliferating said stem cells.

• Storing the stem cell medium, whereby said samples do not appreciably age and can be infused into a mammalian being up to decades or centuries later.

• Periodic infusion into the donor after achieving a minimum age on a global basis, anticipated to be over their entire lifetime, wherein

• The continuation of this periodic stem cell collection and stem cell infusion regime results in an average chronological and/or biological age difference of infused DNA (that is appreciably - at least 5 years younger) compared the chronological age of the donor.

Mammalian cells are pre-programmed, through their genome (DNA mechanism) so as to undergo a finite number of cellular replications - divisions. As the individual cells (which define the total tissue structure) progress along their continuum of number of cellular divisions, their rate of replication slows. Eventually, signs of aging become apparent, as damaged and worn tissue cells are not timely replaced. Cells become worn, exhausted, damaged from environmental insults, and deprived of necessary co-factors and interactive influences from other failing bodily endocrine and physiologic functions. While research efforts are and have been underway to intervene in the aging process, none propose to simply replace the entire body's cells with younger, fresh cells through a self- adaptive (autologous) grafting process, employing the very body and DNA of the recipient.

Human stems cells contain the body's complete genetic information, factors, determinants and capacity for generation of differentiated tissues throughout the entire body of a person into specific end-organ and support tissues such as bone, nerves, muscle, heart, liver, brain, endocrine organs, skin etc. Human stems cell can be collected, stored and re-infused at a later date into the bloodstream of the same person from which they were harvested at an earlier time in his/her life. They will disseminate throughout the entire body, lodging in all tissues. Interacting with factors and determinants specific to differentiated 'resident' tissue cells (already evolved to a specific and distinguishable type of tissue such as bone or muscle, etc.), the stem cells will be induced through interactions with the adopted tissues to 'differentiate' or evolved into the respective distinguishable tissues in which they find residence forming a graft and transient chimera of older and younger DNA-containing cells.

Infusion with sufficient quantities of vital stem cells over regular, adequate and periodic time-frames would result in successive, but eventually diminishing replacement of aging tissue. Because, it is not known what tissues may have an inherent resistance to 'replacement'; it is not known what quantities of stem cells would actually be needed and what time period it would take for total tissue replacement with the younger newly differentiated cells; and while it might be presumed based upon natural statistical truths, it is not known if there is a dose-time response curve (more stem cells infused - the faster they take over by replacing the older tissue cells). In addition, one's supply of viable stem cells would eventually be exhausted, as would their and perhaps society's will and resources. An endless supply of one's younger stem cells could not be obtained to be indefinitely accessed by a person later in his/her life. Prior art research focuses primarily on utilizing human DNA that is obtained in stem cells harvested from donors other than the intended recipient (non-autologous, i.e. embryonic or fetal stem cells). Still other prior art explores autologous DNA obtained from stem cells collected from the donor in real-time or relatively current to the recipient's age and treatment period. Other art discloses both types involve "injecting" whole stem cells (whether autologous or not) into target tissue to promote repair, such as in spinal cord injury or damaged heart.

It is not known or disclosed in the prior art, whether repair occurs due to unique stimulatory factors inherent to stem cells that are exerted upon the resident differentiated damaged tissues or if repair results from the stem cells differentiating themselves into undamaged tissue. To date, the vast focus of autologous stem cell repair employs enucleating the stem cell and transferring this DNA into enucleated cells of the host's tissue to be repaired. This process of enucleating and transferring is mechanically performed.

The applicants instant invention distinguishes over the prior art because it is predicated upon cells being transferred globally "throughout the entire body," neither involving injecting cells or their contents or any portion thereof. Nor does it involve a directed transferring of the contents of cells (i.e. DNA) into either tissues or into enucleated tissue cells. This invention distinguishes because it requires pre-planned protocols, pre-positioned resources for an intended recipient prior to or soon thereafter their birth, and deliberate and periodic collection, storing and deliberate, and periodic autologous whole stem cell infusions (auto- transfusions) on a global whole body basis.

EXAMPLE

A process or method employing: a) periodicity of infusions offset by the deliberate and regular periodic collections of a person's stem cells, under a protocol that would follow a staggered or stair-stepped ascending schedule - the earliest collected stem cells are utilized first; b) where those collected in the early years (such as in the first decade) would be used for the first infusions in the 1st, 2nd, 3rd, 4th and later decades (depending on supply quantities), and where stem cells collected in the 1st, 2nd, 3rd, 4th and later decades would be used in the 5th and 6th decades, and so on - with obvious adjustment as time, supply and need would dictate. EXAMPLE

An anti-aging method or process: a) tanking periodic collection of stem cells from a donor from his birth to his age 500 years or less (until some determinate period prior to this death); b) sorting and concentrating these stem cells in a collection of blood sera contents, such that they would represent a plurality of blood product constituents for use at a future date; c) storing the donor-recipient stem cells in sterile conditions in non-breachable containers under sub-zero temperature sufficient to insure future use; d) thawing a minor portion of said stored stem cells after a period of 1 to 500 years, depending upon the protocol and program of the recipient; e) then making periodic auto-transfusions of the stored stem cells, which would be thawed for use, back into same donor (recipient) starting after a responsible age (likely to be after donor's chronological age 10 to 30 years); f) conducting this protocol in such a manner that the infusion of stem cell would result in an integration of biologically younger stem cells into recipient's tissues throughout his/her entire body

In actuality, the use of Applicants' invention may be elected at any point in a person's life - realizing that optimal benefit of extending youth is achieved using the youngest possible DNA-containing stem cells - (with at least an age differential of 10 years and ideally obtained in the first decade of life). This is the biggest challenge, however - obtaining adequate quantities of stem cells during one's 1st decade to survive years of storage for use later in life. While focus on harvesting stem cells during childhood is emphasized, regular collections should continue, throughout the span of a person's life, in order to assure supplies later on and continue the infusions and anti-aging benefit. Periodic and regular collection makes it possible to store the necessary minimum amounts of autologous whole stem cell (DNA) to practice this invention, and is an essential element.

As contemplated herein periodic collection may begin a birth and continue until prior to the death of the individual. It is anticipated that this invention will practiced over normal life times, which there under will range from birth to age 100, 150, 200, 300, 400, 500, or more years. The ultimate success of the invention has yet to be determined. It is conceivable human life could be extended to age 1000 years.

It is fully expected that processes/devices and/or methods, including protector, promoter, enhancing, inducing, suppressing, and/or inhibiting, and/or other agents, known and/or which will be developed specific for: 1) harvesting of targeted stem cells from the vascular compartment, bone marrow, and the like, 2) screening and concentration, and the like, and 3) their subsequent placement back into donor/recipient's tissues at later date, will be employed.

These processes/devices and/or methods are incorporated into the claims of this invention.

A preferred collection period would include collecting 5 grams to 1000 grams of packed stem cells on a frequently of 1 to every 6 months. Another desirable periodic collection frequency includes at least once every 12 months. Other frequencies include once every 1 to 5 years. Yet another example would be collections of no less than once a year for a donor's age 1 & 2, twice a year for ages 3 to 5, three times a year ages 6 through 15, four times a year 16 years or older. Other periods/frequencies are contemplated. Even so, periodic collection as contemplated in the claims hereto, include periods that are irregular in length and random in nature.

An objective of this invention is to have collected at least 0.01 to 3 kilograms of concentrated stem cell material by a donor's chronological age of 20 to 30 years. Acceptable amounts would be 0.02 to 0.5 kilograms. Similar amounts at chronological age of 30 to 40 years would be acceptable. Concentrated stem cell material of at least 0.2 to 1.0 kilograms would however be more desirable. Concentrations ranging from about 1 kilogram to 6 kilograms and from 1.5 kilograms to 5 kilograms by age 30 would be more desirable. Concentration ranges outside these ranges are expressly contemplated in the practice of this invention.

The invention further distinguishes in that it requires a life-long commitment of the recipient and is for the sole use of the donor-recipient.

The current art employs the technique of auto-transfusions on an as needed basis for whole blood or blood product transfusions, in response to emergency and life threatening incidents or anticipated surgeries for patients with rare blood types, such as AB (-). Collection and storage of blood is sporadic and not conducted over a person's lifetime. The intended product of collection are red blood cells predominately with a smaller percentage of white cells and blood clotting components, as is the case industry wide in blood donation.

Applicants' invention is distinguished because its autologous blood components are concentrated to reflect a plurality of stem cells for a global infusion of younger DNA into the human body. Thus, it is a completely different product than disclosed in the prior art.

EXAMPLE A composition containing DNA in a stem cell carrier, where the DNA is can be delivered in sufficient useable quantity to an autologous donor/recipient to regenerate his existing cell structure on a global body basis.

Instant invention intervenes with the aging process and operates to replace damaged tissue globally, through the use of stem cells and/or their components. It simply recruits the body's own inherent and complex processes to reset its biological clock, through saving its own natural resources (younger stem cells) without complex interference to natural body processes or regulation mechanisms; and re-introduces these said resources to the whole body, through a simply infusion into the blood stream. Claimed invention globally disseminates stem cells that carry younger autologous DNA throughout the entire body that will eventually "replace" the existing older tissue cells.

1. It is an objective of this invention to increase into recipients BM and vascular department "activated" younger DNA containing Primordial SCs at an appreciably higher percentage than they would otherwise exist in recipient within the bone marrow, based upon their chronological/biological age, in light of their sex and weight. For example a 30 year old male weighing approximately 200 lbs could be expected to have some portion of his entire nucleated blood in blood stream (optionally, as measured by CD 34 + nucleated cells or other marker) to be SCs, with some population preferably being primordial stem cells, such as pluripotent SCs. Elevated SCs in blood circulation are likely to be most progenitors generated in the bone marrow. Applicants' compositions and methods are intended to increase these total amounts of blood stream circulated SCs by a minimum of 3x of normal amounts for an extended period. Thus, the minimum percentage of primordial SCs added to a recipient's vascular department from Applicants's invention will be 3x the number of nucleated cells (optionally, as measured by CD 34 + nucleated cells) being SCs compared to normal. Preferred elevations will be much greater, preferably 10x to 100Ox, or more. .

2. A method is contemplated involving periodic treatment is according another systematic regime for bone marrow transplantation known in the art, but periodically repeated to achieve minimum SC concentrations.

3. A method is contemplated wherein after a periodic infusion of SCs into recipient, at least 0.3 to 3.0% by volume blood in circulation contains SCs.

4. As contemplated herein an infusion dosage amount or after an infusion has been made, the number of stem cells within recipient's blood stream (vascular department) increases above normal limits. Age appears to be a factor in the volumes of SC circulated in the vascular department. Applicants' invention contemplates a minimum of a 100% in vascular department stem cell circulation. More desirable increases would represent 2x to 5x normal circulation amounts. It is expected that at some upper limit the body will reject the increased population of such SCs.

5. As a function of total number of stem cells to total nucleated stem cells contained in recipient's blood stream, optionally measured by the number of CD + 34 cells or other indicative marker, circulated stem cells in the blood stream may range from 0.001 to 40.0%, 0.001 to 30.0%, 0.001 to 20.0%, 0.001 to 15.0%, 0.001 to 12.0%, 0.001 to 10.0%, 0.001 to 9.0%, 0.001 to 8.0%, 0.001 to 7.0%, 0.001 to 6.0%, 0.001 to 5.0% 0.001 to 4.0%, 0.001 to 3.0%, 0.001 to 2.0%, 0.001 to 1.0%, 0.001 to 0.50%, 0.001 to 0.40%, 0.001 to 0.30%, 0.001 to 0.25%, 0.001 to 0.20%, 0.001 to 0.25%, 0.001 to 0.20%, 0.001 to 0.15%, 0.001 to 0.010%, 0.001 to 0.05%, 0.30% to 40.0%, 0.30% to 30.0%, 0.30% to 20.0%, 0.30% to 15.0%, 0.30% to 12.0%, 0.30% to 10.0%, 0.30% to 9.0%, 0.30% to 8.0%, 0.30% to 7.0%, 0.30% to 6.0%, 0.30% to 5.0%, 0.30% to 4.0%, 0.30% to 3.0%, 0.30% to 2.0%, 0.30% to 1.0%, 0.30% to 0.50%, 0.30% to 0.40%, 0.50% to 40.0%, 0.50% to 30.0%, 0.50% to 20.0%, 0.50% to 15.0%, 0.50% to 12.0%, 0.50% to 10.0%, 0.50% to 9.0%, 0.50% to 8.0%, 0.50% to 7.0%, 0.50% to 6.0%, 0.50% to 5.0%, 0.50% to 4.0%, 0.50% to 3.0%, 0.50% to 2.0%, 0.50% to 1.0%, 0.750% to 40.0%, 0.750% to 30.0%, 0.750% to 20.0%, 0.750% to 15.0%, 0.750% to 12.0%, 0.750% to 10.0%, 0.750% to 9.0%, 0.750% to 8.0%, 0.750% to 7.0%, 0.750% to 6.0%, 0.750% to 5.0%, 0.750% to 4.0%, 0.750% to 3.0%, 0.750% to 2.0%, 0.750% to 1.0%, 0.7750% to 40.0%, 0.7750% to 30.0%, 0.7750% to 20.0%, 0.7750% to 15.0%, 0.7750% to 12.0%, 0.7750% to 10.0%, 0.7750% to 9.0%, 0.7750% to 8.0%, 0.7750% to 7.0%, 0.7750% to 6.0%, 0.7750% to 5.0%, 0.7750% to 4.0%, 0.7750% to 3.0%, 0.7750% to 2.0%, 0.7750% to 1.0%, 1.0% to 40.0%, 1.0% to 30.0%, 1.0% to 20.0%, 1.0% to 15.0%, 1.0% to 12.0%, 1.0% to 10.0%, 1.0% to 9.0%, 1.0% to 8.0%, 1.0% to 7.0%, 1.0% to 6.0%, 1.0% to 5.0%, 1.0% to 4.0%, 1.0% to 3.0%, 1.0% to 2.0%, 1.5% to 40.0%, 1.5% to 30.0%, 1.5% to 20.0%, 1.5% to 15.0%, 1.5% to 12.0%, 1.5% to 10.0%, 1.5% to 9.0%, 1.5% to 8.0%, 1.5% to 7.0%, 1.5% to 6.0%, 1.5% to 5.0%, 1.5% to 4.0%, 1.5% to 3.0%, 1.5% to 2.0%, 1.75% to 40.0%, 1.75% to 30.0%, 1.75% to 20.0%, 1.75% to 15.0%, 1.75% to 12.0%, 1.75% to 10.0%, 1.75% to 9.0%, 1.75% to 8.0%, 1.75% to 7.0%, 1.75% to 6.0%, 1.75% to 5.0%, 1.75% to 4.0%, 1.75% to 3.0%, 1.75% to 2.0%, 2.0% to 40.0%, 2.0% to 30.0%, 2.0% to 20.0%, 2.0% to 15.0%, 2.0% to 12.0%, 2.0% to 10.0%, 2.0% to 9.0%, 2.0% to 8.0%, 2.0% to 7.0%, 2.0% to 6.0%, 2.0% to 5.0%, 2.0% to 4.0%, 2.0% to 3.0%, 2.25% to 40.0%, 2.25% to 30.0%, 2.25% to 20.0%, 2.25% to 15.0%, 2.25% to 12.0%, 2.25% to 10.0%, 2.25% to 9.0%, 2.25% to 8.0%, 2.25% to 7.0%, 2.25% to 6.0%, 2.25% to 5.0%, 2.25% to 4.0%, 2.25% to 3.0%, 2.5% to 40.0%, 2.5% to 30.0%, 2.5% to 20.0%, 2.5% to 15.0%, 2.5% to 12.0%, 2.5% to 10.0%, 2.5% to 9.0%, 2.5% to 8.0%, 2.5% to 7.0%, 2.5% to 6.0%, 2.5% to 5.0%, 2.5% to 4.0%, 2.5% to 3.0%, 2.75% to 40.0%, 2.75% to 30.0%, 2.75% to 20.0%, 2.75% to 15.0%, 2.75% to 12.0%, 2.75% to 10.0%, 2.75% to 9.0%, 2.75% to 8.0%, 2.75% to 7.0%, 2.75% to 6.0%, 2.75% to 5.0%, 2.75% to 4.0%, 2.75% to 3.0%, 3.0% to 40.0%, 3.0% to 30.0%, 3.0% to 20.0%, 3.0% to 15.0%, 3.0% to 12.0%, 3.0% to 10.0%, 3.0% to 9.0%, 3.0% to 8.0%, 3.0% to 7.0%, 3.0% to 6.0%, 3.0% to 5.0%, 3.0% to 4.0%, 3.25% to 40.0%, 3.25% to 30.0%, 3.25% to 20.0%, 3.25% to 15.0%, 3.25% to 12.0%, 3.25% to 10.0%, 3.25% to 9.0%, 3.25% to 8.0%, 3.25% to 7.0%, 3.25% to 6.0%, 3.25% to 5.0%, 3.25% to 4.0%, 3.5% to 40.0%, 3.5% to 30.0%, 3.5% to 20.0%, 3.5% to 15.0%, 3.5% to 12.0%, 3.5% to 10.0%, 3.5% to 9.0%, 3.5% to 8.0%, 3.5% to 7.0%, 3.5% to 6.0%, 3.5% to 5.0%, 3.5% to 4.0% of total nucleated cells. Minimal preferred circulation amounts after infusion are 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.01,

1.05, 1.1 , 1.15, 1.2, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, 2.00, 1.01, 1.05, 1.1, 1.15, 1.2, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, 2.00, 2.01, 2.05, 2.1, 2.15, 2.2, 2.25, 2.30, 2.35, 2.40, 2.45, 2.50, 2.55, 2.60, 2.65, 2.70, 2.75, 2.80, 2.85, 2.90, 2.95, 3.00, 3.01, 3.05, 3.10, 3.15, 3.2, 3.25, 3.30, 3.35, 3.40, 3.45, 3.50, 3.55, 3.60, 3.65, 3.70, 3.75, 3.80, 3.85, 3.90, 3.95, 4.00, 4.01, 4.05, 4.1, 4.15, 4.2, 4.25, 4.30, 4.35, 4.40, 4.45, 4.50, 4.55, 4.60, 4.65, 4.70, 4.75, 4.80, 4.85, 4.90, 4.95, 5.00, 5.01, 5.05, 5.1 , 5.15, 5.2, 5.25, 5.30, 5.35, 5.40, 5.45, 5.50, 5.55, 5.60, 5.65, 5.70, 5.75, 5.80, 5.85, 5.90, 5.95, 6.00, 6.01, 6.05, 6.1, 6.15, 6.2, 6.25, 6.30, 6.35, 6.40, 6.45, 6.50, 6.55, 6.60, 6.65, 6.70, 6.75, 6.80, 6.85, 6.90, 6.95, 7.0

6. A method is contemplated in which infusions begin no later than after donor's 1st decade of chronological life, no later than after donor's 2nd decade of chronological life, no later than after donor's 3rd decade of chronological life or no later than after donor's 4th decade of chronological life.

7. The method of the present invention contemplates the difference between resulting average biological age of the recipient's transformed body following infusions of DNA-containing stem cells is at least 5 years younger than the recipient's chronological age, and alternatively, at least 10 years, 20 years, 30 years, 40 years, 50 years, 60 years or 100 years. .

8. The anti-aging method of the present invention contemplates collecting stem cells, separating out primordial stem cells, collecting bone marrow, optionally storing said stem cells and bone marrow, augmenting and proliferating said stem cells and bone marrow ex-vivo, periodically infusing into recipient's a DNA compatible composition comprising a:

a. Fraction of recipient compatible or matched stem cells, where said fraction contains a plurality of primordial stem cells, whereby said steam cells are biologically at least 5 years younger than recipient,and

b. Fraction of recipient compatible or matched bone marrow

c. wherein after said infusion the total number of stem cells contained in recipient's blood stream represents at least 0.2 % of the total number of nucleated cells contained in recipient's blood stream.

9. Use of younger bone marrow - younger than host-recipient derived from autologous and 3r party compatible donors is contemplated. Bone Marrow may be extracted from donors under age 5, 10, 15, 20, 25, 30. Bone marrow may also be extracted from host- recipient prior to the infusion. It is contemplated that the use of youthful bone marrow where available will be preferred. To the extent that replication and proliferation technology is available to increase collected marrow, such technologies will be employed.

10. These ranges and other ranges are contemplated as is necessary, on the proviso that dosage together with the frequency of infusions results in the formation of transient chimera tissue, which is biologically younger than the chronological age of recipient. The above infiised percentage amount may be measured as a total of recipient's total nucleated cell blood supply [ optionally as measured by the number of CD + 34 cells or other suitable marker contained in recipient's blood stream, or be measured volumetrically on the basis of total blood contained recipient's body]. Preferred amounts include 0.03% to 0.5%, 0.3 to 0.5%, 0.1 to 1.0%, 0.3 to 1.0%, 1.0 to 4.0%, 2.0 to 3.0%, 2.20% to 3.0%, and about 3.0% of recipient's total nucleated cell blood supply.

a. The infusions can comprise, alternatively, about 0.010 to 20.0 ounces of stem cells, about 0.2 to 1.0 ounce of stem cells, about 0.2 to 0.6 ounce of stem cells, about 2.0 to 20.0 ounces of stem cells, about 0.5 to 10.0 ounces of stem cells, about 0.5 to 5.0 ounces of stem cells, about 0.1 to 4.0 ounces of stem cells, about 0.1 to 3.0 ounces of stem cells, about 0.1 to 2.0 ounces of stem cells, about 0.10 to 1.0 ounce of stem cells, about 0.10 to 0.8 ounces of stem cells, about 0.2 to 0.6 ounces of stem cells.

11. Volumetrically, single stem cell infusion volumes may range from 1 cc to 5,000 cc's. Ranges of 1 to 2 cc's, 2 to 8 cc's, 3 to 10 cc's are contemplated. Other amounts would include approximately 1A to 3A, 1/3 to 1 of the standard volume of a 10 cc hypodermic syringe. Serum carrier materials are expressly contemplated in the delivery of infusions in the practice of this invention.

EXAMPLE

A composition containing DNA in a stem cell carrier and optionally bone marrow, whereby said DNA is delivered in sufficient useable quantity to an autologous donor/recipient to regenerate existing cell structure on a global body basis.

EXAMPLE METHODS

• An autologous anti-aging method comprising:

o Periodic collection of stem cells and/or bone marrow from a donor from after conception to an adult chronological age,

o Providing for the long term storage of said donor-recipient stem cells and/or bone marrow in sterile conditions in non-breachable containers o Thawing a portion of said stored stem cells and/or bone marrow after a period of storage

o Periodic and regular infusions or infusion of said stem cells and/or bone marrow into the donor starting after age 10 years, whereby said periodic infusions result in an average biological age of the new body comprised of replaced tissues from the donor DNA at least 5 years younger than the actual chronological age of said recipient.

• An anti-aging method comprising:

o Periodic collection of stem cells and/or bone marrow from DNA related donor from shortly after his/her conception to chronological age 500 years, whereby said collection provides a plurality of embryonic and/or adult cells selected from the group consisting of totipotent stem cells, pluripotent stem cells, multipotent stem cells, progenitor stem cells and combination thereof

o Optionally, providing for the long term storage of said donor stem cells and/or bone marrow in sterile conditions in non-breachable containers

o Optionally, thawing a portion of said stored stem cells and/or bone marrow after a period of storage

o Periodically transfusing or infusing said stem cells and/or bone marrow into DNA related or compatible recipient, whereby said infusion results in a global integration of biologically younger stem cells into recipient's tissues throughout his/her body.

• An anti-aging method comprising:

o The collection of stem cells and/or bone marrow from a biologically suitable donor from after conception providing embryonic and/or adult cells selected from the group consisting of totipotent stem cells, pluripotent stem cells, multipotent stem cells, progenitor stem cells.

o Optionally, providing for the long term storage of said donor-recipient stem cells and/or bone marrow in sterile conditions in non-breachable containers o Optionally, thawing a portion of said stored stem cells after a period of storage.

o The adaptation of said stem cells to comport with the DNA structure of recipient.

o Periodic transfusing of infusing a quantity of said stem cells into recipient, on the proviso that said transfused stem cells are biologically younger than recipient, whereby said infusion results in the global integration of said stem cells into recipient's tissues throughout his/her body

• In the above example methods, global integration of stem cells results in:

o Biologically younger DNA containing stem cells being integrated into recipient's tissues throughout his/her entire body

o Whereby a transient chimera is formed, characterized by the collective tissue composition of the recipient current chronologically older DNA-containing cells with biologically younger DNA-containing infused stem cells, whereby the biologically age differential of cells comprising the resulting transient tissue chimera is 5 years or greater

Listed below are methodologies contemplated for use in the above example methods either in the alternative or selectively together.

• Infusion includes a means for a global integration of said biologically younger stem cells with chronologically older DNA containing cells.

• Collection and transfusion includes the collection and transfusion or infusion of stem cells.

• Infusion may be by means of a short series of two of more infusions within a period of less than a month.

• Infusion may be by means of an extended long duration infusion of a period ranging from 5 minutes to one week. • Infusion may be by means of massive dosage of at least 0.0000000001 % 0.000000001 % 0.00000001 % 0.00000001 % 0.00001 %, 0. 0001 % 0. 001 % 0.01 % 0. 1 % , 1.0%, 10.0% or more of the body weight of recipient.

• The methods may employ biologically younger non-HLA typing donor stem cells

• The stem cell may pτe-transfusion

• Collected and transfused stem cells are selected from the group consisting of totipotent, pluripotent, multipotent stem cells and combination thereof

• Transfused stem cells are provided in same relative proportion to those collected.

• Transfused stem cells represent a plurality of undifferentiated stem cells.

• Transferred stem cells represent a majority of of undifferentiated stem cells.

• Transferred stem cells are exclusively undifferentiated stem cells.

• Stem cells are embryonic and adult stem cells, and combination thereof.

• Stem cells are embryonic stem cells or proliferated derivatives thereof.

• At any point said stem cells are proliferated in quantity from original quantity collected.

• Adaptation of said stem cells is made to comport with the DNA structure of recipient.

• Donor stem cells or bone barrow are stored in sub-zero cryogenic temperatures in sterile non-breachable containers with means to preserve said stem cells or marrow indefinitely.

• Collection is from harvesting stem cells collection means.

• Means of collecting said stem cells is selected from an embryonic, umbilical, bone marrow, sterile mass blood, cellular dialysis, continuous circulating cell technique means and combination.

• Collection of said stem cells is by means specific for harvesting targeted cells from the vascular compartment. • Collection of said stem cells is by concentration means specific for harvesting targeted cells from bone marrow.

• Concentration of said stem cells, is by means sufficient to result in a plurality of stem cells, which are in combination with blood product constituents.

• Collection obtains at least on the order of 10.sup.6 total nucleated cells per Kg. weight of the person in a single collection session.

• Each unit comprises a dosage of a fraction of the total nucleated cells required to be transfused or infused.

• The units collected or transfused contain equal or different dosages.

• The preserving or storage step is independent of tissue or HLA typing of the collected stem cells prior to storing in a stem cell bank.

• The preserving or storage step comprises the step of determining from the collected stem cells at least a distinctive property associated with the person prior to storing in a the stem cell bank, so as to provide a means of secured identification to match the collected stem cells with the person at the time of use.

• The typing step includes providing indicia with each unit representing information of said at least one distinctive property.

• The indicia is embodied in at least one of a label, bar code, magnetic strip, and microchip.

• The stem cells are collected during a pre-disease stage in which the person may be diagnosed with a health condition.

• Collection and proliferation result in storage of at least 1000 grams of stem cells or material containing a plurality to a majority of said stem cells.

• Collection and proliferation results in storage of at least 500 grams of stem or material containing a plurality to a majority of said stem cells.

• Collection and proliferation results in storage of at least 250 grams of stem cells or material containing a plurality to a majority of said stem cells. • Collection and proliferation results in storage of at least 50 grams of stem or material containing a plurality to a majority of said stem cells.

• An amount is the result of a single collection session or one or more collection sessions and which exists in inventory.

• The mass is collected by donor's chronological age of 30, 40 or 50.

• Collection is no more frequently than every three months, every six months or every 5 years.

• Collection is no less than once a year for a donor chronological age 1 & 2, no less than twice a year for a donor ages 3 to 5, three times a year ages 6 through 15, and annually for ages 16 years or older.

• A plurality to a majority of said collection occurs during the first 50 years of donor's chronological life.

• A plurality to a majority of collection occurs during the first 10 to 40 years of said donor's chronological life.

• Agents increase the quantity, quality and accessibility of stem cells within the circulation.

• After said concentration, said stem cells are pooled, packed and batched according to incremental age time frames of collection.

• Said incremental time frame of stem cell and/or bone marrow collection is from immediately after conception to birth (umbilical cord), birth to 2 years, 3 to 5 years, 5 to 10 years, 11 to 15 years, and in 5-year increments thereafter.

• Collection after conception does not interfere in any way with embroyonic development.

• The minimum mass of stem cells collected is equivalent to 10% to 100% of a standard unit of red blood cells (no less than 25 grams) standard in the blood banking industry. • The storing of said donor-recipient stem cells is under sterile conditions in non-

breachable containers at temperature below O0F, -5O0F, -2000F, -3000F, or -4000F.

• The temperature is a cryogenic freezing temperature sufficient to insure long term storage of said stem cells.

• The storage is under liquid nitrogen.

• The storage of said stem cells comprises:

o Securely enclosing said stem cells in non breachable containers suspended in bio-compatible nutrient media.

o Labeling, dating & entering into a secure data base (with redundancy and system controls for accuracy and backup in alternate locations)

o Storing said containers under liquid nitrogen.

• The storing said stem cells is within a bio-compatible media suspension.

• The periodic infusion results in integration of biologically younger stem cells into recipient tissues throughout his/her body at least once every five years, which are comprised of the recipient current chronologically older aged DNA-containing cells.

• The infusion results in the creation of a transient tissue chimera- age difference between said infused stem cells (collected and stored years earlier) and recipient's currently aged DNA containing cells is 20 years or greater.

• The infusion incorporates harvested stem cells, which have been collected at least 5, 10, 20, 30, 40, or 100 years earlier.

• The infusion contains a mixture of stem cells collected over various dates.

• The infusion incorporates means for selecting stem cells for maximizing anti-aging response.

• The infusion is by means of transfusions or grafting of earlier collected stem cells or bone marrow.

• The infusion delivers said stem cells or bone marrow by means of indirect • The infusion delivers said stem cells or bone marrow by means of direct transportation via direct infusion into recipient's bone marrow.

• The infusion is indirect by single or multiple access ports of entry into the blood stream, whereby said infused stem cells find their way to all body tissue sites.

• The infusion is coupled with bone marrow infusion.

• The stem cells, lodge and become integrated into said donor's cell structure (integrated component).

• The integration is by self means.

• The infusion of said stem cells throughout said body's tissues are enhanced with respect to delivery, placement and integration within said donor/recipient's tissues, by means of preparing, promoting, and inducing agents.

• The infusions begin no later than in said donor's 1st, 2nd, 3rd , 4th, or 5th decade of chronological life.

• Theinfusions are made at intervals ranging from once every month to once every 5 years.

• The infusion into said donor is under a periodicity of no less than once every 5-year.

• The infusion into said donor is of a period of no less than at 10-year intervals.

• The stem cells, which are collected after said donor has reached a chronological age of 40 years or greater, are characterized as being of a biological age no greater than 25 years.

• The stem cells, which are collected after said donor has reached a chronological age of 50 years or greater, are characterized as being of a biological age no greater than 35 years.

• The stem cells, which are collected after said donor has reached a chronological age of 100 years or greater, are characterized as being of a biological age no greater than 55 years.

• The infusion continues until at least chronological age of 100 years • The stem cell samples are collected within a collection period prior to donor's first infusion, whereby said stem cells have an average chronological age equivalent to their biological DNA age.

• The stem cell samples are collected within a collection period prior to donor's infusion, whereby upon infusion said stem cells have an average biological DNA age at least 5 years less than the chronological age of said donor/recipient

• The difference between the infused biological DNA stem cell age and the chronological age of the donor/recipient increases, as the chronological age of the donor/recipient increases.

• The said stem cells are collected prior to donor's chronological age of 10 years, 20 years, 30 years, 40 years, 50 years or 60 years.

• The difference between resulting average biological age of the recipient's transformed body following infusions of DNA-containing stem cells is at least 5 years, 20 years, 30yeras, 40 years, 50 years or 60 years younger than the recipient's chronological age.

• A composition containing DNA in a stem cell carrier and optionally bone marrow, whereby said DNA is delivered in sufficient useable quantity to an autologous donor/recipient to regenerate existing cell structure on a global body basis.

• The composition of wherein said regeneration provides a 5 year differential between the donor/recipient's chronological age and his biological age.

• The composition wherein the age difference is no less than 20 years.

• The composition wherein concentrated stem cells represent a plurality to a majority of the composition.

• The activity resulting from said infusion into said recipient results in global cellar replacement of biologically younger DNA.

• The method is allogeneic.

• The allogeneic method wherein umbilical cord, embroyonic stem cell are employed. • The allogeneic method wherein embroyonic stem cells are employed taken from cloned embroyonic stem cells

• At least 3 billion nucleated stem cells are collected per session.

• The periodic collection and infusion methods are pro-active, absent response or in anticipation of any disease.

• The harvesting/collection of stem cells comprises making one or more insertions of a syringe means into the donor's hip or pelvic bone to extract bone marrow as said biological specimen containing stem cells.

• The stem cells comprise adult hematopietic stem cells.

• Introducing a stem cell growth stimulating agent into the donor prior to the harvesting in a manner effective to increase the population of the stem cells in the peripheral blood of the donor before harvesting the specimen.

• An anti-aging method comprising of collecting stem cells and/or separating out primordial stem cells selected from the group consisting of totipotent and pluripotent stem cells, optionally storing said stem cells, proliferating said primordial stem cells ex-vivo, infusing a stem cell product comprising a plurality of primordial stem cells into recipient in an amount representing 0.001 to 20.0% of the total nucleated cells contained in recipient's blood stream.

• An anti-aging method comprising periodically infusing a product containing a plurality of ex-vivo proliferated primordial stem cells selected from the group consisting of totipotent and pluripotent stem cells and combination, in an amount representing 0.001 to 20.0% of the total nucleated cells contained in recipient's blood stream.

• An autologous anti-aging method comprising:

o Periodic collection of whole stem cells from donor-recipient from shortly after his/her conception to chronological age 500 years, whereby said collection of stem cells provides a plurality of embryonic and/or adult cells selected from the group consisting of totipotent stem cells, pluripotent stem cells, multipotent stem cells, progenitor stem cells and combination thereof

o Providing for long term storage of said donor-recipient stem cells and/or bone marrow in sterile conditions in non-breachable containers

o Thawing a portion of said stored stem cells and/or bone marrow after a period of storage

o Proliferating said stem cells

• Periodic infusion of stem cells and/or bone into recipient results in an integration of biologically younger DNA containing stem cells and/or bone marrow throughout recipient's bone marrow & body tissues.

• A plurality of said infused stem cells are pluripotent.

• A majority of all said infused stem cells are pluripotent.

• Substantially all said infused stem cells are pluripotent.

• The stem cells are ex-vivo derived from an embroyonic stem cell.

• The stem cell are ex-vivo derived from an from umbilical cord stem cell

• The stem cell is an ex-vivo cell derived from a donor under age 10.

• Donor stem cells are matched to recipient's DNA.

• Donor stem are absent a HLA typing.

• Infusion product contains recipient's own DNA, stem cell and/or derivative

• The stem cell is an autogolous stem cell.

• The methods above being allogeneic, wherein said infusion product results in recipient experiencing no immune sensitivity/response.

• Infusion is vascular, targets cellular renewal/regeneration of recipient's bone marrow, infusion represents 0.1 to 5.0% of the total nucleated cells contained in recipient's blood stream, wherein said infusion represents 0.1 to 1.0% of the total nucleated cells contained in recipient's blood stream, wherein said infusion represents 0.3 to 4.0% of the total nucleated cells contained in recipient's blood stream, wherein said infusion represents 1.0 to 3.0% of the total nucleated cells contained in recipient's blood strea, wherein said infusion represents approximately 2.0% to 3.0% of the total nucleated cells contained in recipient's blood stream, and/or wherein said infusion represents approximately 3.0% of the total nucleated cells contained in recipient's blood stream, wherein said infusion is made on an average frequency 1 to 4 times/month over a period ranging from 2 to 12 months

• The recipient is under chronological age 20 and said infusion is made on an average frequency of 1 to 4 times/month over a period ranging from 2 to 9 months

• The average frequency is at least once a month over a period of 3 to 6 months

• The recipient is over chronological age 20 and said infusion is made on an average frequency of 1 to 3 times/month over a period ranging from 2 to 9 months

• The average frequency is once a month over a period of 6 months

• The periodic treatment is on average once to four times a month for a period of at least two to nine consecutive months in any single calendar year

• At least one or more infusions are repeated within the year following said infusions.

• The series of infusions is repeated at least once every other year for recipient's age less than 40.

• The recipient is over chronological age 30 and said infusion is made on an average frequency of once a month for the balance of recipient's life.

• The series of 2 to 12 consecutive infusions averaging 1 to 4 infusions/month is repeated at least once every 5 years for recipients under age 40.

• The recipient is over chronological age 40 and said infusion is made on an average frequency of once a month for the balance of recipient's life.

• The infusion product contains DNA in a stem cell carrier and optionally bone marrow, whereby said DNA is delivered in sufficient useable quantity to a recipient to regenerate existing cell structure on a global body basis. • The periodic infusion results in a global integration of biologically younger stem cells into recipient's tissues throughout his/her entire body, characterized by the formation of a transient chimera having a collective biological tissue composition age younger than recipient's chronological age.

• The biologically age differential of cells comprising the resultant transient tissue chimera is at least 5 years younger or 10 years younger than the chronological age of recipient.

• Periodic collection of stem cells and/or bone marrow from a recipient from the period shortly after his/her conception to chronological age 500 years,

• Separating, augmenting, proliferating said stem cells, independently or in combination with allogeneic donor cells under conditions where DNA matching exists

• The plurality of said stem cells are pluripotent stem cells.

• The recipient's blood contains on average of at least 0.3% to 3.0.0% volume of said pluripotent stem cells on a total nucleated cell basis for at least 72 hours.

• The allogeneic method comprises means of converting donor's stem cell's to the DNA characterization of recipient

• The stem cells are embroyonic stem cells, cord stem cells and proliferations thereof.

• The infusion product is the result of ex-vivo means.

• The infusion is by means of engraftment.

• The collection and transfusion includes the collection and transfusion or infusion of stem cells.

• The infusion is by means of a short series of two of more infusions within a period of less than a month.

• The infusion is by means of an extended long duration infusion of a period ranging from 5 minutes to one week. • The infusion is by means of massive dosage of at least 0.0000000001 % 0.000000001 % 0.00000001 % 0.00000001 % 0.00001 %, 0. 0001 % 0. 001 % 0.01 % 0. 1 % , 1.0%, 10.0% or more of the body weight of recipient.

• A combination of recipient's DNA with biologically younger non-HLA typing donor stem cells, resulting from an in-vitro or in-vivo process, whereby a younger identical DNA, DNA compliant or an otherwise sufficiently similar stem cell to recipient is produced, such that it is able to effect said global renewal/integration of recipient's cellular tissue with biologically compatible younger DNA.

• The method employs biologically younger non-HLA typing donor stem cells

• The stem cell is pτe-transfusion

• The collection and transfusion includes both the collection and transfusion or infusion of bone marrow and stem cells.

• The collection and transfusion or infusion includes the collection and infusion of bone marrow.

• The transfusion comprises infusing younger bone marrow into recipient's bone marrow

• The collected and transfused stem cells are selected from the group consisting of totipotent, pluripotent, multipotent stem cells and combination thereof

• The transfused stem cells are provided in same relative proportion to those collected.

• The transfused stem cells represent a plurality of undifferentiated stem cells.

• The transferred stem cells represent a majority of of undifferentiated stem cells.

• The transferred stem cells are exclusively undifferentiated stem cells.

• The stem cells are embryonic and adult stem cells, and combination thereof.

• The stem cells are embryonic stem cells or proliferated derivatives thereof.

• Wherein at any point said stem cells are proliferated in quantity from original quantity collected. • A recipient autoimmune suppression means.

• The adaptation of said stem cells is made to comport with the DNA structure of recipient.

• The additionally containing a signaling means to encourage generalized adsorption and usage of transfused stem cells.

• The undifferentiated stem cell is characterized by containing an Oct-4 protein.

• The collected stem cells are from the umbilical cord

• The collected stem cells are concentrated, augmented or proliferated

• The collected stem cells are sorted, concentrated, augmented or proliferated, and combination.

• The stem cells in their collected sera contents represent a plurality of blood product constituents.

• The donor stem cells or bone barrow are stored in sub-zero cryogenic temperatures in sterile non-breachable containers with means to preserve said stem cells or marrow indefinitely.

• The collection is from harvesting stem cells collection means.

• The means of collecting said stem cells is selected from an embryonic, umbilical, bone marrow, sterile mass blood, cellular dialysis, continuous circulating cell technique means and combination.

• The collection of said stem cells is by means specific for harvesting targeted cells from the vascular compartment.

• The collection of said stem cells is by concentration means specific for harvesting targeted cells from bone marrow.

• The concentration of said stem cells, is by means sufficient to result in a plurality of stem cells, which are in combination with blood product constituents. • The supply of said stem cells collected and transfused is sufficient to achieve periodic global integration of biologically younger stem cells into donor-recipient's bone marrow & tissues throughout his/her body.

• The collection obtains at least on the order of 10.sup.6 total nucleated cells per Kg. weight of the person in a single collection session.

• The unit comprises a dosage of a fraction of the total nucleated cells required to be transfused or infused.

• The units collected or transfused contain equal or different dosages.

• The preserving or storage step is independent of tissue or HLA typing of the collected stem cells prior to storing in a stem cell bank.

• The typing step includes providing indicia with each unit representing information of said at least one distinctive property.

• The indicia is embodied in at least one of a label, bar code, magnetic strip, and microchip.

• The stem cells are collected during a pre-disease stage in which the person may be diagnosed with a health condition.

• Securely enclosing said stem cells in non breachable containers suspended in bio¬ compatible nutrient media.

• Labeling, dating & entering into a secure data base (with redundancy and system controls for accuracy and backup in alternate locations)

• Storing said containers under liquid nitrogen.

• The storing said stem cells is within a bio-compatible media suspension. • The periodic infusion results in integration of biologically younger stem cells into recipient tissues throughout his/her body at least once every five years, which are comprised of the recipient current chronologically older aged DNA-containing cells.

• The composition of claim of 127, wherein said regeneration provides a 5 year differential between the donor/recipient's chronological age and his biological age.

• The composition of claim of 127, wherein the age difference is no less than 20 years.

• The composition of claim 127, wherein concentrated stem cells represent a plurality to a majority of the composition.

• The activity resulting from said infusion into said recipient results in global cellar replacement of biologically younger DNA.

• The collection methods of the aforementioned claims where at least 3 billion nucleated stem cells are collected per session.

• The periodic collection and infusion methods of the aforementioned claims are pro¬ active, absent response or in anticipation of any disease.

• The stem cells comprise adult hematopietic stem cells.

• Infusion constitutes routine cellular/tissue maintenance and administered prior to the onset of any disease, which may be contracted by recipient • An anti-aging blood serum composition comprising pluripotent stem cells, and normal blood constituents whereupon transfusion of said blood serum that recipient's blood contains 1.0% to 4.0.0% said stem cells compared to total nucleated cells contained in the blood stream, measured by the total number of CD + 34 cells contained.in recipient's blood stream.

• An anti-aging method comprising:

o periodic collection of stem cells from donor from birth to age 500 years

o sorting and concentrating said stem cells of step 1 in the collected sera contents, such that they represent a plurality of blood product constituents

o storing said donor-recipient stem cells of step 2 in sterile conditions in non- breachable containers under sub-zero temperature (equal or less than O0F)

o thawing a minor portion of said stored stem cells of step 3 after a period of 1 to 500 years

o periodic auto-transfusions of said thawed stem cells back into same donor (recipient) starting after said donor's chronological age 10,

o whereby said infusion results in an integration of biologically younger stem cells into recipient tissues throughout his/her body

• The collection is from harvesting stem cells collection means.

• The means is selected from a sterile mass blood collection, cellular dialysis, continuous circulating cell screening technique and combination.

• The collection of said stem cells is by means specific for harvesting targeted cells from the vascular compartment

• The collection of said stem cells is by concentration means specific for harvesting targeted cells from bone marrow.

• The concentration of said stem cells, is by means sufficient to result in a plurality of stem cells, which are in combination with blood product constituents. • The collection results in storage of at least 50, 250, 500 or 1000 grams of stem cells or material containing a plurality to a majority of said stem cells

• The mass is collected by donor's chronological age of 10, 20, 30, 40 or 50.

• The periodic collection occurs one (1) to twelve (12) times annually.

• The collection is no more frequently than every three months

• The periodic collection is no more frequently than every 6 months

• The collection of said stem cells is no less than every 5 years.

• The collection is no less than once a year for a donor chronological age 1 & 2, no less than twice a year for a donor ages 3 to 5, three times a year ages 6 through 15, and annually for ages 16 years or older.

• The a plurality to a majority of said collection occurs during the first 50 years of donor's chronological life.

• The collection yield is increased through the use of agents selected from the group consisting of donor preparatory, promotion, induction, and circulation enhancers.

• The agents increase the quantity, quality and accessibility of stem cells within the circulation.

• After said concentration, said stem cells are pooled, packed and batched according to incremental age time frames of collection.

• The incremental time frame is from birth to 2 years, 3 to 5 years, 5 to 10 years, 11 to

15 years, and in 5-year increments thereafter

• The minimum mass of stem cells collected is equivalent to 10% to 100% of a standard unit of red blood cells (no less than 25 grams) standard in the blood banking industry.

• The temperature is a cryogenic freezing temperature sufficient to insure long term storage of said stem cells.

• The storage is under liquid nitrogen. • The periodic infusion results in integration of biologically younger stem cells into recipient tissues throughout his/her body, which are comprised of the recipient current chronologically older aged DNA-containing cells

• Infusion incorporates harvested autologous stem cells, which have been collected at least 5, 10, 20, 30 or 40 years earlier

• The infusion contains a mixture of stem cells collected over various dates

• The infusion incorporates means for selecting stem cells for maximizing anti-aging response.

• The infusion is by means of auto-transfusions (autologous infusions) of the owner donor- recipient of earlier collected stem cells

• The infusion delivers said stem cells by means of indirect transportation via said donor's blood stream

• The infusion is by single or multiple access port of entry into the blood stream, whereby said infused stem cells find their way to all body tissue sites

• The stem cells, lodge and become integrated into said donor's cell structure (integrated component).

• The integration is by self means

• The infusion is characterized as being global, whereby autologous stem cells are delivered to all sites, throughout the recipient body, wherein

o biologically younger stem cells are integrated into recipient's tissues throughout his/her entire body

o a transient chimera is formed, characterized by the collective tissue composition of the recipient current chronologically older DNA-containing cells and biologically younger DNA-containing infused autologous stem cells, which were collected and stored years earlier

o the age differential of cells comprising the resulting transient tissue chimera is 5 years or greater

• An anti-aging method comprising:

o Periodic collection of stem cells from a donor from birth to adult,

o Sorting and concentrating said stem cells in the collected sera contents, such that said stem cells represent a plurality of blood product constituents,

o storing said donor-recipient stem cells in sub-zero temperatures in sterile non- breachable containers as means to preserve said stem cells indefinitely,

o periodic and regular infusions of said stem cells into the donor starting after age 10 years, whereby,

o the continuation of said periodic collections and periodic infusions result in an average biological age of the new body comprised of replaced tissues from the donor DNA at least 5 years younger than the actual chronological age of said donor/recipient

• The stem cell samples are collected within a collection period prior to donor's first infusion, whereby said stem cells have an average chronological age equivalent to their biological DNA age

• The difference between the infused biological DNA stem cell age and the chronological age of the donor/recipient increases, as the chronological age of the donor/recipient increases.

• The difference between resulting average biological age of the recipient's transformed body following infusions of DNA-containing stem cells is at least 5, 10, 20 30 40 50 60 or 100 years younger than the recipient's chronological age • A composition whereby said DNA is delivered in sufficient useable quantity to an autologous donor/recipient to regenerate existing cell structure on a global body basis.

• The composition wherein said regeneration provides a 5 year differential between the donor/recipient's chronological age and his biological age.

• The composition wherein the age difference is no less than 20 years.

• The composition wherein concentrated stem cells represent a plurality to a majority of the composition.

• The activity resulting from said infusion into said donor results in global cellar replacement.

Bone Marrow Methodologies • An in-vivo, optionally human, inchoate-transient tissue chimera structure comprised of tissue cells with at least two distinct DNA chronological ages [optionally with two distinct genomes], [optionally, whereby the age differential between DNA tissue age is at least 3 months, 6 months, 9 months, 1,2,3,4,5, 8, 10, 12, 15, 20, 30, 40, 60, 80, 90, or more, years]. • The following methods and conditions are applicable in the alternative and/or selectively combined. o The whole of the said transient chimera tissue structures is defined by the statistical preponderance of younger DNA containing cells incorporated throughout the majority of the recipient's body organs and tissues, thus establishing a 'mature' chimera. o The younger tissues transmogrifies the recipient's initial older tissues o The transmogrification is result of a proactive periodic series of introductions/infusions containing SC and/or BM into a recipient's body structures and/orsera. • An ex-vivo bone marrow (BM) composition includes: o a fraction of bone marrow selected from a matched donor, or recipient, and combination, and o A fraction of stem cells (SC) selected from compatible or altered [NGvHR - non graft-versus-host rejecting, matched or HLA typing free] donor stem cells or recipient (autologous) stem cells, and combination, ■ Optionally wherein said SCs comprise a plurality of primordial SCs selected from the group consisting of totipotent, pluripotent, omnipotent (multipotent) SCs, and combination. ■ Optionally, wherein said plurality is at least or greater than 5%, of primordial stem cells. ■ Optionally, being pluripotent and/or omnipotent (multipotent) ■ Optionally, containing normal blood constituents or serum ■ Optionally, wherein said SC are proliferated SCs ■ Optionally, said SCs are derived from donor under the age of 30, optionally collected from donor blood, donor bone marrow, umbilical cord, placenta or undamaged embryo. o Optionally, wherein said composition after the combination of both BM and SCs fractions becomes an activated composition, characterized as enjoying/generating greater anti-aging potency, regeneration capability and/or compatibility with recipient than prior to said combination.. An ex-vivo [recipient compatible or altered] bone marrow composition comprising a fraction of bone marrow selected from a matched donor and from recipient, o Optionally, wherein recipients bone marrow is younger, and the age differential between donor's BM and recipient's BM is at least five years o Optionally, wherein said age differential between 3rd party donor and recipient is as of date of infusion a product containing said BM into recipient. o Optionally wherein the ratio of the fraction of Donors BM to recipient's BM is more than 2:1 o Optionally, wherein said formulation/ratio enhances DNA compatibility with recipient o Optionally, reducing/eliminating graft v host rejection o Optionally, wherein said formulation/ratio enhances anti-aging potency. o Optionally, wherein said donor BM is collected from recipient matched donor [optionally using HLA matching procedure] o A fraction of stem cells (SC) selected from compatible or altered, NGvHR, matched or HLA typing free donor stem cells to recipient, recipient (autologous) stem cells, and combination, Optionally wherein said SCs comprise a plurality of primordial SCs selected from the group consisting of totipotent, pluripotent, omnipotent (multipotent) SCs, and combination. ■ Optionally, wherein said SCs are proliferated SCs , optionally enhanced, augmented, proliferated o Optionally, wherein said composition after the combination of both BM and SCs fractions becomes an activated composition, characterized as generating greater anti-aging potency, capability and/or compatibility than prior to said combination. • An ex-vivo [recipient compatible or altered] bone marrow composition comprising a bone marrow from a compatible or altered, NGvHR, or matched to recipient donor, wherein the age differential between donor's BM and recipient's chronological age is on average at least five years [or 50. 40, 30, 20, 10 years]. o Optionally wherein said BM is collected from compatible or altered donor , who is HLA type-matched or otherwise matched with recipient; or donated SCs are altered to be compatible or altered to recipient's DNA o A fraction of stem cells (SC) selected from compatible or altered, NGvHR, matched, or HLA typing free donor stem cells, recipient (autologous) stem cells, and combination, ■ Optionally wherein said SCs comprise a plurality of primordial SCs selected from the group consisting of totipotent, pluripotent, omnipotent (multipotent) SCs, and combination. • In vivo recipient compatible or altered BM composition comprising: o Recipient's in-vivo BM, o Optionally, compatible or altered 3rd party donor BM Optionally, wherein said BM is donor collected from compatible or altered, NGvHR, or matched donor, [or Autologous/recipient Donor], and combination, o Optionally, BM collected from autologous/recipient prior to [or approximately concurrent with] an infusion incorporating said recipient's BM. o And, a fraction of stem cells selected from compatible or altered, NGvHR, matched, or HLA typing free donor SCs, or recipient (autologous) stem cells, and combination, ■ Optionally, wherein said SC are proliferated ex-vivo SCs, and ■ Optionally wherein a plurality of said SCs comprise totipotent, pluripotent, omnipotent SCs, and combination. • An recipient compatible or altered or NGvHR BM composition comprising: o In-vivo recipient (autologous) bone marrow, o Younger aged DNA containing Donor or Autologous SCs. • An ex-vivo [recipient own] bone marrow composition comprising: o A fraction of bone marrow selected from a donor-recipient [autologous BM], ■ Optionally, wherein donor-recipient bone marrow is chronologically younger than chronological age of recipient on date of an infusion by least five years] o A fraction of stem cells (SC) selected from a 3rd party donor compatible or altered, NGvHR, matched or HLA typing free donor stem cells, recipient (autologous) stem cells, and combination, ■ Optionally wherein said SCs comprise a plurality of primordial SCs selected from the group consisting of totipotent, pluripotent, omnipotent SCs, and combination. • Optionally, wherein said SC are proliferated SCs • An ex-vivo or in-vivo recipient compatible or altered composition comprising o a fraction of bone marrow comprising donor and/or recipient BM, and o a fraction of proliferated recipient's autologous SCs, matched, NGvHR, or HLA typing free 3rd party donor SC, and combination, wherein said fraction contains at least a plurality of 5% primordial stem cells, selected from totipotent, pluripotent, omnipotent SCs, and combination. ethod of preparing an ex-vivo BM product, comprising: o Collecting BM, from a 3rd party donor, or (autologous) recipient, and combination

• Optionally said BM is collected from matched donor;.

• Optionally, wherein recipient's bone marrow is younger, and the age differential between donor's BM and recipient's BM is at least five years.

• Optionally, wherein said BM collected from autologous/recipient

• Optionally, wherein said BM is collected at a period prior to an infusion incorporating said BM, which is sufficient for its useful incorporation in this product.

• Optionally, said BM collected from recipient is at least 4-7 days, prior to date of an infusion incorporating said BM.

• Optionally, said BM collected from recipient is at least 1 years younger than chronological age of recipient.

• Optionally, said BM collected from recipient is prior to his age of 30 years or younger.

o Collecting SC from a matched 3rd party donor, or source where SCs do not require HLA typing (embryonic, umbilical cord, placenta), or autologous source (recipient), and combination,

■ Optionally, wherein said SCs are collected from an embryo (absent damage) or umbilical cord or placenta and combination, and/or 3rd party donor age 10 years or younger than recipient.

■ Optionally where said SCs are 3rd party SCs at least 10 biological or chronological years younger than recipi nt o Separating said stem cells into their primordial fraction, selected from the group consisting of totipotent, pluripotent and omnipotent SCs, and combination,

optionally, selecting pluripotent and omnipotent SCs

optionally pluripotent SCs only

Optionally preparing a SC fraction wherein said SCs comprise a plurality of primordial SCs selected from the group consisting of totipotent, pluripotent, omnipotent SCs, and combination.

Optionally, wherein said SC plurality is at least or greater than 5%, of the total of SCs contained in the fraction. o Proliferating separated/collected primordial SCs

Optionally proliferating totipotent, pluripotent, omnipotent SCs, only o Combining a fraction of said proliferated SCs and BM together to create a product.

■ Optionally, wherein said BM fraction is a combination of both donor and recipient BM and wherein said combination provides for a ratio of donor's BM fraction to fraction of recipient's BM greater than 2:1

• Optionally, a ratio wherein said formulation/ratio enhances DNA compatibility with recipient

• Optionally, a ratio wherein said formulation/ratio enhances anti-aging potency.

• Optionally in the activation step below.

■ Optionally, wherein said BM fraction to SC fraction is greater than 2:1

• Optionally, a ratio wherein said formulation/ratio enhances DNA compatibility with recipient

• Optionally, a ratio wherein said formulation/ratio enhances anti-aging potency. • Optionally in the activation step below.

Optionally, activating, nurturing, stimulating and/or shocking ["Activating"] said product containing fractions of SCs and BM by exogenous and/or endogenous means (Activated Composition).

Optionally, said activation is by exogenous means including:

Signaling or stimulation via biological & chemical agents or means and physical agents or means, such as sound (harmonic resonance, ultra sonic or other), light or electronic wave (various spectrum including UV, radio, high to low wave length to infrared, etc.), electronic, magnetic, chemical, agitation/mixing, pressure changes , vacuum applications, temperature - thermo or cryo manipulations,

other means (as provided in the referenced art), and combination

■ Optionally, wherein said activation includes introduction of other inputs, including nutrients, preservation, augmentation ingredients or other means as provided in the referenced art :

• Providing matched or autologous blood cell and other components, constituents, minerals, elements, sera, protein and/or other product sufficient to nourish said composition

• Optionally, additionally providing normal blood cell and other constituents or sera.

■ Optionally, wherein said activation occurs naturally as condition of combining BM and SC fraction

■ Optionally, wherein said activation includes method to

■ Preserve said BM and SC composition

■ Enhance SC and/or BM growth and proliferation

• Inhibit or Enhance SC differentiation, if deemed desirable. Or other techniques such as may employ enhancement and augmentation of, through prior and/or ongoing xp u e t i d i d/ ti l ti biological, chemical, physical agents to include nanotechnology, radio, photonic, magnetic, electronic chemical, harmonic, thermo or cryogenic, hormonal agents and/or techniques and combinations thereof

Optionally, wherein after the introduction of said primordial SC into the composition, and/or after said activation, said SC and/or BM [independently] proliferate and/or differentiate

• Optionally, wherein said SC proliferation or differentiation is independent of earlier SC proliferation [after collection prior to combination with BM].

• Optionally, wherein said activation proliferation is dependent upon method employed for earlier SC proliferation.

■ Optionally, wherein said proliferation and/or differentiation of activation further results in stem cells selected from the group consisting of totipotent, pluripotent, omnipotent or progenitor SCs, and combination.

• Optionally, pluripotent SCs

■ Optionally, wherein after activation said product increases in volume and/or potency.

• Optionally, wherein total SC population increases 2 to 6 times ionally, incubating/growing said activation product for a period of time,

■ Optionally, said incubation period is sufficient to generate maximum potency, recipient compatibility, or product (SC and/or BM) volume, and combination.

■ Optionally, said incubation is for 1hour to 1 year.

• Optionally, at least 5 to 6 days [or other acceptable period ranging from 2 hours to 6 months, 1-6 hours, 4-6 hours, 2-24 hours, 1-5 days, 1-7 days, 3-8 days, 1-5 weeks, 4-6 weeks, 1-8 months, 4-6 months].

• Optionally, processing, adopting, augmenting and/or preparing said product, as required, to become a compatible or altered infusion product for recipient, whereby infusion is made absent "graft v host" or immune sensitivity response

• Optionally, wherein DNA attributes of recipient are conferred to the composition.

• Optionally storing said product, any component or subcomponent of said product at any step or point prior to infusion.

■ Storage of any component or subcomponent may optionally be at any step after collection of SC and/or bone marrow, prior to infusion/injection.

o Preparing a final composition/product suitable for infusion into recipient.

The following elements apply alternatively or selectively together to the above bone marrow methodologies

• The composition contains bone marrow selected from both recipient and a compatible or altered or NGvHR donor.

• A fraction of recipient bone marrow and a fraction of compatible or altered or NGvHR donor bone marrow.

• An ex-vivo bone marrow composition includes altered or NGvHR donor bone marrow, recipient bone marrow, and a quantity of compatible or altered, NGvHR or HLA typing free donor primordial stem cells.

• An ex-vivo bone marrow composition includes compatible or altered/matched or NGvHR donor bone marrow, recipient bone marrow and optionally a fraction of stem cells.

• An ex-vivo bone marrow composition includes compatible or altered donor bone marrow of recipient, recipient bone marrow, and a quantity of compatible or altered pluripotent and/or omnipotent stem cells. • The bone marrow composition is augmented to become recipient compatible or altered or enhanced bone marrow.

• The pluripotent and/or omnipotent stem cells are at least 10 years younger than recipient's chronological age, optionally being embroyonic, cord stem cells or placental SCs,.

• The bone marrow is harvested from a donor, optionally augmented and then expanded or proliferated

• The volume of bone marrow to stem cells is greater than 1:1.

• The volume of bone marrow to stem cells is at least 2 :1

• The volume of bone marrow to stem cells is at least 3:1

• The combination has incubated for a period of at least 4 hours to 5 hours, days or weeks.

• The composition becomes active after the combination of BM and SC fractions due to a change of temperature or other physical or biological factors or use of novel technologies to include nanotechnology and bio-engineering.

• The composition becomes active after an endogenous stimulation.

• After said primordial stem cells are introduced into a composition of proliferated SCs, said composition is activated, characterized by greater anti-aging potency than prior to said combination.

• The activation is characterized by a volume increase in the total number of SCs.

• An composition avoids "graft versus host" rejection or immune response of recipient.

• The BM or SC are collected from compatible or altered, NGvHR, matched or HLA typed donors to recipient or other means suitable to insure compatibility to include altering the donated cellular DNA by various current and emerging technologies.

• The 3rd party donor BM and/or SCs are employed, where said BM and SCs are introduced/combined with recipient's BM and/or SCs under conditions and for a sufficient period of time to acquire recipient DNA characterization • The bone marrow fraction is collected from a matched 3rd party donor at least 10 years younger than recipient

• The ex-vivo and in-vivo composition of claims , wherein said bone marrow fraction is collected from 3rd party donor, who is as of date of collection at least 30 years younger than recipient.

• The ex-vivo composition of claims , wherein said BM fraction is solely or contains in part recipient's bone marrow

• The ex-vivo composition of claims , wherein said BM fraction is solely or contains in part matching 3rd party donor bone marrow.

• The ex-vivo composition of claims , wherein said BM fraction has been augmented, refined, separated, and/or proliferated, in preparation for infusion into recipient and may employ selective inhibition methods and/or enhancements of- through prior and/or ongoing exposure to inducing and/or stimulating biological (including organisms - re-engineered retroviruses, other similar means), antibodies, immune reactants - stimulants, homeopathic chemicals, cellular growth or other hormonal agents; physical agents to include nanotechnology, radio, photonic, electronic, magnetic, chemical, harmonic, thermo or cryogenic and/or techniques, and combinations thereof

The Appendix attached hereto includes definitions and other information applicable to and incorporated herein. DEFINITIONS [TO BE ALPHABETIZED AND EDITED]

INTRODUCTION as provided herein includes transfusion, infusion, injection, transportation, engraftment, inoculation, adsorption, absorption, intercalation, nano-continuous/nano-based or supported transport, physical transport, and other means, whereby Applicants' compositions are provided into the body, body tissue, bone marrow, skeletal system, vascular system of recipient . As provided herein and in Applicants' claims and specification the terms "introduction," "transportation" and "infusion" may be generally interchanged.

ADULTSTEM CELLS

"Adult stem cells" refer to multipotent stem cells such as those found in the blood stream. They can be derived from living persons or umbilical cords and would not involve destruction of an early embryo. Here again, we note that the term "adult" may be misleading, for the just-born infant is considered an "adult" for purposes of adult stem cell research.

Others argue that adult stem cells will not solve the ethical issues. Adult stem cells are already partially differentiated, already designated for a limited range of tissue types. They are not pluripotent. To date, no credible experiments on adult stem cells have demonstrated that their value to regenerative medicine is equal to that of embryonic stem cells. Some studies have suggested that adult stem cells from one tissue type can migrate to and integrate into other tissues. However, it has not been demonstrated that these stem cells actually become the new tissue type; that is, it has not been demonstrated that they function as a stem cell of this new tissue type - they do not produce daughter cells of that tissue type nor do they appear to regenerate that tissue. In order for transplanted stem cells to be valuable for regenerative medicine they need to be capable of three things: l) they must lodge in the host tissue, 2) they must become that tissue type, and 3) they must regenerate that tissue. As of this writing, only embryonic stem cells have demonstrated all three capabilities. Most scientists recommend that adult stem cell research continue, to be sure; but they recommend that embryonic stem cell research also be continued.

Germ Cells

Cells comprising actual reproductive components of an organism (specifically, eggs and sperm, and their precursors). hES: Human Embryonic Stem Cells

Pluripotent stem cells that differentiate into various tissues in the body.

Pluripotent Stem-Cells

Stem cells are cells that can develop into any of the three major tissue types: endoderm (interior gut lining), mesoderm (muscle, bone, blood), and ectoderm (epidermal tissues and nervous system). Pluripotent stem cells can eventually specialize in any bodily tissue, but they cannot themselves develop into a human being. These, like totipotent SCs are uncommitted. Omnipotent (multipotent) are also uncommitted, but often being close to progenitor status, which are committed SCs.

On the top are totipotent (totally potent) stem cells, which are capable of forming every type of body cell. Each totipotent cell could replicate and differentiate and become a human being. All cells within the early embryo are totipotent up until the 16 cell stage or so.

Next are the pluripotent stem cells which can develop into any of the three major tissue types: endoderm (interior gut lining), mesoderm (muscle, bone, blood), and ectoderm (epidermal tissues and nervous system). Pluripotent stem cells can eventually specialize in any bodily tissue, but they cannot themselves develop into a human being. Other stem cells include multipotent/omnipotent and progenitor. Progenitor are committed stem cells.

ES Cells

Embryonic stem cells. Adult Stem Cell

Any stem cell taken from mature tissue, regardless of the age of the donor.

Chimera: In medicine, a person composed of two genetically distinct types of cells. Human chimeras were first discovered with the advent of blood typing when it was found that some people had more than one blood type. Most of them proved to be "blood chimeras" - non- identical twins who shared a blood supply in the uterus. Those who were not twins are thought to have blood cells from a twin that died early in gestation. Twin embryos often share a blood supply in the placenta, allowing blood stem cells to pass from one and settle in the bone marrow of the other. About 8% of non-identical twin pairs are chimeras.

In Greek mythology, the Chimera was an awesome fire-breathing monster with the head of a lion, the body of a goat, and the tail of a serpent. The Chimera was killed by the hero Bellerophon mounted, in most versions of the tale, on Pegasus, the winged horse.

Tissue Chimera: Tissue composed of two genetically distinct types of cells. Human chimeras were first discovered with the advent of blood typing when it was found that some people had more than one blood type. Most of them proved to be "blood chimeras" —non-identical twins who shared a blood supply in the uterus. Those who were not twins are thought to have blood cells from a twin that died early in gestation. Twin embryos often share a blood supply in the placenta allowing blood stem cells to pass from one and settle in the bone marrow of the other. About 8% of non-identical twin pairs are chimeras.

Many more people are microchimeras and carry smaller numbers of foreign blood cells that may have passed from mother across the placenta, or persist from a blood transfusion. In vitro fertilization (IVF) is also contributing to the number of human chimeras. To improve success rates, two or more embryos are placed in the uterus so women who have IVF have more twin pregnancies than usual. More twins mean more chimeras. In Greek mythology, the Chimera was an awesome fire-breathing monster with the head of a lion, the body of a goat, and the tail of a serpent. The Chimera was killed by the hero Bellerophon mounted, in most versions of the tale, on Pegasus, the winged horse.

In the practice of this invention a "Tissue Chimera" or "transient tissue chimera" is a tissue structure of two distinct intertwined DNA aged tissue having two distinctive aged genomes. The blended or interspersed dual DNA aged tissue is heterogeneously interwoven.. The age differential of the tissue chimera is eventually diminished with the continuation of periodic infusion with the younger DNA aged tissue ultimately surviving. In biology the genome of an organism is its whole hereditary information and is encoded in the DNA (or, for some viruses, RNA) . This includes both the genes and the non-coding sequences of the DNA. The term was coined in 1920 by Hans Winkier. Professor of Botany at the University of Hamburg . Germany , as a portmanteau of the words gene and chromosome.

More precisely, the genome of an organism is a complete DNA sequence of one set of chromosomes ; for example, one of the two sets that a diploid individual carries in every somatic cell. The term genome can be applied specifically to mean the complete set of nuclear DNA (i.e., the "nuclear genome") but can also be applied to organelles that contain their own DNA, as with the mitochondrial genome or the chloroplast genome. When people say that the genome of a sexually reproducing species has been "sequenced ." typically they are referring to a determination of the sequences of one set of autosomes and one of each type of sex chromosome , which together represent both of the possible sexes. Even in species that exist in only one sex, what is described as "a genome sequence" may be a composite from the chromosomes of various individuals. In general use, the phrase "genetic makeup" is sometimes used conversationally to mean the genome of a particular individual or organism. The study of the global properties of genomes of related organisms is usually referred to as genomics, which distinguishes it from genetics which generally studies the properties of single genes or groups of genes.

Gene Splicing

The cell process by which a gene is cut into different parts, exons and introns. The exons are the coding region and are put back together to make the gene that is transcribed and translated into a protein. Sometimes the same gene can be spliced different ways to give rise to different proteins.

Gene Expression

Genes are activated or deactivated throughout life. Activated genes are expressed by being transcribed and translated.

Gene Mutation

A change in the DNA base pair arrangement. It can be a single nucleotide that is altered, or it can be a large deletion or insertion of bases. graft-versus-tumor

An immune response to a person's tumor cells by immune cells present in a donor's transplanted tissue, such as bone marrow or peripheral blood.

Graft versus host rejection(disease)

• GVHD. A reaction of donated stem cells against the patient's tissue. Also called GVHD. It may be characterized as an anaphylactic reaction, or negative immune response.

Previous grade 2 follicular lymphoma, grade 3 follicular lymphoma. Definitions: grade IV astrocytoma, grading, graft Next graft-versus-tumor . gram, granisetron. granulocyte . Definitions: granulocyte colony-stimulating factor

Stem Cells

Stem cells are essentially undifferentiated cells. There are many kinds of stem cells, some more differentiated than others. When they divide, their progeny mature and specialize into a specific type of cell (i.e. heart, blood, liver). These differentiated cells form an embryo. Stem cells also exist in adults (Adult Stem (AS) cells) and are used to repair and regenerate damaged organs and tissues throughout life. However, in adults the repair and regeneration by stem cells is limited to only certain cell types. In contrast, embryonic stem (ES) cells are not limited in there potential to differentiate into every cell type. Embryonic germ (EG) cells have the same potential as ES cells. It is the versatility and nonspecifically of these cells that gives them the potential to have therapeutic applications.

hES: Human Embryonic Stem Cells

Pluripotent stem cells that differentiate into various tissues in the body. Transmogrification trans'mog'ri'fied, trans mogτ i'fying, trans-mog'ri'fies To change into a different shape or form, especially one that is fantastic. See Synonyms at convert .

In Vitro

Outside the living body and in an artificial environment.

In Vivo

In the living body of a plant or animal.

Primordial Germline Cells

The source of embryonic germ cells. In normal development, these are the cells that give rise to eggs or sperm.

Somatic Cells

Cells from the body that compose the tissues, organs, and parts of that individual other than the germ (sex) cells.

Totipotent Stem Cells

Stem cells which are capable of forming every type of body cell. Each totipotent cell could replicate and differentiate and become a human being. All cells within the early embryo are totipotent up until the 16 cell stage or so.

ES Cells

Embryonic stem cells.

Proliferation—Expansion of cells by the continuous division of single cells into two identical daughter cells.

Adult (or somatic) stem cell—An undifferentiated cell found in a differentiated tissue that can renew itself and differentiate (with certain limitations) to give rise to all the specialized cell types of the tissue from which it originated. It is important to note that scientists do not agree about whether or not adult stem cells may give rise to cell types other than those of the tissue from which they originate.

Adult stem cells are cells capable of dividing and replacing damaged tissue. They exist in tissues such as the bone marrow, brain, muscle and liver. Unlike their neighbors, which are already differentiated into specialized cell types, adult stem cells remain immature. When tissue becomes damaged, the adult stem cells divide (a process called self-renewal) to produce new cells. Some of the resulting cells divide, but unlike stem cells they mature to take over for the damaged cells. Within any organ or tissue, generally only the stem cells have the ability to self-renew and appear to be the only cells that regenerate tissues when they become damaged. In the bone marrow, blood- forming stem cells make up only about 1 in 20,000 cells.

Hematopoietic stem cells (HSC) are stem cells and the early precursor cells which give rise to all the blood cell types that include both the myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets and some dendritic cells) and lymphoid lineages (T-cells, B-cells, NK-cells, some dendritic cells). The definition of hematopoietic stem cells have undergone considerable revision in the last two decades. The hematopoietic tissue have cells with long term and short term regeneration capacities and committed multipotent, oligopotent and unipotent progenitors.

Astrocyte—a type of supporting (glial) cell found in the nervous system.

Adult Stem Cell —"Adult stem cells" refer to multipotent stem cells such as those found in the blood stream. They can be derived from living persons or umbilical cords and would not involve destruction of an early embryo. Here again, we note that the term "adult" may be misleading, for the just-bom infant is considered an "adult" for purposes of adult stem cell research.

Blastocoel—The fluid-filled cavity inside the blastocyst of the developing embryo. Blastocyst—A preimplantation embryo of about 150 cells produced by cell division following fertilization. The blastocyst is a sphere made up of an outer layer of cells (the trophoblast), a fluid-filled cavity (the blastocoel), and a cluster of cells on the interior (the inner cell mass).

Bone marrow stromal cells—A mixed population of stem cells found in bone marrow that does not give rise to blood cells but instead generates bone, cartilage, fat, and fibrous connective tissue.

Cell division—Method by which a single cell divides to create two cells. There are two main types of cell division: mitosis and meiosis.

Cell-based therapies—Treatment in which stem cells are induced to differentiate into the specific cell type required to repair damaged or destroyed cells or tissues.

Cell culture—Growth of cells in vitro in an artificial medium for experimental research.

Clone—Generate identical copies of a molecule, cell, or organism.

1. When it is used to refer to cells grown in a tissue culture dish, a clone is a line of cells that is genetically identical to the originating cell. This cloned line is produced by cell division (mitosis) of the originating cell. 2. The term clone may also be used to refer to an animal produced by somatic cell nuclear transfer (SCNT).

Cloning—See Somatic cell nuclear transfer (SCNT).

Cord blood stem cells—See Umbilical cord blood stem cells.

Culture medium—The liquid that covers cells in a culture dish and contains nutrients to feed the cells. Medium may also include other growth factors added to produce desired changes in the cells.

Differentiation—The process whereby an undifferentiated embryonic cell acquires the features of a specialized cell such as a heart, liver, or muscle cell. Directed differentiation—Manipulating stem cell culture conditions to induce differentiation into a particular cell type.

DNA—Deoxyribonucleic acid, a chemical found primarily in the nucleus of cells. DNA carries the instructions or blueprint for making all the structures and materials the body needs to function.

Ectoderm—Outermost germ layer of cells derived from the inner cell mass of the blastocyst; gives rise to the nervous system, sensory organs, skin, and related structures.

Embryo—In humans, the developing organism from the time of fertilization until the end of the eighth week of gestation, when it is called a fetus.

Embryoid bodies—Rounded collections of cells that arise when embryonic stem cells are cultured in suspension. Embryoid bodies contain cell types derived from all 3 germ layers.

Embryonic germ cells—Pluripotent stem cells that are derived from early germ cells (those that would become sperm and eggs). Embryonic germ cells (EG cells) are thought to have properties similar to embryonic stem cells.

Embryonic stem cells—Primitive (undifferentiated) cells derived from a 5-day preimplantation embryo that have the potential to become a wide variety of specialized cell types.

Embryonic stem cell line—Embryonic stem cells, which have been cultured under in vitro conditions that allow proliferation without differentiation for months to years.

Endoderm—Innermost layer of the cells derived from the inner cell mass of the blastocyst; it gives rise to lungs, other respiratory structures, and digestive organs, or generally "the gut".

Enucleated — A cell with its nucleus removed.

Feeder layer—Cells used in co-culture to maintain pluripotent stem cells. For human embryonic stem cell culture, typical feeder layers include mouse embryonic fibroblasts (MEFs) or human embryonic fibroblasts that have been treated to prevent them from dividing.

Fertilization—The joining of the male gamete (sperm) and the female gamete (egg).

Fetus—A developing human from approximately eight weeks after conception until the time of its birth.

Gamete—An egg (in the female) or sperm (in the male) cell. See also Somatic cell.

Gene—A functional unit of heredity that is a segment of DNA found on chromosomes in the nucleus of a cell. Genes direct the formation of an enzyme or other protein.

Germ layers—Fertilization of an egg stimulates cell division, and the resulting cells are organized into three different layers, called germ layers. The three layers are the ectoderm, the mesoderm, and the endoderm.

Hematopoietic stem cell—A stem cell that gives rise to all red and white blood cells and platelets.

Human embryonic stem cell (hESC)—A type of pluripotent stem cell derived from the inner cell mass (ICM) of the blastocyst.

In vitro—Latin for "in glass"; in a laboratory dish or test tube; an artificial environment.

In vitro fertilization—A technique that unites the egg and sperm in a laboratory, instead of inside the female body.

Inner cell mass (ICM)—The cluster of cells inside the blastocyst. These cells give rise to the embryo and ultimately the fetus. The ICM cells are used to generate embryonic stem cells.

Long-term self-renewal—The ability of stem cells to renew themselves by dividing into the same non-specialized cell type over long periods (many months to years) depending on the specific type of stem cell. Mesenchymal stem cells—Cells from the immature embryonic connective tissue. A number of cell types come from mesenchymal stem cells, including chondrocytes, which produce cartilage.

Meiosis—Cell division of a gamete to reduce the chromosomes within it to half the normal number. This is to ensure that fertilization restores the full number of chromosomes rather than causing aneuploidy, or an abnormal number of chromosomes.

Mesoderm—Middle layer of a group of cells derived from the inner cell mass of the blastocyst; it gives rise to bone, muscle, connective tissue, kidneys, and related structures.

Microenvironment—The molecules and compounds such as nutrients and growth factors in the fluid surrounding a cell in an organism or in the laboratory, which play an important role in determining the characteristics of the cell.

Mitosis—Cell division that allows a population of cells to increase its numbers or to maintain its numbers.

Multipotent—Ability of a single stem cell to develop into more than one cell type of the body. See also pluripotent and totipotent. Multipotent stem cells such as those found in the blood stream. They can be derived from living persons or umbilical cords and would not involve destruction of an early embryo.

Neural stem cell—A stem cell found in adult neural tissue that can give rise to neurons and glial (supporting) cells. Examples of glial cells include astrocytes and oligodendrocytes.

Neurons—Nerve cells, the structural and functional unit of the nervous system. A neuron consists of a cell body and its processes—an axon and one or more dendrites. Neurons function by starting and conducting impulses. Neurons transmit impulses to other neurons or cells by releasing neurotransmitters at synapses.

Oligodendrocyte—A supporting cell that provides insulation to nerve cells by forming a myelin sheath (a fatty layer) around axons.

Passage —Around of cell growth and proliferation in cell culture. Plasticity—The ability of stem cells from one adult tissue to generate the differentiated cell types of another tissue.

Pluripotent—Ability of a single stem cell to give rise to all of the various cell types that make up the body. Pluripotent cells cannot make so-called "extra-embryonic" tissues such as the amnion, chorion, and other components of the placenta.

Preimplantation —With regard to an embryo, preimplantation means that the embryo has not yet implanted in the wall of the uterus. Human embryonic stem cells are derived from preimplantation stage embryos fertilized outside a woman's body (in vitro).

Proliferation—Expansion of cells by the continuous division of single cells into two identical daughter cells.

Regenerative medicine—A treatment in which stem cells are induced to differentiate into the specific cell type required to repair damaged or destroyed cell populations or tissues. (See also cell-based therapies).

Reproductive cloning—The goal of reproductive cloning is to create an animal being identical to the animal that donated the somatic cell nucleus. The embryo is implanted in a uterus and develops into a live being. The first animal to be created by reproductive cloning was Dolly the sheep, born at the Roslin Institute in Scotland in 1996. See also Somatic cell nuclear transfer (SCNT).

Signals—Internal and external factors that control changes in cell structure and function.

Somatic cell—any body cell other than gametes (egg or sperm). See also Gamete.

Somatic cell nuclear transfer (SCNT) —A technique that combines an enucleated egg (nucleus removed) and the nucleus of a somatic cell to make an embryo. SCNT is the scientific term for cloning. SCNT can be used for therapeutic or reproductive purposes, but the initial stage that combines an enucleated egg and a somatic cell nucleus is the same. See also therapeutic cloning and reproductive cloning. Somatic stem cells—Non-embryonic stem cells that are not derived from gametes (egg or sperm cells).

Stem cells—Cells with the ability to divide for indefinite periods in culture and to give rise to specialized cells.

Stromal cells—Non-blood cells derived from blood organs, such as bone marrow or fetal liver, which are capable of supporting growth of blood cells in vitro. Stromal cells that make the matrix within the bone marrow are also derived from mesenchymal stem cells.

Subculturing— Transferring cultured cells, with or without dilution, from one culture vessel to another.

Surface markers—Proteins on the outside surface of a cell that are unique to certain cell types, which are visualized using antibodies or other detection methods.

Teratoma—Scientists verify that they have established a human embryonic stem cell (hESC) line by injecting putative stem cells into mice with a dysfunctional immune system. Since the injected cells cannot be destroyed by the mouse's immune system, they survive and form a multi-layered benign tumor called a teratoma. Even though tumors are not usually a desirable outcome, in this test, the teratomas serve to establish the ability of a stem cell to give rise to all cell types in the body. This is because the teratomas contain cells derived from each of the three embryonic germ layers.

Therapeutic cloning—The goal of therapeutic cloning is to create cells that exactly match a patient. By combining a patient's somatic cell nucleus and an enucleated egg, a scientist may harvest embryonic stem cells from the resulting embryo that can be used to generate tissues that match a patient's body. This means the tissues created are unlikely to be rejected by the patient's immune system. See also Somatic cell nuclear transfer (SCNT).

Totipotent —A totipotent stem cell can give rise to all the cell types that make up the body plus all of the cell types that make up the extraembryonic tissues such as the placenta. (See also Pluripotent and Multipotent). Transdifferentiation —The process by which stem cells from one tissue differentiate into cells of another tissue. See also Plasticity.

Trophectoderm—a term used to refer to trophoblast cells in mice.

Trophoblast—The extraembryonic tissue responsible for implantation, developing into the placenta, and controlling the exchange of oxygen and metabolites between mother and embryo.

Umbilical cord blood stem cells—stem cells collected from the umbilical cord at birth that can produce all of the blood cells in the body (hematopoietic). Cord blood is currently used to treat patients who have undergone chemotherapy to destroy their bone marrow due to cancer or other blood-related disorders.

Undifferentiated—A cell that has not yet generated structures or manufactured proteins characteristic of a specialized cell type.

Glossary of Terms

adult stem cells: Undifferentiated cells that are found in various specialised (differentiated) tissues of the body (eg in bone marrow, skin, intestine).

bioinformatics: The use of computing technologies to discover and manage systematic biological information by translating genetic and protein data into manageable forms that can be analysed and interpreted, often in conjunction with rapidly growing databases.

biomass: All the living organisms of a particular region, considered collectively.

bioprospecting: The practice of screening samples of plants, animals and microorganisms (often collected from the wild) for active chemical compounds or molecules that can be developed into patented and marketable commodities.

biotechnology: The understanding and use of biological processes and organisms for health, social, environmental and economic applications or outcomes. biotechnology industry: A collection of industries - eg pharmaceutical, food processing, plant and animal agriculture, environmental management or minerals processing - which utilise some of the techniques of biotechnology. blastocyst: A hollow ball of 50 - 100 cells reached after 4-5 days embryonic development just before implantation in the uterus. cell: The smallest structural unit of living organisms that is able to grow and reproduce independently. cell line: Cells of common descent and type cultured in the laboratory. clone: A cell, group of cells, or organism produced from one individual cell through asexual processes that do not involve the interchange or combination of genetic material. The word 'clone' may be used as a noun or a verb. cytotechnology: 'diagnostic cytopathology' = Involves the interpretation of cells that spontaneously exfoliate or are removed from tissues by abrasion or fine needle aspiration, eg specimens from the cervix (Pap tests), breast, thyroid, lymph node, liver, etc

de-differentiation: The process of inducing a specialised cell to revert towards pluripotency.

deoxyribonucleic acid (DNA): The chemical compound that constitutes the hereditary material of living organisms, ie the genetic code. The DNA in human beings is grouped into approximately 35,000 genes.

differentiation: The process by which less specialised cells develop into more specialised cell types.

embryo: A general term applied to the developing organism from the completion of fertilisation, until 8 weeks when the organism becomes known as a foetus.

enucleated: A cell from which the nucleus has been removed (usually an egg). foetus: The term used for a developing human after the eighth week of development until birth. functional genomics: The study of the functional consequences for a cell or organism of the presence, absence or modification of a gene. gamete: A mature male or female germ cell, a sperm or egg. gene: The carrier of hereditary characteristics, a piece of DNA that codes for the production of a particular molecule (usually a protein) used to make a part of machinery or tissue of an organism genetically modified organism (GMO): An organism in which characteristic/s have been altered by a modification of the genome (for example, by the introduction of a modified gene from another organism). genome: The collection of all the genes in a cell in an organism genomics: The study of the organisation, structure and control of genes genotype: The entire genetic constitution of an individual gene therapy: Treating, diagnosing or preventing disease by introducing specific alterations in the genetic material of the human body germ cell: A sexual reproductive cell (sperm or egg). germ line: Cells from which the next generation of eggs or sperm will be derived

germplasm: The total genetic variability of an organism, represented by the total available pool of germ cells or seed

Intellectual Property: Can be defined as any product of the human intellect that is unique, novel and unobvious (has some value in the marketplace)

In vitro fertilisation (IVF): Technologies by which eggs and sperm are collected and united to achieve fertilisation outside the body. monoclonal antibody: A highly specific antibody that is derived from only one clone of cells. multipotent: Stem cells that are already partially differentiated but capable of producing cells of a certain type. Eg stem cells from bone marrow are capable of producing the different cell types present in blood. nanotechnology: Functioning devices with moving parts that are only molecules in size, such as a biosensor with a tiny molecular switch as its central component nuclear magnetic resonance (NMR): A technique that provides information on the structural behaviour of complex molecules in their environments nuclear replacement: see somatic cell nuclear transfer

nucleus: The cell structure that houses the genetic information (chromosomes).

nutraceutical: A plant or natural product that when consumed orally, confers a health benefit

oocyte: The female germ cell.

pluripotent: Cells with the capacity to develop into every cell type in the human body but not the placenta and umbilical cord. Pluripotent cells are not capable of developing into an entire organism.

primitive streak: A collection of cells which appears about 14 days after fertilisation from which the central nervous system eventually develops.

protein: A complex organic compound composed of numerous amino acids. Proteins occur in all living organisms and their production is coded for by genes

proteome: As the genome is the genetic complement of an organism, so the proteome is the complement of all proteins in an organism. Proteins may differ in the sequence of their amino acids or in chemical modifications which result in changed properties that can be identified proteomics: The high-throughput separation, identification and characterisation of proteins from a biological sample - a complementary technology to genomics, but starting with the protein rather than the gene

SCNT: Somatic cell nuclear transfer is a technique that involves transferring the nucleus of a somatic cell into an enucleated egg.

somatic cell: Any body cell apart from a sperm or egg.

somatic cell nuclear transfer (SCNT): A technique that involves transferring the nucleus of a somatic cell into an enucleated egg.

sperm: The mature male germ cell.

stem cell: A cell with the ability to divide indefinitely and to give rise to specialised cells as well as new stem cells with identical potential.

stem cell line: Stem cells that are cultured in the laboratory and divide to give rise to more stem cells.

Therapeutic Cloning: (same as SCNT)

totipotent: Cells that have the capacity to differentiate into the embryo and into extra embryonic membranes and tissues. Totipotent cells contribute to every cell type of the adult organism.

transgenic: The introduction of a modified gene from one organism into another.

vector: The agent used to carry new genes into cells.

xenotransplantation: The implantation of an organ or limb from one species to another organism in a different species. When performed in animals 'rejection' of the transplant by the recipient's immune system is a common response. zygote: The single cell formed when the male sperm fertilises the female egg. • TFCentral - A portal dedicated to transformation. Hosting, forums, image gallery, story archives, and chat. • Portal of Transformation - Site with sections on the folklore behind a number of different shapeshifters from around the world. • Stories set in fictional town "Barken. TX" • The Siren Song - Started back in 1997 - this site is home to original TG transformation/shapeshifting art, comics and animated shorts. • Shapeshifters in Love - A series of articles about shapeshifting characters in romance and speculative fiction

Decrease Paαe Size Novel Treatment for GVHD Cell therapy for complications arising from bone marrow transplant www.osiris.com Dictionary

Library > Words > Dictionary

graft-ver sus-host disease (graft1vύr'sas-hOst1, -saz-) n.

A pathological condition in which cells from the transplanted tissue of a donor initiate an immunologic attack on the cells and tissue of the recipient.

Medical IfOUGIlTON " * AIIFKlIX O COMPANY Library > Health > Medical Dictionary graft-versus-host disease n.

A type of incompatibility reaction of transplanted cells against host tissues that possess an antigen not possessed by the donor. Also called graft versus host reaction. O Library > Reference > Wikipedia graft-versus-host disease Graft-versus-host disease 1 Classification & external , resources ; ICD-IQ T86.0 ICD-9 996.85 DiseasesDB 5388

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Graft-versus-host disease (GVHD) is a common complication of allogeneic bone marrow transplantation in which functional immune cells in the transplanted marrow recognize the recipient as "foreign" and mount an immunologic attack. After bone marrow transplantation, T cells present in the graft, either as contaminants or intentionally introduced into the host, attack the tissues of the transplant recipient after perceiving host tissues as antigenically foreign. The T cells produce an excess of cytokines, and the fierce interplay of these cytokines, including TNF alpha and interleukin- 1 (IL-I), was first classified as a cytokine storm by Ferrara and co-workers in 1993. A wide range of host antigens can initiate graft-versus-host-disease, among them the HLAs. However, graft-versus-host disease can occur even when HLA-identical siblings are the donors. HLA-identical siblings or HLA-identical unrelated donors (called a minor mismatch as opposed to differences in the HLA antigens, which constitute a major mismatch) often still have genetically different proteins that can be presented on the major histocompatibility complex (MHC) .

While donor T-cells are undesirable as effector cells of graft-versus-host-disease, they are valuable for engraftment by preventing the recipient's residual immune system from rejecting the bone marrow graft (host-versus-graft). Additionally, as bone marrow transplantation is frequently used to cure cancer, mainly leukemias. donor T-cells have proven to have a valuable graft-versus-tumor effect. A great deal of current research on allogeneic bone marrow transplantation involves attempts to separate the undesirable graft-vs-host-disease aspects of T-cell physiology from the desirable graft-versus-tumor effect. Types

Clinically, graft-versus-host-disease is divided into acute and chronic forms. The acute or fulminant form of the disease is observed within the first 100 days post-transplant, and the chronic form of graft-versus-host-disease is defined as that which occurs after 100 days. This distinction is not arbitrary: acute and chronic graft-versus-host-disease appear to involve different immune cell subsets, different cytokine profiles, and different types of target organ damage. Classically, acute graft-versus-host-disease is characterized by selective damage to the liver, skin and mucosa, and the gastrointestinal tract. Newer research indicates that other graft-versus-host-disease target organs include the immune system (the hematopoietic system —e.g. the bone marrow and the thymus) itself, and the lungs in the form of idiopathic pneumonitis . Chronic graft-versus-host-disease damages the above organs, but also causes changes to the connective tissue (e.g. of the skin and exocrine glands).

GVHD of the GI tract can result in liters of watery diarrhea per day [[not all people will get diarrhea, I have never gotten diarrhea and I am a chronic GVHD patient]], abdominal pain, nausea, vomiting. This is diagnosed via intestinal biopsy. Liver GVHD is measured by the bilirubin level in acute patients. Skin GVHD results in a diffuse macularpapular rash sometimes in a lacy pattern.

Acute GVHD is staged like the following - overall grade (skin-liver-gut) with each organ staged individually from a low of 1 to a high of 4. Patients with grade 4 GVHD usually have a poor prognosis. If the GVHD is severe and requires intense immunosuppression involving steroids and additional agents to get under control, the patient may develop severe infections as a result of the immunosuppression and may die of infection. Transfusion-Associated GvHD

Main article: Transfusion-associatedsraft versus host disease

This type of GvHD is associated with transfusion of un-irradiated blood to immunocompromised recipients. It can also occur in situations, where the blood donor is homozygous and the recipient is heterozygous for an HLA haplotvpe. It is associated with higher mortality (80-90%) due to involvement of bone marrow lymphoid tissue, however the clinical manifestations are similar to GvHD resulting from bone marrow transplantation. Prevention

Graft-versus-host-disease can largely be avoided by performing a T-cell depleted bone marrow transplant. These types of transplants result in reduced target organ damage and generally less graft-versus-host-disease, but at a cost of diminished graft-versus-tumor effect, a greater risk of engraftment failure, and general immunodeficiency, resulting in a patient more susceptible to viral, bacterial, and fungal infection. Methotrexate and ciclosporin are common drugs used for GVHD prophylaxis. In a multi-center study (Lancet 2005 Aug 27-Sep 2;366(9487):733-41), disease-free survival at 3 years was not different between T cell depleted and T cell replete transplants. Media p Bibliography

• Graft-vs.-Host-Disease by Ferrara et al. (3rd ed.[2005]) published by Marcel Dekker is a nice bound volume that is full of useful information. • Examples of journals that publish current research on graft-versus-host-disease include The Biology of Blood and Marrow Transplantation, Journal of Clinical Investigation, Journal of Experimental Medicine, Blood, Journal of Immunology, Nature Immunology, Nature Medicine, Immunity, Bone Marrow Transplantation, and Transplantation. See also

• Transplantation o Transplant rejection, also known as "host-versus-graft disease" • Immunology o Immunosuppression • Cancer External links

• Graft versus Host Disease, from the National Marrow Donor Program

• DOR BioPharma. Inc.

Organ transplants

Types of Transplants: Allograft - Alloplant - Allotransplantation - Autotransplantation - Xenotransplantation

Tissue and Organs Transplanted: Organ transplant - Bone grafting - Bone marrow - Corneal - Face - Hand - Heart - Heart-Lung - Kidney - Liver - Lung - Pancreas - Penis - Skin grafting - Spleen

Related issues: Cellular memory - Biomedical tissue - Edmonton protocol - Eye bank - Graft- versus-host disease - Immunosuppressive drugs - Islet cell transplantation - Living donor liver transplantation - Lung allocation score - Machine perfusion - Medical grafting - Non-heart beating donation - Organ donation - Post-transplant lvmphoproliferative disorder - Total body irradiation - Transplant reiection

Organizations related to Transplants: Human Tissue Authority - National Marrow Donor Program - United Network for Organ Sharing

People related to transplants: Christiaan Barnard - Isabelle Dinoire - Jean-Michel Dubemard - Gregory Scott Johnson - List of notable organ transplant donors and recipients

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The activation of cellular genes in transformed cell*

Rv P. W.J. RiQHV, P. M . BiiiRKELL, D . S. LATCH MAN, D . M UR P HY, K.-H. WESTPH ΛL AND K. WII.MJOK 1 Cancer Rutarck Campaign, Eukaryotic MoUtular Gtnetks Rtstartk Group, Department of Biochemistry, Imperial CoIUp a/Stum* m i Ttd υtologg, LmdoΛ SWl IAZ, U.K. 1 Chain Beaity Laboratories, InuituU of Center Research, FuUUm Road, Union $W$ &/£, U.K.

We have used differential cDNA cloning techniques to isolate a number of genes that are activated at a result of transformation by the PNA tumour virus Simian virus 40. From the nucleotide sequences of ihe cDNA clones we have been able to identify some of these genes. One of them derives from the major histocompatibility complex and contains a repetitive clement that identifies a large number of RN Λs present In pluripoteniial embryonic cells.

I NTRODUCTI ON The translbrmaiion of established lines of cultured cells in vitro requires the action of only one gene product, be it of viral or cellular origin (Cooper 198a; Tooze 1981; Weiss el at. 198a}. However, the resultant transformed cell lines differ from their normal parents in a large number of biological and biochemical properties. It ii unlikely that these changes all result from dirτcl actions or the transforming protein and it therefore seems necessary that one of the functions of such proteins is to reprogrammc the cell's pattern of gene expression. We have sought to identify cellular genes that are activated a* a result of transformation by the DNA tumour virus Simian virtu 40 (SV40). There were a number of reason! for choosing SV40 as the model system for this kind of experiment. Transformation by SV40 depends on the gene encoding large T-aniigen (Martin 1981) which is a nuclear DKA-binding protein known to be able tn regulate transcription (Rigby & Lane 1983). Large T-antigcn represses the transcription of its own mRNA (Hansen et at. 1981; Rio & Tjian 1983) and activates transcription of the late region of the viral genome (Keller & Alwine 1984). Furthermore, functional large T-antigcn is required for the induction of cellular enzyme synthesis which is the first detectable response to SV40 infection (Postel & Levine 1976) and an activity of large T-anrigcn is involved in the transcriptional reactivation of lhe silent rDN Λ complement in human-mouse somatic cell hybrids (Soprano We/. 1979). Moreover, Williams ti at. (1977) used the technique of cDNA-mRNA hybridization in solution to show that there are differences between the cytoplasmic mRN Λ populations of an SV40-transformed human cell line and its normal parent. It therefore seemed sensible to u*e SV40-transformed crib to search for cellular genes thai are activated by the viral transforming protein. We have developed a differential cDNA cJotiing protocol which has allowed us to isolate a number of genes activated as a mult of transformation by SV40 (Scott el at. 19830, b). We shall here consider the identity ofseveral of these genes and discuss possible roles for a repetitive element contained within one of them.

( Hepatocyte Lineage Cells Derived from Pluripotent Stem Cells Patent No: 92561 Date Issued: 03/31/05 Country: Singapore Selective Antibody Targeting of Undifferentiated Stem Cells Patent No: 6,921,665 Date Issued: 07/26/05 Country: US Differentiated Stem Cells Suitable for Human Therapy Patent No: 2386120 Date Issued: 03/9/05 Country: United Kingdom Tolerizing Allografts of Pluripotent Stem Cells Patent No.: 2386125 Date Issued: 2/23/05 Country: United Kingdom Neural Progenitor Cell Populations Patent No: 92904 Date Issued: 12/30/04 Country: Singapore Neural Progenitor Cell Populations Patent No: 2379447 Date Issued: 12/29/04 Country: United Kingdom Hepatocyte Lineage Cells Derived from Pluripotent Stem Cells Patent No: 2380490 Date Issued: 12/29/04 Country: United Kingdom Making Neural Cells for Human Therapy or Drug Screening from Human Embryonic Stem Cells, Neural Progenitor Cell Populations Patent No: 6,833,269 Date Issued: 12/21/04 Country: US Method of Making Embryoid Bodies From Primate Stem Cells Feeder-Free Culture Patent No: 90904 Date Issued: 11/30/04 Country: Singapore Methods and Materials for the Growth of Primate-Derived Primordial Stem Cells in Feeder-Free Culture Patent No: 6,800,480 Date Issued: 10/05/04 Country: US Method of Making Embryoid Bodies From Primate Embryonic Stem Cells Patent No: 520700 Date Issued: 08/12/04 Country: New Zealand Country: New Zealand Selective Antibody Targeting of Undifferentiated Stem Cells Patent No: GB 2374076 Date Issued: 02/25/04 Patent Origin: United Kingdom cDNA Libraries Reflecting Gene Expression During Growth and Differentiation of Human Pluripotent Stem Cells Patent No: 6,667,176 Date Issued: 12/23/03 Patent Origin: US Conditioned Media for Propagating Human Pluripotent Stem Cells Patent No: 6,642,048 Date Issued: 11/4/03 Patent Origin: US Hematopoietic Differentiation of Human Embryonic Stem Cells Patent No: 6,613,568 Date Issued: 09/02/03 Patent Origin: US Methods of Making Embryoid Bodies From Primate Embryonic Stem Cells Patent No: 6,602,71 1 Date Issued: 08/05/03 Patent Origin: US Differentiated Cells Suitable for Human Therapy Patent No: 6,576,464 Date Issued: 06/10/03 Patent Origin: US Differentiation of Human Embryonic Germ Cells Patent No: 6,562,619 Date Issued: 05/13/03 Patent Origin: US Hepatocyte Lineage Cells Derived From Pluripotent Stem Cells Patent No: 6,506,574 Date Issued: 01/14/03 Patent Origin: US Techniques for Growth and Differentiation of Human Pluripotent Stem Cells Patent No: 751 321 Date Issued: 12/05/02 Patent Origin: Australia Hepatocyte Lineage Cells Derived From Pluripotent Stem Cells Patent No: 6,458,589 Date Issued: 10/01/02 Patent Origin: US Human Embryonic Germ Cell Line and Methods of Use Patent No: 745976 Date Issued: 06/25/02 Patent Origin: Australia Hematopoietic Differentiation of Pluripotent Human Embryonic Stem Cells g Human Embryonic Germ Cell Line and Methods of Use Patent No: 6,245,566 Date Issued: 06/12/01 Patent Origin: US Methods and Materials for the Growth of Primate-Derived Primordial Stem Cells in Feeder-Free Culture Patent No: 729377 Date Issued: 05/17/01 Patent Origin: Australia Primate Embryonic Stem Cells Patent No: 6,200,806 Date Issued: 03/13/01 Patent Origin: US Human Embryonic Pluripotent Germ Cells Patent No: 6,090,622 Date Issued: 07/18/00 Patent Origin: US Primate Embryonic Stem Cells Patent No: 5,843,780 Date Issued: 12/01/98 Patent Origin: US Human Embryonic Germ Cell and Methods of Use Patent No: 6,33 1,406 Date Issued: 12/18/01 Patent Origin: US Human Embryonic Germ Cell Line and Methods of Use Patent No: 68251 Date Issued: 10/29/01 Patent Origin: Singapore

John Gearhart , "Derivation of pluripotent stem cells from cultured human primordial germ cells" Derived hPG / hEG cells (human primordial germ cells / embryonic germ cells) from human fetal tissue (5-9 weeks

gestation) PNAS: 1998 95 (23) • 13726-13731

Proc Natl Acad Sci U S A. 1998 November 10; 95(23): 13726-13731. Copyright © 1998, The National Academy of Sciences Developmental Biology Derivation of pluripotent stem cells from cultured human primordial germ cells

Michael J. Shamblott, * Joyce Axelman, * Shunping Wang, * Elizabeth M. Bugg, * John W. Littlefield, Peter J. Donovan, Paul D. Blumenthal, § George R. Huggins, § and John D. Gearhart • Young HE • Steele TA. • Bray RA. • Hudson J. • Flovd JA . • Hawkins K. • Thomas Kf • Austin T. • Edwards Cf • Cuzyourt J, • Duenzl M. • Lucas PA. • Black AC Jr.

Division of Basic Medicai Science, Mercer University School of Medicine, Macon, Georgia 31207, USA. young_he~~mercer.edu~~

This study details the profile of 13 cell surface cluster differentiation markers on human reserve stem cells derived from connective tissues. Stem cells were isolated from the connective tissues of dermis and skeletal muscle derived from fetal, mature, and geriatric humans. An insulin/dexamethasone phenotypic bioassay was used to determine the identity of the stem cells from each population. All populations contained lineage-committed myogenic, adlpogenic, chondrogenic, and osteogenic progenitor stem cells as well as lineage- uncommitted plurlpotent stem cells capable of forming muscle, adipocytes, cartilage, bone, fibroblasts, and endothelial cells. Flow cytometric analysis of adult stem cell populations revealed positive staining for CD34 and CD90 and negative staining for CD3, CD4, CD8, CDlIc, CD33, CD36, CD38, CD45, CD117, Glycophorin-A, and HLA DR-II. Copyright 2001 Wlley-Liss, Inc.

~~PMID: 11505371 [PubMed - Indexed for MEDLINE] Patents~~

Methods I'nieni for CuUuring Hrimate Also disclosed by the new ρ:κc i is u incihod of Embryonic Stem CcIh Issues to Ctnin using the cells, wlielher ixi π reil. j^eiiciically π iπiiip- ulatcd. υ r rciiwrally jlTecietl hy a genetic disease, to — ,SV. ix ihe a.«άgnee. The patent discloses the culture of celts from prl- α Λ β ι male niDTula* <ιr hlustiKy- φ i in Midi U way Ihπ i ai I r CtoM Kcccivnt ntiangiogcrwid l** ent least half of lbcm will remain totipotent * capable of —One ΛtiiitMkfy Ua.* Ik Readyfor Cllnkut Trinh differential ing inli) :aιy lypc of cell ifcxircd, TIM; ceJts may cumuin one or more genetic alterations. NfiW YOKK. NY I irMVM— fmCl αntf Systems, The Siintmary uf the Invention includes tin: jititc- lac. hns received a U.S. patcM covering lhc Hicra- Λ iiKiii that " I ϊ |*i ab Vt embodiment*, ilie primordial µcuik UM of vttseular diul αiheliul y ι>w ιh Aickir K - slcin veils iirc derived From human embryonic cells." ceptoc (VECFR) und ami- VECFR antibody in com- ι Claim y ) and i .% depcndeiM claims 4(1 through 44 bitiiMHHi with r.xllminn pr vhuinirtlic π ipy ιt > iretil specify that ιhι- ccllx arc hurnun. (Gcr αn ;ιlso has a cancer. pending paieni pppiic αiinii specillcuilly directed to TIw cwπ ijtiiiy's nmi-epidcrnul g rϋ wih-r^cuir re¬ ihe isυluikm and cuhiirc υ f Inintuii pπ ni υrd'ml Mem ceptor untibody cetuxiniab (£rbi ιu.\) wait appro\*ed cclLi.) i n February 20O4 fur ilic ireatmenl o f advsiiK α l eul- Much of the paicni describes culture nπ din with orcctaJ eunccr but probably I* best known for ihe υ υ υ l w ttMMOiic |ircs$ur and \nw ϋiidiiioxin c ntcnlr.i- role i l played in :ι fedcnil caw ihai >c ιιi ]mClonc'$ iioii to whii'h noo-cssejiiul amino ucids. an anliox- former head. Samuel Widual, to fithou (or insider π π KUIIII. se im. :MMJ CIIH: iw iinjn: jj .mih faciixs sw.li lRuling and Waksal'y friend, gracipu» living expert as nucleosides. inin.slOniilng. j{ruwih facior^beiu. in< Manh ύ Sicwart. to pri ϋoii for lyioy abotii tar trad¬ π υ ς α icrlcuki .N r iiitcri ukin rcccplixs. and v scnlnr en¬ ing i n Ijndonv Mock (sec 22 BLR 139 2003). dothelial, epidermal, or plaielctntcrived growth fac» The βew piMeot it No. 6.81 1.77V. lor havu been added. A nteiliuil IV>r idenlifying The company has developed antibodies ihni bind suiiabte ^rowih factors is disclosed. TIM - substrate two ιy \κ x of VECFR and is testing ihtnt for iheir iπ υy eoπ »iyt uf feeder cells sueh as πi υαsc embry¬ ability to inhibit the formnioD o f new blood ves¬ onic fibroblasts o r extracellular matrix laid down hy sel*. TuniuTv nxjuirc new vt» κclx Io µrovkl α the fibrobla-su that are then killed. Notably, it mny also large amou ni5 o f nut ricnta lhey need t o su ppon iheir

l>e created frcuM known exu^ce ϋ ubr-ncnrix compo¬ rapid growth, :ιικ l uHgkij^ncyiv has beiai .1 t.-irgci nents .such as collagen, fibroncctin. Laminin. and forc αitcer researchers since Judith Folkniun and hi.< clKiiid π xlin suH'aie. i l)eruby avoiding ihe us: u f bbnratury ide π iificd ihe process and iis mecha¬ feeder cells. nism*. A n ol)jcciira ι raised to lite use ι/ ilw lnuam em¬ linCkme row filed ;ιπ Ins-csiiginiumil Ne«v Dnig bryonic stein cell lilies whose w e Ls perniiiied in applic αiion for one of the antibodicui and bα pei io π α fcdO illv fiiinled -suircli is ihat nil of iliein \vcrc begin cliiiicnl trύ ils t:ιiu in 2OfM \>r enrly in 20O5. established o n mouse feeder cvlli and ihutc nuy bύ- cuιM:ιιιιiιuιlc

~~Characteristics of Human Embryonic Stem Cells: (scroll down) roduced~~

~~2. Self-renewing. Using in vitro cell culture, hES cells can propagate indefinitely in the undifferentiated state without losing pluripotency.~~

3. Express Telomerase and Oct4. Telomerase is an RNA-dependent DNA polymerase whose function diminishes in aging cells and whose expression, when re-activated in normal cells, allows their continual proliferation. hES cells express telomerase. The continual telomerase expression conveys replicative immortality. Other stem cells express telomerase at low levels and thus stop dividing, age, and die with time. Thompson fig: Oct-4 is a "master regulator" of ES cell pluripotency that activates or inhibits a host of target genes and maintains ES cells in a proliferative, non-differentiating state.

4. Normal chromosome structure (karyotype). hES cells maintain a normal set of chromosomes (46, XX or XY) even after prolonged tissue culture. They do not have Injection o f pluripotent SC into recipient is conducted on proviso recipient is cancer free, as pluripotent SC may accelerate cancer or tumor growth.

~~Collection and use of of proliferated pluripotent SCs is an express embodiment o f this invention.~~

~~Q&A on the Institute for Cancer/Stem Cell Biology and Medicine~~

~~What is the goal of the Institute for Cancer/Stem Cell Biology and Medicine?~~

The institute will study the basic biology of stem cells and translate discoveries from this research into treatments for diseases such as cancer, Parkinson's disease, Lou Gehrig's disease, diabetes, cardiovascular disease, autoimmune diseases, allergies and neurodegenerative disorders. Work at the institute could help the scientific community understand fundamental processes in biology such as how adult stem cells self-renew and differentiate to regenerate tissues and organs, what genetic processes are disrupted in cancer cells, and how cells with disease-causing genetic mutations develop into those diseases.

Stem cells can also be coaxed to form many different cell types of the body, providing researchers with a way to better understand the function of these cells when healthy and diseased. A primary goal of the institute is to use this knowledge to help treat cancer and to understand the processes involved in cancer development. Stem cells might also be able to replenish healthy cells that are lost because of aggressive cancer treatment. Who is the director of the institute?

Irving Weissman, MD, the Karel and Avice Beekhuis professor of cancer biology, will direct the institute. In 1988, Weissman became the first to isolate an adult stem cell by developing a method to identify adult blood-forming stem cells found in the bone marrow of mice. He later isolated blood-forming stem cells from human bone marrow and helped identify adult brain-forming stem cells. Weissman's research has focused on using purified blood-forming stem cells to treat cancers. Adult stem cells are cells capable of dividing and replacing damaged tissue. They exist in tissues such as the bone marrow, brain, muscle and liver. Unlike their neighbors, which are already differentiated into specialized cell types, adult stem cells remain immature. When tissue becomes damaged, the adult stem cells divide (a process called self-renewal) to produce new cells. Some of the resulting cells divide, but unlike stem cells they mature to take over for the damaged cells. Within any organ or tissue, generally only the stem cells have the ability to self-renew and appear to be the only cells that regenerate tissues when they become damaged. In the bone marrow, blood-forming stem cells make up only about 1 in 20,000 cells. What can be learned by studying adult stem cells?

Studying adult stem cells can teach researchers about processes that lead to cancer and other diseases. Most mature cells of the body no longer divide. In many cancerous tissues, genes that block the cell from dividing and direct cells to mature are turned down or off, and genes that stimulate division are turned on. In many cancers studied thus far, a small population of cells, called cancer stem cells, self-renew to replenish the growing cancer. These cancer stem cells can use the same genes that adult stem cells use in the process of self-renewal. By studying adult stem cells to learn more about the genes involved in self-renewal, it might be possible to identify new molecular targets for drug and immune therapies that destroy the self-renewing cancer stem cells.

One important area of research involves learning whether all cancers have cancer stem cells. For each type of cancer, it is also important to learn which genes are used in self- renewing adult stem cells compared with cancer stem cells in that same tissue. Both of these cells are capable of self-renewing, but only the cancer cells go on to grow indefinitely and spread to other organs through the blood stream. If researchers can learn which genes are mutated or used differently in the cancer cells they can develop drugs to block that behavior.

In addition to studying cancer biology, adult stem cells can be used to learn more about the adult tissues from which those stem cells are derived. Researchers have already identified adult stem cells in the brains of mice and humans, and can now use those stem cells to understand how cells of the developing brain differentiate into the many different cell types found in the adult brain. By studying the cells in the lab, researchers may be able to understand what processes go awry in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Researchers at the institute hope to identify adult stem cells from other tissues such as lungs or liver to understand how those tissues develop and what goes wrong when those tissues become diseased. How can adult stem cells be used to treat disease?

Adult stem cells can be used to replenish damaged tissue. One example of this is in bone marrow transplants, where blood-forming stem cells regenerate the blood of transplant recipients who receive otherwise lethal doses of chemotherapy to destroy all the cancer cells in the body. Stanford was the first institution in the United States to use purified patients with their own cancer cells.

Researchers at Stanford are also pioneering the use of blood-forming stem cells to treat type 1 (juvenile) diabetes. In this form of diabetes, cells of the immune system attack pancreas cells that produce insulin. Working in mice, the researchers replaced the cells of the immune system that are prone to autoimmune reactions with healthy adult blood- forming stem cells from a related donor. These cells matured in the bone marrow into immune cells, but lacked the autoimmune reaction. This type of research in laboratory animals could eventually lead to new diabetes treatments in humans and might be extended to other autoimmune diseases such as multiple sclerosis, lupus, and rheumatoid arthritis.

Researchers at the institute hope to isolate adult stem cells from a variety of tissues in addition to the blood and brain stem cells identified so far. Doctors could then give high doses of radiation to destroy tumors in tissues such as brain, lungs or liver, and inject tissue-specific stem cells to replace radiation-damaged cells. Similarly, tissue-specific stem cells could replenish cells damaged by Parkinson's disease, Alzheimer's disease, multiple sclerosis or diabetes. What are pluripotent (embryonic) stem cells? How are they different from adult stem cells?

Pluripotent stem cells have the ability to become any type of cell in the body; "pluri" means many, and "potent" means potential. These cells have the potential to become many different kinds of cells in the body.

Like adult stem cells, pluripotent stem cells are capable of self-renewal, but are unique because they can form specialized cells in any tissue type, whereas adult stem cells from a given tissue appear to only form cells found in that tissue.

Pluripotent stem cells can come from a very early stage of an animal's development called the blastocyst stage. A blastocyst is a ball of cells that forms after the fertilized egg undergoes seven to nine divisions. It cannot give rise to a developing embryo or fetus unless it is implanted in the uterus. About 17 years ago, scientists learned how to take pluripotent stem cells from a mouse blastocyst and grow them in a lab. These cells could divide continuously in a test tube and go on to form cells from all tissue types. The cell lines that have been created by using this method in mice are also called embryonic stem cell lines. What can be learned by studying pluripotent stem cells?

Studying mouse pluripotent stem cells carrying disease-causing mutations has already greatly enhanced scientific and medical knowledge of how genetic diseases develop. The hope is that a similar knowledge explosion will take place by studying human pluripotent , g , y

By studying stem cells that carry DNA with disease-causing mutations, researchers might leam more about how these mutations cause the cell to become diseased. They may also leam how the proteins made by the mutated genes fail to function properly, leading to an understanding of the molecular basis of the disease. This may enable researchers to generate drugs or therapies that make up for the genetic defect and treat the disease. Although this work has great promise, at this time only mouse pluripotent stem cell lines exist that carry disease-causing mutations.

~~How can pluripotent stem cells be used to treat disease?~~

At Stanford, pluripotent stem cells have already been used experimentally to treat mice with diabetes. Researchers found a set of growth factors that induced pluripotent stem cells to develop into insulin-producing cells normally found in the pancreas. When they implanted these cells into diabetic mice that have lost the ability to produce insulin, the implanted cells produced insulin in a biologically normal way and treated the diabetes. This work is still in the early stages of being tested in animals, but could one day lead to new ways of treating diabetes in people.

Pluripotent stem cells, like adult brain stem cells, might also replace nerves damaged in spinal cord injuries or cells lost in neurodegenerative diseases. For any of these treatments to work, researchers have to first learn how to grow the stem cells in a lab so they take on the characteristics of the cells they are meant to replace. At this time it isn't clear whether pluripotent or adult stem cells will be best in this type of therapy.

Inducing stem cells to develop into mature cells in a lab dish might also reduce some of the side effects of using stem cells to treat disease. In early animal experiments using immature pluripotent stem cells to treat disease in animals, the cells often formed tumors called teratocarcinomas. This happened because the cells still had the ability to self- renew and did not all mature into non-dividing cells. In current laboratory experiments, researchers induce the cells to mature into the appropriate cell type before injecting these cells into animals. In the diabetes experiments, the mature, insulin-producing cells produced no tumors in the mice, whereas early experiments with less mature cells produced deadly tumors within a matter of weeks. What are pluripotent cell lines and how are they created?

A pluripotent stem cell line comes from the pluripotent cells isolated from one blastocyst. These cells can divide in a lab dish and produce new cells - each of which is an exact replica of the original isolated cells- that the researchers can divide into multiple test tubes. A stable, dividing pool of identical cells is called a cell line. Researchers can share these cell lines so that labs around the world can conduct experiments on identical cells. If the cell line comes from a blastocyst with a genetic defect, then many researchers can Researchers at the institute hope to learn the best way to create new pluripotent stem cell lines. They will first test several different methods in mice, then apply the most successful technique to human cells. At this time there are two likely approaches to generating the new cell lines:

1. Transplant an adult nucleus into an egg that has had its nucleus removed, stimulate the cell to divide as if the egg had been fertilized, and culture the blastocyst-stage pluripotent cells. This process, called nuclear transplantation, has been successful in mice but has not yet been shown to be successful using human cells.

2. Remove the nucleus from an existing but highly modified pluripotent stem cell line and replace it with a nucleus from an adult cell that carries genetic mutations implicated in human disease. This process has not yet been successful in any organism, but does show promise in mice.

In both approaches, the goal is to take a nucleus from an adult cell and reprogram it to behave like the nucleus of a pluripotent stem cell. The adult nucleus from a skin cell, for example, will use the genes that are needed by a normal skin cell. To become a nucleus in a pluripotent stem cell, that nucleus will need to stop using skin-specific genes and begin using those genes found in pluripotent cells. The first goal of the institute in this research area is to study and accomplish such nucleus reprogramming first in mice, then using human cells. Why are new pluripotent cell lines needed?

About 70 existing human embryonic stem cell lines are said to exist worldwide. These cell lines came from blastocysts at fertility clinics and not from blastocysts created through nuclear transplantation. While these lines may be useful for some research, they do not carry disease-specific mutations that are of interest to many researchers. By creating human pluripotent stem cell lines that contain the genetic information that predisposes an individual to develop a specific disease, scientists can study the multi-step progression of that disease in many different tissue types. Scientists from around the world, as well as at Stanford, have identified this line of research as a critical part of developing therapies for diseases including cancer, neurodegenerative disorders, diabetes and others.

~~If human eggs are used to create new pluripotent stem cell lines, where will the eggs come from?~~

It is too early to know whether Stanford researchers will use human eggs to create new pluripotent stem cell lines. If researchers do choose this method, that work would be subject to review by an internal review board made up of doctors and bioethicists who would analyze the risks and benefits before deciding whether the researchers could move forward and where researchers could get human eggs. If the researchers do move forward with nuclear transplantation into human eggs, one possible source would be immature The institute will not use any human eggs, embryos or subjects without a thorough review process and without the donor's fully informed consent. No biological materials - eggs or embryos - that Stanford currently has in its possession will be used in this research.

~~Why is creating pluripotent stem cell lines sometimes called therapeutic cloning?~~

Researchers at the institute refer to the creation of pluripotent stem cell lines as "nuclear transplantation to produce human pluripotent stem cell lines," while others refer to the procedure as "therapeutic cloning," "research cloning" or "cloning for biomedical research."

The wording used by the institute's researchers has been endorsed by two panels convened by the National Academies to debate cloning and stem cell research, one of which was chaired by Irving Weissman, MD, the director of the institute. The National Academies is composed of the National Academy of Sciences, the National Academy of Engineering, the Institute of Medicine of the National Academies and the National Research Council.

Both panels chose not to use the words "embryo" and "cloning" because of confusion in the public over their meaning. In scientific terms, all stages of development from fertilization up to organ development constitute the embryonic period. However, most people asked to draw an embryo instead draw a fetus with head, limbs, eyes and other identifiably human traits. Likewise, scientists use the word "cloning" every day to describe how they isolate genes; how cancer cells develop from a single cancer stem cell; or to characterize the progeny of a single blood-forming stem cell, or bacterium or virus. But to most people the word "cloning" conjures up images of mad scientists producing fully grown human clones.

For this reason, both National Academies panels chose to use language that accurately and dispassionately describes the nuclear transplantation technique. This language was also supported in a Science article written by the presidents of the National Academies of Science and of the Institute of Medicine.

1. National Research Council, Stem Cells and the Future of Regenerative Medicine (National Academy Press, Washington, DC, 2001). Bert Vogelstein, chair. http://books.nap.edU/books/0309076307/html/l.html

2. National Research Council, Scientific and Medical Aspects of Human Reproductive Cloning (National Academy Press, Washington, DC, 2002). Irving Weissman, chair. httD://books.nap.edu/books/0309076374/html/l.html

3. Support for dispassionate language to describe nuclear transfer by the presidents of the National Academies of Science and of the Institute of Medicine along with Dr Bert o y. ι http://www.sciencemag.org/cgi/content/full/295/5S58/1237

~~How is creating pluripotent stem cell lines different from reproductive cloning?~~

Human reproductive cloning is an effort to create new humans with genetic material identical to that of the donor. The National Academies panel convened to study the issue of cloning and a special State of California panel both voted unanimously for a legally enforceable ban on implanting blastocysts created by nuclear transfer into the uterus of a woman, and therefore against allowing all reproductive cloning. In addition, all researchers involved in Stanford's institute are vehemently opposed to human reproductive cloning.

In both reproductive cloning and in one method of creating new stem cell lines, the first step involves replacing the nucleus of an egg with a nucleus from an adult cell and stimulating the egg to divide seven to nine times to form a blastocyst. In generating a pluripotent stem cell line, the researchers remove pluripotent cells from the blastocyst. After this procedure neither the blastocyst nor the stem cell line can go on to become an adult animal. In reproductive cloning, such as that used to create Dolly the sheep and other cloned animals, the blastocyst is implanted into the uterus of an adult animal where it can develop.

Reproductive cloning has been used in several different animal species, but in all cases the technique has not been very efficient. In fact, on average more than 99 percent of the implanted blastocysts result in embryonic or fetal death. Offspring born from reproductive cloning also tend to have deformities and, according to some evidence, may age faster than other animals. What is the controversy over stem cell research?

Research with pluripotent stem cell lines is controversial because, for religious and ethical reasons, some people feel that the blastocysts used to generate the cell lines are fully human and should not be the source of pluripotent cells. They hold this view whether those blastocysts come from fertility clinics or are created through nuclear transfer. If researchers at the institute are able to create new pluripotent stem cell lines by transferring a nucleus into an existing stem cell line, then they will not be destroying a blastocyst. These cell lines would not carry the same controversy as those created through nuclear transfer.

The issue about when personhood develops in an individual cannot at this time be settled scientifically, and so it will remain the subject of controversy and debate. What are the federal and state laws regarding stem cell research? cell lines. However, there are no laws in place that relate to the use of private funding to create new stem cell lines. Both National Academies panels voted that nuclear transplantation to produce human pluripotent stem cell lines was sufficiently important that it should not be banned and should be the subject of a broad debate.

In the fall of 2002, California passed and Gov. Davis signed State Senate Bill 253, calling for research involving the derivation and use of human embryonic (pluripotent) stem cells, human embryonic germ cells and human adult stem cells from any source, including nuclear transplantation, with full consideration of the ethical and medical implication of this research, including a requirement for overview by an approved institutional review board. The legislation promotes the kind of research being conducted at the institute and has created a favorable climate for this type of work in California.

~~Other states have formed or are in the process of forming their own laws regarding nuclear transplantation to produce new stem cell lines.~~

~~1. Report of the California Advisory Committee on Human Cloning http://www.scu.edu/ethics/pubiications/adbdreport.html~~

~~2. California Senate Bill 253 http://www.aab.org/california%20sb%20253%20stem%20cells%209%2022%2002.pdf What are the federal and state laws regarding reproductive cloning?~~

There are no federal or California laws relating to reproductive cloning. Both National Academies panels and the California panel voted unanimously that human reproductive cloning should be banned. How will work at the institute be funded?

AUwork relating to the creation of new embryonic stem cell lines will be privately funded or funded by the state of California. The institute began with $12 million in seed funding and hopes to continue recruiting new sources of private funding. With so much controversy in this field, why is Stanford increasing its efforts in stem cell research?

The formation of the new institute is consistent with Stanford's history of embracing new technologies to aid in the discovery of treatments for catastrophic diseases. Researchers at the institute will be guided by a team of scientists working with legal experts and ethicists at the Center for Biomedical Ethics to continue putting respect for patients first and foremost in the mission of advancing knowledge and improving the lives and health of our world community. y g g Institute for Cancer/Stem Cell Biology and Medicine at Stanford.

Incorporate by reference in its entirety "Z701408 Human Embryonic Stem Cells," General description A discussion of all the key issues in the use of human pluripotent stem cells for treating degenerative diseases or for replacing tissues lost from trauma. On the practical side, the topics range from the problems of deriving human embryonic stem cells and driving their differentiation along specific lineages, regulating their development into mature cells, and bringing stem cell therapy to clinical trials. Regulatory issues are addressed in discussions of the ethical debate surrounding the derivation of human embryonic stem cells and the current policies governing their use in the United States and abroad, including the rules and conditions regulating federal funding and questions of intellectual property.

~~Identifiers ISBN-IO 1-58829-31 1-4~~

~~ISBN- 13 978-1-58829-31 1-4~~

~~Properties publication info A. Chiu and M. Rao, ed., Humana Press, 2003, 464 pp., hard cover~~

Table of Contents Part I: POLICY 1. Ethical Issues Associated with Pluripotent Stem Cells 2. A Researcher's Guide to Federally Funded Human Embryonic Stem Cell Research in the United States 3. Intellectual Property of Human Pluripotent Stem Cells Part II: TYPES OF PLURIPOTENT CELLS 4. Embryonal Carcinoma Cells 5. Human Pluripotent Stem Cells from Bone Marrow 6. Protocols for the Isolation and Maintenance of Human Embryonic Stem Cells 7. Subcloning and Alternative Methods for the Derivation and Culture of Human Embryonic Stem Cells Part III: DIFFERENTIATION 8. Differentiation of Neuroepithelia from Human Embryonic Stem Cells 9. Pancreatic Differentiation of Pluripotent Stem Cells 10. Human Embryonic Stem Cell-Derived Cardiomyocytes: Derivation and Characterization 13. Human Embryonic vs Adult Stem Cells 14. Genetic Manipulation of Human Embryonic Stem Cells 15. Human Therapeutic Cloning 16. Therapeutic Uses of Embryonic Stem Cells 17. Human Embryonic Stem Cells and the Food and Drug Administration: Assuring the Safety of Novel Cellular Therapies 18. Studies of a Human Neuron-Like Cell Line in Stroke and Spinal Cord Injury: Preclinical and Clinical Perspectives Appendix I: Cell Lines and Companies Involved with Human Embryonic Stem Cell Research Appendix II: Useful Websites Appendix III: Research Agreements and Material Transfer Agreements Between Investigator and Stem Cell Provider Appendix IV: Stem Cell Patents

~~Isolating Pluripotent SCs from bone marrow is known. See for example~~

~~http ://www. ortecinternational .com/fibrin microbeads .html~~

Fibrin Microbβads, or Fibrin MB, (typical diameter: 50-250 micrometers) are fabricated from clinical grade human fibrinogen and thrombin. Fibrin MB have numerous qualities, some of which are noted below: • Fibrin MB can bind, select and grow responsive cells such as stem cells from mixed cell types, such as found in bone marrow or peripheral blood. • Cells on Fibrin MB can be transferred without trypsinization. • Fibrin MB can be used to isolate pluripotent stem cells from bone marrow and other tissue sources. • Fibrin MB can be sterilized by gamma-irradiation. • Fibrin MB are biodegradable and non-immunogenic.

Click on the links below to view the United States Patent: Patent Number: 6.150.505 Patent Number: 6.552.172 B2 Patent Number. 6.737.074 B2 Patent Number: 6.503.731 B2 http://en.wikipedia.org/wiki/Bone_marrow_transplant

Bone marrow transplantation is a medical procedure in the field of hematology and oncology that involves transplantation of hematopoietic stem cells (HSC). It is most often performed for people with diseases of the blood or bone marrow, or certain types of cancer.

Bone marrow transplantation was pioneered at the Fred Hutchinson Cancer Research Center from the 1950s through the 1970s by E. Donnall Thomas, whose work was later recognized with a Nobel Prize in Physiology and Medicine. Dr. Thomas 1work showed that bone marrow cells infused intravenously could repopulate the bone marrow and produce new blood cells. His work also reduced the likelihood of developing a life- threatening complication called Graft-versus-host disease. p p g p gg graft, and does not require that the donor be subjected to general anesthesia to collect the graft.

~~Hematopoeitic stem cell transplantation remains a risky procedure and has always been reserved for patients with life threatening diseases. Principles~~

Most recipients of HSCTs are leukemia patients or others who would benefit from treatment with high doses of chemotherapy or total body irradiation . Other patients who receive bone marrow transplants include pediatric cases where the patient has an inborn defect such as severe combined immunodeficiency or congenital neutropenia and was bom with defective stem cells. Children or adults with aplastic anemia have lost their stem cells after birth and may not require such high doses of chemotherapy and irradiation prior to a transplant. In this case there is a greater need for immunosuppressive agents. Other conditions that bone marrow transplants are considered for include thalassemia major, sickle-cell disease, mvelodvsplastic syndrome, neuroblastoma, lymphoma. Hodgkin's disease, and multiple myeloma. More recently non-myeloablative, or so-called "mini transplant," procedures have been developed which do not require such large doses of chemotherapy and radiation. This has allowed HSCT to be conducted in older patients and without the need for hospitalization. [edit] Stem cell collection

~~feditI Types of donors~~

There are two major types of stem cell transplantation maneuvers. Autologous HSCT involves isolation of HSC from a patient, storage of the stem cells in a freezer, high-dose chemotherapy to eradicate the malignant cell population at the cost of also eliminating the patient's bone marrow stem cells, then return of the patient's own stored stem cells to their body. Autologous transplants have the advantage of a lower risk of graft rejection, infection and graft-versus-host disease.

Allogeneic HSCT involves two people, one is the (normal) donor and one is the (patient) recipient. Allogeneic HSC donors must have a tissue (HLA) type that matches the recipient and, in addition, the recipient requires immunosuppressive medications. Allogeneic transplant donors may be related (usually a sibling) or unrelated volunteers. Allogeneic transplants are also performed using umbilical cord blood as the source of stem cells.

~~fedit] Donor selection~~

A major limitation of allogeneic bone marrow transplantation is a shortage of donors. To avoid rejection of the transplanted stem cells or severe graft-versus-host disease, the donor should have the same human leukocyte antigens (HLA) as the recipient. About 25 can be used. However, the use of mismatched donors may increase the risk of graft rejection or severe graft-versus-host disease .

A compatible donor is found by doing additional HLA-testing from the blood of potential donors. The HLA genes fall in two categories (Type I and Type II). In general, mismatches of the Type-I genes (i.e. HLA-A, HLA-B, or HLA-C) increase the risk of graft rejection. A mismatch of an HLA Type II gene (i.e. HLA-DR, or HLA-DQBl) increases the risk of graft-versus-host disease. In addition a genetic mismatch as small as a single DNA base pair is significant so perfect matches require knowledge of the exact DNA sequence of these genes for both donor and recipient. Leading transplant centers currently perform testing for all five of these HLA genes before declaring that a donor and recipient are HLA-identical.

~~"Race " and ethnicity are known to play a major role in donor recruitment drives, as members of the same ethnic group are more likely to have matching genes, including the genes for HLA. [1]~~

~~Sources of HSC~~

In the case of a bone marrow transplant, the HSC are removed from a large bone of the donor, typically the pelvis , through a large needle that reaches the center of the bone. The technique is referred to as a bone marrow harvest and is performed under general anesthesia because hundreds of insertions of the needle are required to obtain sufficient material.

Peripheral blood stem cells are now the most common source of stem cells for HSCT. They are collected from the blood through a process known as apheresis . The donor's blood is withdrawn through a sterile needle in one arm and passed through a machine that removes white blood cells . The red blood cells are returned to the donor. The peripheral stem cell yield is boosted with daily subcutaneous injections of Granulocvte-colonv stimulating factor, which mobilizes stem cells from the donor's bone marrow into the peripheral circulation.

Umbilical cord blood is obtained when parents elect to harvest and store the blood from a newborn's umbilical cord and placenta after birth. Cord blood has a higher concentration of HSC than is normally found in adult blood. The quality of the cord blood harvest is operator-dependent, and many delivery room nurses have not had training in collecting and storing umbilical cord blood. However, the small quantity of blood obtained from an umbilical cord (typically about 50 mL) makes it more suitable for transplantation into small children than into adults. Newer techniques using ex-vivo expansion of cord blood units or the use of 2 cord blood units from different donors are being explored to allow cord blood transplants to be used in adults.

[edit] Storage ofHSC case of allogeneic transplants fresh HSC are preferred in order to avoid cell loss that might occur during the freezing and thawing process. Allogeneic cord blood is stored frozen at a cord blood bank because it is only obtainable at the time of childbirth . To cryopreserve HSC a preservative, DMSO, must be added and the cells must be cooled very slowly in a control rate freezer to prevent osmotic cellular injury during ice crystal formation. HSC may be stored for years in a cryofreezer which typically utilizes liquid nitrogen because it is non-toxic and it is very cold (boiling point -1960C.)

~~[edit] Conditioning regimens~~

The chemotherapy or irradiation given immediately prior to a transplant is called the conditioning or preparative regimen. The purpose is to help eradicate the patient's disease prior to the infusion of HSC and to suppress immune reactions. Chemotherapy drugs and radiation both damage DNA in the cell nucleus which kills rapidly dividing cells by triggering a self-destruct mechanism called apoptosis. Bone marrow cells divide frequently and are particularly sensitive to this treatment. The bone marrow can be ablated at doses that cause minimal injury to other tissues. In allogeneic transplants a combination of cyclophosphamide with busulfan or total body irradiation is commonly employed. This treatment also has an immunosuppressive effect which prevents rejection of the HSC by the recipient's immune system. Autologous transplants may also use these conditioning regimens but many other chemotherapy combinations can be used depending on the type of disease.

Non-myeloablative allogeneic HSCT is a newer treatment approach which uses lower doses of chemotherapy and radiation which are too low to eradicate all of the bone marrow cells of a recipient. Instead, non-myeloablative transplants exploit the graft versus tumor effect for their benefit. They do require high doses of immunosuppressive agents in the early stages of treatment. This leads to a state of mixed chimerism early after transplant where both recipient and donor HSC coexist in the bone marrow space. Decreasing doses of immunosuppressive therapy then allows donor T-cells to eradicate the remaining recipient HSC and to induce graft-versus-host disease and the graft versus tumor effect.

Non-myeloablative (or "mini") allogeneic transplants, because of their gentler conditioning regimens, are associated with a lower risk of transplant-related mortality and therefore allow patients who are considered too high-risk for conventional allogeneic HSCT (because of age or other comorbidities) to undergo potentially curative therapy for their disease. These new transplant strategies are experimental, for the most part, and available at academic research centers. [edit] Transplantation and engraftment

HSC are infused into the blood stream of the recipient through an intravenous (i.v.) catheter, like any other i.v. fluid. The HSC briefly circulate in the blood stream and then populate the recipient's bone marrow spaces where they grow and start to produce blood cells After several weeks of growth in the bone marrow expansion of HSC and their g p g p known as stem cell plasticity.

~~[edit] Side effects and complications~~

HSCT is associated with a fairly high mortality in the recipient (10% or higher), which limits its use to conditions that are themselves essentially life-threatening. Major causes of complications are sepsis, graft-versus-host disease and veno-occlusive disease.

~~[edit] Regimen-related toxicity~~

~~Regimen-related toxicities are side-effects of the high dose chemotherapy or irradiation used in ablative HSCT.~~

Severe liver injury is termed hepatic veno-occlusive disease (VOD). Elevated levels of bilirubin, hepatomegaly and fluid retention are clinical hallmarks of this condition. Initially thought to be a specific form of Budd-Chiari syndrome (i.e. thrombosis of the liver veins). There is now a greater appreciation of the generalized cellular injury and obstruction in hepatic vein sinuses, and it has thus been referred to as sinusoidal obstruction syndrome (SOS). Severe case are associated with a high mortality. Anticoagulants or defibrotide may be effective in reducing the severity of VOD but may also increase bleeding complications. Ursodiol has been shown to help prevent VOD, presumably by helping the flow of bile.

Mucositis is the injury of the mucosal lining of the mouth and throat and is a common regimen-related toxicity following ablative HSCT regimens. It is usually not life- threatening but is very painful, and prevents eating and drinking. Mucositis is treated with pain medications plus intravenous infusions to prevent dehydration and malnutrition.

~~feditl Infection~~

Bone marrow transplantation usually requires that the recipient's own bone marrow is destroyed ("myeloablation"). Prior to "engraftment" patients may go for several weeks without appreciable numbers of white blood cells to help fight infection. This puts a patient at risk of infections, sepsis and septic shock despite prophylactic antibiotics. The immunosuppressive agents employed in allogeneic transplants for the prevention or treatment of graft-versus-host disease further increase the risk of opportunistic infection. Immunosuppressive drugs are given for a minimum of 6-months after a transplantation, or much longer if required for the treatment of graft-versus-host disease. Transplant patients lose their acquired immunity, for example immunity to childhood diseases such as measles or polio. For this reason transplant patients must be re-vaccinated with childhood vaccines once they are offof immunosuppressive medications.

~~[edit l Graft versus host~~

feditl Graft-versus-host disease (GVHD) p p because the immune system can still recognize other differences between their tissues. It is aptly named graft-versus-host disease because bone marrow transplantation is the only transplant procedure in which the transplanted cells must accept the body rather than the body accepting the new cells. Acute graft-versus-host disease typically occurs in the first 3 months after transplantation and may involve the skin, intestine, or the liver. Corticosteroids such as prednisone are a standard treatment. Chronic graft-versus-host disease may also develop after allogeneic transplant and is the major source of late complications. In addition to inflammation, chronic graft-versus-host disease may lead to the development of fibrosis, or scar tissue, similar to scleroderma or other autoimmune diseases and may cause functional disablity, and the need for prolonged immunosuppressive therapy. Graft-versus-host disease is usually mediated by T cells when they react to foreign peptides presented on the MHC of the host. Removal of these T cells before donation can lessen the risk of this disease.

~~[editl Graft versus tumor effect~~

The beneficial aspect of the Graft-versus-Host phenomenon is known as the "graft versus tumor" or "graft versus leukemia" effect. For example, leukemia patients with chronic graft-versus-host disease after an allogeneic transplant have a lower risk of leukemia relapse. This is due to a therapeutic immune reaction of the grafted donor lymphocytes. more specifically, the Natural Killer cells, against the diseased bone marrow of the recipient. This lower rate of relapse accounts for the increased success rate of allogeneic transplants compared to transplants from identical twins, and indicates that allogeneic HSCT is a form of immunotherapy. Graft vs tumor is also the major mechanism of benefit of non-myeloablative transplants which do not employ high dose chemotherapy or radiation. edit] Conditions treated with bone marrow or HSC transplantation

~~feditl Acquired~~

• Acute lymphocytic leukemia • Acute myelogenous leukemia • Aplastic anemia • Chronic myelogenous leukemia - accelerated phase or blast crisis • Hodgkin's disease • Multiple myeloma • Myelodysplasia • Non-Hodgkin's lymphoma • Paroxysmal nocturnal hemoglobinuria (PNH) - severe aplasia • Radiation poisoning • Kahler's disease • chronic lymphocytic leukemia • AL amyloidosis p • Amegakaryocytic Thrombocytopenia • Sickle cell disease • Griscelli syndrome type II • Hurler syndrome • Kostmann syndrome • Krabbe disease • Metachromatic leukodystrophy • Thalassemia • Hemophagocytic lymphohistiocytosis (HLH) • Wiskott-Aldrich syndrome • neuroblastoma • Some inborn errors of metabolism [edit] References

• Thomas ED, Lochte HL, Lu WC et al. Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. N EnglJMed 1957; 157: 491-496. PMID 13464965. Google Scholar • Sociέ, Gerard, et. al. (December 2001). "Busulfan plus cyclophosphamide compared with total-body irradiation plus cyclophosphamide before marrow transplantation for myeloid leukemia: long-term follow-up of 4 randomized studies". Blood 98(13): 3569-3574. Fulltext . PMID 11739158. • Richardson PG, et. al. (December 2002). "Multi-institutional use of defibrotide in 88 patients after stem cell transplantation with severe veno-occlusive disease and multisystem organ failure: response without significant toxicity in a high-risk population and factors predictive of outcome". Blood 100(13): 4337-43. PMID 12393437 • Guglielmi, PT, et. al. (December 1995). "Autologous bone marrow transplantation as compared with salvage chemotherapy in relapses of chemotherapy-sensitive non-Hodgkin's lymphoma". New England Journal of Medicine 333(23): 1540-5. PMID 7477169. • Barker IN, et. al. (December 2005). "Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy". Blood 105(3): 1343-1347. Fulltext . PMID 15466923 . [edit] External links

~~[edit] Marrow Donor Registries~~

• Australian Bone Marrow Donor Registry • Bone Marrow Donors Worldwide • U.S. National Marrow Donor Program • Europdonor Foundation • ZKRD German Bone Marrow Donor Registry • The British Bone Marrow Registry Th Gift f Lif B M F d ti p p p • Korea Marrow Donor Program • The Anthony Nolan Trust

~~[editl Bone Marrow Recruitment Groups~~

• Asian American Donor Program • Asians for Miracle Marrow Matches • Because I Care • Cammy Lee Leukemia Foundation • International Bone Marrow Transplant Association • IcIa da Silva Foundation, Inc. • Kids Beating Cancer. Inc. • South Asian Marrow Association of Recruiters

~~[editl Education for Patients~~

• The Bone Marrow Foundation • The Merck Manual of Diagnosis and Therapy bone marrow transplantation page. • Cancer Medicine online textbook. • Bone Marrow Transplantation and Peripheral Blood Stem Cell Transplantation: Questions and Answers. National Cancer Institute. • Blood & Marrow Transplant Information Network • Anthony Nolan Trust • Bone Marrow Transplant and Medical Oncology at Washington University • Universtiy of Maryland Greenbaum Cancer Center Info • Infant Bone Marrow Transplant Journal

~~[editl Experiences of Donors~~

• Donating Bone Marrow: A detailed experience of donating marrow using traditional harvest • Mike's Marrow Donor Story • Bob's Donations • Lorenz's Donations • The Right Thing to Do • My Personal Experience as a Bone Marrow Donor • A Gift of Life • My Experience as a Bone Marrow Donor • Bone Marrow Donation: A Donor's Journal • My Marrow Donation Experiences

~~\editl Transplantation and Immunology~~

~~• Website of Geneva University about transplantation immunology From Wikipedia, the free encyclopedia~~

~~Jump to: navigation, search~~

Transplant rejection occurs when the immune system of the recipient of a transplant attacks the transplanted organ or tissue. This is because a normal healthy human immune system can distinguish foreign tissues and attempts to destroy them, just as it attempts to destroy infective organisms such as bacteria and viruses .

~~Contents~~

~~[hide]~~

• 1Types of rejection o 1.1 Hyperacute rejection o 1.2 Acute rejection o 1.3 Chronic rejection • 2 Rejection Mechanisms • 3 Prevention of rejection • 4 Treatment of rejection • 5 External links

~~[edit] Types of rejection~~

~~[edit] Hyperacute rejection~~

Hyperacute rejection is a complement -mediated response in recipients with pre-existing antibodies to the donor (for example, ABO blood type antibodies). Hyperacute rejection occurs within minutes and the transplant must be immediately removed to prevent a severe systemic inflammatory response. Rapid coagulation of the blood occurs. This is a particular risk in kidney transplants, and so a prospective cytotoxic crossmatch is performed prior to kidney transplantation to ensure that antibodies to the donor are not present. For other organs, hyperacute rejection is prevented by transplanting only ABO- compatible grafts. Hyperacute rejection is the likely outcome of xenotransplanted organs.

~~[edit ] Acute rejection~~

~~Main article: Histocompatibility~~

Acute rejection is generally acknowledged to be mediated by T cell responses to proteins from the donor organ which differ from those found in the recipient. Unlike antibody- mediated hyperacute rejection, development of T cell responses first occurs several days after a transplant if the patient is not taking immunosuppressant drugs. Since the development of powerful immunosuppressive drugs such as cyclosporin , tacrolimus and and treated appropriately. Episodes occur in around 60-75% of first kidney transplants, and 50 to 60% of liver transplants. A single episode is not a cause for concern if recognised and treated promptly and rarely leads to organ failure, but recurrent episodes are associated with chronic rejection of grafts. The bulk of the immune system response is to the Major Histocompatibility Complex (MHC) proteins. MHC proteins are involved in the presentation of foreign antigens to T cells, and receptors on the surface of the T cell (TCR) are uniquely suited to recognition of proteins of this type. MHC are highly variable between individuals, and therefore the T cells from the host recognize the foreign MHC with a very high frequency leading to powerful immune responses that cause rejection of transplanted tissue. Identical twins and cloned tissue are MHC matched, and are therefore not subject to T cell mediated rejection. The first successful organ transplant was performed between identical twins by Dr. Joseph Murray at the Peter Bent Brigham Hospital in Boston. This transplant was successful because no T cell mediated responses were generated to the transplanted organ. Dr. Murray later received a Nobel prize for his work.

~~leditl Chronic rejection~~

Chronic rejection was a term used to describe all long term loss of function in organ transplants associated with fibrosis of the internal blood vessels of the transplant, but this is now termed chronic allograft vasculopathy and the term chronic rejection is reserved for those cases where the process is shown to be due to a chronic alloreactive immune response. It can be caused by a member of the Minor Histocompatibility Complex such as the H-Y gene of the male Y chromosome. This usually leads to need for a new organ after a decade or so. [edit] Rejection Mechanisms

Rejection is an adaptive immune response and is mediated through both T cell mediated and humoral immune (antibodies) mechanisms. The number of mismatched alleles determines the speed and magnitude of the rejection response. Different grafts usually have a proclivity to a certain mechanism of rejection. Bonemarrow CMI

~~Cornea Usually accepted unless vascularised, CMI~~

~~[edit] Prevention of rejection~~

~~Rejection is prevented with a combination of drugs including:~~

• Calcineurin inhibitors o Ciclosporin o Tacrolimus • mTOR inhibitors o Sirolimus o Everolimus • Anti-proliferatives o Azathioprine o Mycophenolic acid • Corticosteroids o Prednisolone o Hydrocortisone • Antibodies o Monoclonal anti-IL-2R receptor antibodies Basiliximab Daclizumab o Polyclonal anti-T-cell antibodies • Anti-thvmocvte globulin (ATG) Anti-lymphocyte globulin (ALG)

Generally a triple therapy regimen of a calcineurin inhibitor, an anti-proliferative, and a corticosteroid is used, although local protocols vary. Antibody inductions can be added to this, especially for high-risk patients and in the United States. mTOR inhibitors can be used to provide calcineurin-inhibitor or steroid-free regimes in selected patients.

A bone marrow transplant allows the chimeric body's immune system to adapt and accept a new organ. This requires that the bone marrow, which produces the immune cells, be from the same person as the organ donation (or an identical twin or a clone). Bone marrow is not attacked by the body's immune system, and is the only known type of transplant that has this quality. However, there is a risk of graft versus host disease (GVHD) in which the immune cells arising from the bone marrow transplant recognise the host tissues as foreign and attack and destroy them accordingly.

An FDA approved immune function test from Cylex has shown effectiveness in minimizing the risk of infection and rejection in post-transplant patients^ by enabling j g p p y of immunosuppressive drugs, lowering drug therapy expenses while reducing the morbidity associated with liver biopsies, improve the daily life of transplant patients, and could prolong the life of the transplanted organ. [edit] Treatment of rejection

Acute rejection is normally treated initially with a short course of high-dose methylprednisolone . which is usually sufficient to treat successfully. If this is not enough, the course can be repeated or ATG can be given. Acute rejection refractory to these treatments may require plasma exchanges to remove antibodies to the transplant.

The monoclonal anti-T cell antibody OKT3 was formerly used in the prevention of rejection, and is occasionally used in treatment of severe acute rejection, but has fallen out of common use due to the severe cytokine release syndrome and late post-transplant lymphoproliferative disorder , which are both commonly associated with use of OKT3; in the United Kingdom it is available on a named-patient use basis only.

Acute rejection usually begins after the first week of transplantation, and most likely occurs to some degree in all transplants (except between identical twins). It is caused by mismatched HLA antigens that are present on all cells. HLA antigens are polymorphic therefore the chance of a perfect match is extremely rare. The reason that acute rejection occurs a week after transplantation is because the T-cells involved in rejection must differentiate and the antibodies in response to the allograft must be produced before rejection is initiated. These T-cells cause the graft cells to lyse or produce cytokines that recruit other inflammatory cells, eventually causing necrosis of allograft tissue. Endothelial cells in vascularized grafts such as kidneys are some of the earliest victims of acute rejection. Damage to the endothelial lining is an early predictor of irreversible acute graft failure. The risk of acute rejection is highest in the first 3 months after transplantation, and is lowered by immunosuppressive agents in maintenance therapy. The onset of acute rejection is combatted by episodic treatment.

Chronic rejection is irreversible and cannot be treated effectively. The only definitive treatment is re-transplantation, if necessary. This would typically be ten years after a transplant, and this may entail returning to a transplant queue.

Hematopoietic stem cell transplantation (HSCT) or Bone marrow transplantation is a medical procedure in the field of hematology and oncology that involves transplantation of hematopoietic stem cells (HSC). It is most often performed for people with diseases of the blood or bone marrow , or certain types of cancer.

Bone marrow transplantation was pioneered at the Fred Hutchinson Cancer Research Center from the 1950s through the 1970s by E. Donnall Thomas, whose work was later p p g threatening complication called Graft-versus-host disease.

Since the early 1990s and the availability of the stem cell growth factors GM-CSF and G CSF. most hematopoeitic stem cell transplantation procedures have been performed with stem cells collected from the peripheral blood. Collecting stem cells provides a bigger graft, and does not require that the donor be subjected to general anesthesia to collect the graft.

~~Hematopoeitic stem cell transplantation remains a risky procedure and has always been reserved for patients with life threatening diseases.~~

~~Source~~

~~Sketch of bone marrow and its cells~~

HSC are found in the bone marrow of adults, which includes femurs, hip, ribs, sternum. and other bones. Cells can be obtained directly by removal from the hip using a needle and syringe, or from the blood following pre-treatment with cytokines, such as G-CSF (granulocyte colony stimulating factors), that induce cells to be released from the bone marrow compartment. Other sources for clinical and scientific use include umbilical cord blood, placenta molilized peripheral blood. For experimental purposes, fetal liver, fetal spleen and AGM (Aorta-gonad-mesonephros) of animals are also useful sources of HSCs. [edit] Functional Characteristics

~~[edit! multipotency and self-renewal~~

~~As stem cells, they are defined by their ability to form multiple cell types (multipotency) and their ability to self-renew.~~

Multipotency: Individual HSC have the ability to give rise to any of the end-stage blood cell types. During differentiation, daughter cells derived from HSC undertake a series of commitment decisions, retaining differentiation potential for some lineages while losing Self-Renewal: Some kinds of stem cells are thought to undertake asymmetric cell division, generating one daughter cell that remains a stem cell and one daughter cell that differentiates. For Hematopoietic Stem Cells, however, whether asymmetric cell division occurs during self-renewal is not known with certainty. It is instead possible that hematopoiesis occurs via symmetrical divisions, that sometimes give rise to two daughter HSC, and that at other times give rise to progeny that are committed to differentiate. The balance between self-renewal versus differentiation would therefore be regulated by the control of these two kinds of symmetrical cell division.

It is known that a small number of HSC can expand to generate a very large number of progeny HSC. This phenomenon is used in bone marrow transplant when a small number of HSC reconstitute the hematopoietic system. This indicates that at least during bone marrow transplant, symmetrical cell divisions that give two progeny HSC must occur, as expansion in HSC numbers seen during bone marrow transplant cannot occur in any other way.

Stem cell self-renewal is thought to occur in the stem cell niche in the bone marrow, and it is reasonable to assume that key signals present in this niche will be important in self- renewal. There is much interest in the environmental and molecular requirements for HSC self-renewal, as understanding the ability of HSC to replenish themselves will eventually allow the generation of expanded populations of HSC ex vivo that can be used therapeutically.

~~[edit] Functional Assays~~

• Cobble stone area forming Cell (CFAC) assay: This is a cell culture based empirical assay. When plated onto a confluent culture of stromal feeder layer, a fraction of HSCs creep between the gaps (even though the stromal cells are touching each other) and eventually settle between the stromal cells and the substratum (here the dish surface) or trapped in the cellular processes between the stromal cells. Emperipolesis is the in vivo phenomenon in which one cell is completely engulfed into another (eg thymocytes into thymic nurse cells'); on the other hand, when, in vitro, lymphoid lineage cells creep beneath nurse like cells it is called pseudoemperipolesis . This similar phenomeonon is more commonly known in HSC field by the cell culture terminology cobble stone area forming cells (CFAC) which means areas of cluster of cells which look dull cobblestone - like under phase contrast microscopy, compared to the other HSCs which are refractile. This happens because the cells which are folating loosely on top of the stromal cells are spherical and thus refractile. However, the cells which creep beneath the stromal cells are flattened and thus not refractile. The mechanism of pseudoemperipolesis is only recently coming to light it may be mediated by interection through CXCR4 (CD 184) the receptor for CXC Chemokines (eg SDFl) and aAβ\ integπng.111. [edit] Physical characteristics p p p , p y p above description is based on the morphological characteristics of a heterogeneous population of which PHSC are a component.

~~[edit Markers~~

Hematopoeitic stem cells are phenotypically identified by their small size, lack of lineage (Hn) markers, low staining (side population) with vital dyes such as rhodamine 123 (rhodamine DULL, also called rho or Hoechst 33342, and presence of various antigenic markers on their surface many of which belongs to the cluster of differentiation series, like: CD34 . CD38. CD90. CD133. CD105. CD45 and also c-kit - the receptor for stem cell factor. The hematopoietic stem cells are negative for the markers which are used for detection of lineage commitment and are thus called Lin-, and during their purification by FACS. a bunch of upto 13 to 14 different mature blood-lineage marker eg CD13 & CD33 for myeloid, CD71 for erythroid, CD19 for B cells, CD61 for megakaryocyte etc for humans; and, B220 (murine CD45) for B cells . Mac-1 (CDUb/CD18) for monocytes. Gr-I for Granulocytes . Terl l 9 for erythroid cells, 117Ra. CD3. CD4 . CD5. CD8 for T cells etc for mice) antibodies are used as a mixture to deplete the lin+ cells or late multipotent progenitors (MPP)s.

~~There are a lot of differences between the human and mice hematopoietic cell markers for the commonly accepted type of hematopoietic stem cells.fl ]~~

~~. .~~

However not all stem cells are covered by these combinations which nonetheless have become pupular. In fact even in humans there are hematopoietic stem cells which are CD34 7CD38 ". . Also some later studies suggested that earliest stem cells may lack c- kit on the cell surface^. For human HSCs use of CD133 was one step ahead as both CD34* and CD34 HSCs were CD]U +.

Traditional purification method used to yield a reasonable purity level of mouse hematopoietic stem cells generally requires a large(~10-12) battery of markers, most of which were surrogate markers with little functional significance and thus partial overlap with the stem cell populations and sometimes other closely related cells which are not stem cells. Also some of these markers 9eg Thyl) are not conserved across mouse species, and use of markers like CD34 " for HSC purification requires mice to be at least 8 weeks old. Alternative methods which could give rise to similar or better harvest of stem cells is a hot area of research and are presently emerging. One such method uses a signature of SLAM family of cell surface molecules. SLAM (Signaling lymphocyte activation molecule ) family is a group of >10 molecules whose genes are mostly located tandemly in a single locus on chromosome 1 (mouse), all belonging to a subset of immunoglobulin gene superfamily, and originally thought to be involved in T-cell The signature SLAM code for the hemapoietic higherchy are:

~~• Hematopoietic stem cells (HSC) : CD150 +CD48 ' CD244 ' • Multipotent progenitor cells (MPPs) : CD150 ' CD48 ' CD244 + • Lineage-restricted progenitor cells (LRPs) : CD 15O' CD48 +CD244 +~~

For HSCs CD1 50+CD48 ' was sufficient instead of CD150 +CD48 ' CD244 ' because CD48 is a ligand for CD244 and both would be positive only in the activated lineage restricted progenitors. This code was more efficient than the more tedious earlier set of the large number of markers and are also conserved across the mouse strains. . CD150 +CD48 ~ gave stem cell purity comparable to Thyl '°Sca- l +lin~c-kit + in mice. 1

Irving Weissman 's group at Stanford University that was the first to isolate mouse hematopoietic stem cells in 1988, was also the first to work out the markers to distinguish the mouse long term (LT-HSC) and short term (ST-HSC) hematopoietic stem cells (self renew capable), and the Multipotent progenitors (MPP, low or no self renew capability — the later the developmental stage of MPP, the lesser the self renewal ability and the more of some of the markers like CD4 and flk2):

~~[edit] Nomenclature of hematopoietic colonies and lineages~~

Between 1948 and 1950, the Committee for Clarification of the Nomenclature of Cells and Diseases of the Blood and Blood-forming Organs issued reports on the nomenclature of blood cells.™* 1An overview of the terminology is shown below, from earliest to final stage of development:

~~• [rootjblast • pro[root]cyte • [rootjcyte • meta[root]cyte • mature cell name~~

The root for CFU-E is "rubri", for CFU-GM is "granule" or "myelo" and "mono", for CFU-L is "lympho" and for CFU-Me is "megakaryo". According to this terminology, the stages of red blood cell formation would be: rubriblast, prorubricyte, rubricyte, metarubricyte and finally erythrocyte. However, the following nomenclature seems to be, t t th t l t Osteoclasts also arise from haemopoietic cells of the monocyte/neutrophil lineage, specifically CFU-GM. [edit] Colony-forming units

~~There are various kinds of colony-forming units:~~

~~• Colony-forming unit lymphocyte (CFU-L) y g g ( ) • Colony-forming unit Basophil (CFU-B) • Colony-forming unit Eosinophil (CFU-Eo)~~

The above CFUs are based on the lineage. Another CFU, the colony-forming unit-spleen (CFU-S) was the basis of an in vivo clonal colony formation, which depends on the ability of infused bone marrow cells to give rise to clones of maturing hematopoietic cells in the spleens of irradiated mice after 8 to 12 days. It was used extensively in early studies, but is now considered to measure more mature progenitor or Transit Amplifying Cells rather than stem cells. edit] References

1. Burger JA, Spoo A, Dwenger A, Burger M, Behringer D. CXCR4 chemokinc receptors (CDl 84) and alpha4betal integrins mediate spontaneous migration of human CD34+ progenitors and acute myeloid leukaemia cells beneath marrow stromal cells (pseudoemperipolesis). Br J Haematol. 2003 Aug;122(4):579-89. PMID: 12899713 2. Bhatia, M., D. Bonnet, B. Murdoch, O.I. Gan and J.E. Dick, A newly discovered class of human hematopoietic cells with SCID-repopulating activity, 4(9), 1038, 1998. u o 3. . >Yalin »Lubbert, Michael , Engelhardt, Monika CD34- Hematopoietic Stem Cells: Current Concepts and Controversies Stem Cells 2003; 21: 15-20; First published online ; doi: 10. 1634/stemcells.2 1-1-15 4. H. Doi et al. (1997) Proc. Natl. Acad. Sci. USA 94, 2513-2517 5. Gary Van Zant Stem cell markers: less is more! Blood 107: 855-856. 6. Kiel et al, Cell, Vol. 121, 1109-1 121, July 1, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.cell.2005.05.026 7. (1948) "First report of the Committee for Clarification of the Nomenclature of Cells and Diseases of the Blood and Blood-forming Organs.". AmerJ CHn Pathol 18: 443-450. 8. (1950) "Third, fourth and fifth reports of the committee for clarification of the nomenclature of cells and diseases of the blood and blood-forming organs.". Am J Clin Pathol 20 (6): 562-79. PMID 15432355.

~~Transplant - bone marrow~~

~~Definition Return to top~~

A bone marrow transplant is a procedure that transplant healthy bone marrow into a patient whose bone marrow is not working properly. A bone marrow transplant may be done for several conditions including hereditary blood diseases, hereditary metabolic diseases, hereditary immune deficiencies, and various forms of cancer. Description Return to top

Bone marrow is a soft, fatty tissue inside the bones. This is where blood cells (red blood cells, platelets, and white blood cells) are produced, and where they develop. In a disease of the blood cells —especially cancers such as leukemia - high doses of chemotherapy may be required to destroy the cancer. However, this also destroys normal blood cells.

In other cases in which hereditary or acquired disorders cause abnormal blood cell production, transplantation of healthy bone marrow may correct these problems. Transplanted bone marrow will restore production of white blood cells, red blood cells, and platelets.

~~Bone marrow transplant patients are usually treated in specialized centers. The patient stays in a special nursing unit - a bone marrow transplant unit, or BMT —to limit exposure to infections.~~

Donated bone marrow must match the patient's tissue type. It can be taken from the patient, a living relative (usually a brother or a sister), or from an unrelated donor (found through the national marrow donor program). Donors are matched through special blood tests called HLA tissue typing. (See HLA antigens .)

Bone marrow is taken from the donor in the operating room while the donor is unconscious and pain-free (under general anesthesia). Some of the donor's bone marrow is removed from the top of the hip bone. The bone marrow is filtered, treated, and transplanted immediately or frozen and stored for later use. Transplant marrow is transfused into the patient through a vein (IV) and is naturally carried into the bone cavities where it grows to replace the old bone marrow.

Alternatively, blood cell precursors, called stem cells, can be made to move from the bone marrow to the blood stream using special medications. These stem cells can then be taken from the bloodstream through a procedure called leukapheresis.

The patient is prepared for transplant by administering high doses of chemotherapy or radiation (conditioning). This serves 2 purposes. First, it destroys the patient's abnormal blood cells or cancer. Second, it slows the patient's immune response against the donor bone marrow (graft rejection).

Following conditioning, the patient is ready for bone marrow infusion. After infusion, it takes 10 - 20 days for the bone marrow to establish itself. During this time, the patient requires support with blood cell transfusions.

~~Indications Return io top~~

~~Bone marrow transplant may be recommended for:~~

• Bone marrow deficiency disease caused by: o Abnormal red blood cell production, such as thalassemia or sickle cell disease o Aggressive cancer treatments (chemotherapy, radiation therapy), especially for leukemia or lymphoma σ Lack of normal blood cell production (aplastic anemia) • Immune system disorders (immunodeficiencies) such as: σ Congenital neutropenia o Severe combined immunodeficiency syndrome • Specific forms of cancer: o Leukemias o Lymphomas o Myeloma

~~BSTITUTE ) • Patients with other diseases that may limit survival~~

~~Risks Return Io lop~~

~~The risks for any anesthesia are:~~

~~• Reactions to medications • Problems breathing~~

Chemotherapy given pπor to bone marrow transplant (conditioning) may cause significant toxicity, such as mouth sores, diarrhea, liver damage, or lung damage. While waiting for bone marrow to grow, the patient is at high risk for infection.

The major problem with bone marrow transplants —when the marrow comes from a donor, not the patient - - is graft-versus-host disease . The transplanted healthy bone marrow cells may attack the patient's cells as though they were foreign organisms. In this case, drugs to suppress the immune system must be taken, but this also decreases the body's ability to fight infections.

~~Expectations after surgery Return to too~~

Ideally, bone marrow transplant lengthens the life of a patient who would otherwise die. Relatively normal activities can be resumed as soon as the patient feels well enough, and after consulting with the doctor.

Other significant problems with a bone marrow transplant are those of all major organ transplants - the finding of a donor, and the cost. The donor is frequently a sibling with matching tissue. The more siblings the patient has, the more chances there are of finding a matching donor.

~~Convalescence Return to top~~

The hospitalization period is from 4 - 6 weeks, dunng which time the patient is isolated and under strict monitoring because of the increased πsk of infection. The patient will require attentive follow-up care for 2 - 3 months after discharge from the hospital. It may take 6 months to a year for the immune system to fully recover from this procedure.

~~http ://www .cancerbackup .org .uk/Treatments/Stemcellbonernarro wtransplants/Generalinformation/Stemcellsfromadonor~~

~~I Stem cells from a donor~~

In this type of transplant, stem cells are donated by another person (a donor). Doctors call this an allogeneic transplant or an allograft. It is sometimes used as part of the treatment for cancers that are in the bone marrow, such as leukaemia and myeloma . It can also be used to treat some rare non¬ cancerous diseases of the bone marrow or the immune system.

~~The treatment Why donor stem cell transplants are used Risks of a donor transplant The treatment~~

Treatment with high-dose chemotherapy is given to destroy the cancer cells in your bone marrow. This also destroys your healthy stem cells. After the treatment, stem cells donated by someone else (a donor) are given to you by drip. The stem cells go into the bone marrow and start to produce blood cells. This helps you to recover from the high-dose treatment.

~~The most suitable donor is usually a brother or sister whose bone marrow is a close match to your own.~~

Occasionally it is possible to use stem cells from a person who is not related to you, if tests have shown that their white blood cells are a good match with yours. This is known as a matched unrelated donor (MUD) transplant.

The stem cells from a donor will contain immune cells. These immune cells can sometimes attack some of the cells of the person who has received the transplant (the recipient). This can cause a reaction in the body known as qraft-versus-host disease (GvHD). As well as attacking healthy cells, the immune cells from the donor may also attack any cancer cells that are left. This is known as graft- versus-disease (GvD).

~~An allogeneic transplant is a very serious and complicated procedure. This intensive treatment is earned out in specialised transplant units in larger university hospitals.~~

~~back to top~~

~~Why donor stem cell transplants are used~~

Donor stem cell transplants may be used to treat some types of leukaemia, lymphoma and myeloma. These allogeneic stem cell transplants are used to improve the chances of curing the disease or prolonging a remission. A transplant may be earned out in the early stage of treatment when the disease is in remission. A transplant may also be done if the illness comes back after treatment. There are different types of leukaemia and lymphoma and a donor transplant is not a suitable treatment for everyone. Whether there is a suitable donor is one of the important factors for doctors to consider.

Other factors your doctor will take into account when considering whether to recommend a transplant for you are your age and general health. There are no lower age limits, but guidelines recommend that for donor transplants the upper age limit is 45-50. This is because the risk of severe graft-versus-host disease is much higher after that age However, sometimes a donor transplant may be given to people older than 45-50. This depends on your general health and the nsk of recurrence of the disease.

A donor stem cell transplant procedure is extremely demanding, both physically and emotionally. You may need to stay in hospital for 4-6 weeks or longer. For most of that time you will usually be in a room of your own, and you will probably feel very ill.

~~back to top~~

~~Risks of a donor transplant~~

~~An allogeneic stem cell transplant is a complicated and specialised treatment It has many side effects and possible complications.~~

The main risk comes during the time after the high-dose treatment when your bone marrow is recovering. At that time, you are at risk of possible life-threatening infections and bleeding. There is also the risk of graft-versus-host disease, in which the donor's marrow or stem cells react against your own tissue Very occasionally, the donated marrow or stem cells do not start to produce new blood Your age and general state of health are key factors to take into account. A transplant takes a long time and may make you very ill. If you are at the upper age limit and your general health is not good, you are more likely to have complications.

You need to weigh up the benefits and risks of this treatment very carefully. The treatment may give a greater chance of curing the cancer than any other type of treatment. However, this has to be weighed against the possible side effects and the fact that some people die during the procedure. You will want to discuss this in detail with your doctor and your family. Most hospitals that carry out blood stem cell transplants have a transplant counsellor you can talk to about any fears or worries.

~~back to top~~

~~Fertility~~

One issue that your doctor should discuss with you, if it is relevant for you, is fertility. Unfortunately, infertility is usually a side effect of the intensive treatment, for both men and women. There is a higher risk of infertility with an allogeneic (from a donor) transplant than with high-dose treatment with stem cell support. See the section on life after high-dose treatment for more information.

~~http ://www. cancerbackup .org .uk/Treatments/Stemcellbonemarro wtransplants/Generalinformation/Findingadonor~~

~~Finding a match~~

To reduce the risk of your body rejecting the donated stem cells (graft rejection), the donor's tissue type has to be closely matched to yours. The matching process involves a blood test, and is done by looking for specific proteins known as markers on the surface of the cells. The markers are called human leukocyte antigens (HLAs). Once the HLA type of your bone marrow has been found, other people can be tested to see whether their bone marrow and stem cells are the same type as yours. The closeness of a match needed for a good result depends on which particular tissue markers are the same as yours. For some markers, doctors may decide to accept small differences, to improve the chances of finding an acceptable donor. In this situation, the transplant is known as a mismatched transplant.

Donors need to be in good health. They will be given a thorough medical check-up to make sure that there will be no risk to their own health from the procedure. The donor will have to go to the hospital for a couple of outpatient visits for these checks. They then have either their bone marrow or stem cells collected at the hospital.

~~back to top~~

~~Related donor~~

Usually brothers and sisters (your siblings) are tested first, as they are likely to have the best match. Because of the way that your siblings inherit the 'HLA markers' from your parents, some will be more likely to be a good match, others will be less likely. If one of your siblings is completely matched to you, they are known as an HLA identical donor. The match is unlikely to be perfect unless you have an identical twin. A transplant using bone marrow or stem cells from an identical twin is called a syngeneic transplant. Parents, half-brothers and half-sisters will not usually be a good match, although they may be tested if you have no other siblings, or if your doctors have not been able to find a complete match for you. If you have no suitable relatives it may be possible to find a matched unrelated donor (known as MUD). The National Blood Service (England and Wales) , the National Blood Transfusion Service (Scotland) and some charities have lists of volunteer unrelated donors. Your doctor will have access to these and can search for a close match for you. Remember, however, that it may be very difficult and time-consuming to find a good match. Overall, only about 1 in 10 searches will find a closely matched, unrelated donor.

People from ethnic groups often have difficulty finding a good match from the volunteer registries. This is because most people who register as potential bone marrow donors are from the white population, and tissue types rarely match across different ethnic groups.

~~http ://www. cancerbackup .org .uk/Treatments/Stemcellbonemarro wtransplant s/Be ingtreated/Col leetingstemcel Is~~

~~Collecting stem cells~~

~~Taking stem cells from the blood Collecting a donor's stem cells~~

~~Taking stem cells from the blood~~

First the stem cells are made to move from the bone marrow into the blood. To do this you will usually be given some chemotherapy , followed by a course of daily injections of a growth factor (G-CSF). The growth factor is given as an injection under the skin. Sometimes stem cells can be made to spill over into the blood using growth factors alone. You or a relative can be taught to give these injections, or you can go to your GP or the hospital to have them. Your blood will be tested regularly, and when there are enough stem cells in the blood, they will be collected.

Collecting the stem cells takes 3-4 hours. You will be asked to He down on a couch and a drip will be put into the vein of each arm. Blood will be taken from one arm, through the drip, into a machine called a cell separator. The separator spins the blood to separate out the stem cells. These are collected, and the remaining blood is given back to you through the drip in your other arm. Sometimes a tube may be put into a vein at the top of your leg (Instead of your arm) to collect the stem cells. If you already have a central line, or a PICC line, going into the main vein in your chest this can be used. The stem cells are then frozen until you have had the high-dose treatment.

~~back to top~~

~~Collecting a donor's stem cells~~

Your donor will have their stem cells taken directly from the bloodstream in the same way except they are not given chemotherapy drugs. They will be given a short course of injections of a growth factor (G-CSF). This encourages the production of stem cells so that they spill over from the bone marrow into the blood. The stem cells are then collected from the blood, usually in one session, which takes 3- 4 hours. Occasionally, if not enough stem cells are collected, the donor may have to come in for another collection. This can all be done as a day patient. -CSF (Neupogen®, Granocyte®, Neulasta®)

~~What is G-CSF? What it looks like How G-CSF is given How often it is given Possible side effects Additional information References~~

~~What is G-CSF?~~

G-CSF (granulocyte-colony stimulating factor) is an haematopoietic growth factor that works by encouraging the bone marrow to produce more white blood cells. Growth factors are special proteins which are produced naturally in the body. They can also be made as a drug.

One of the main side effects of chemotherapy drugs is a reduction in the number of white blood cells. This makes your body less able to Tight infection. There is a risk that you could develop a serious infection, which may have to be treated In hospital. If the number of blood cells (your blood count) in your blood is low when your next dose of chemotherapy is due, the chemotherapy may have to be delayed, or the dose lowered.

In this situation, G-CSF can be given to stimulate the bone marrow to produce new white cells more quickly after chemotherapy. This can shorten the period during which you are at risk of developing a serious infection. G-CSF is not needed with all types of chemotherapy treatment, as the white blood cell count can often recover on its own.

G-CSF may sometimes be used before high-dose chemotherapy to make the bone marrow produce more stem cells. These extra stem cells can then be collected and given back to you after high-dose chemotherapy treatment. The stem cells then go back into the bone marrow and produce blood cells.

~~There are three different types of G-CSF:~~

~~lenograstim (Granocyte®) filgrastim (Neupogen®) pegylated filgrastim (Neulasta®).~~

~~These drugs all work In similar ways. The molecules of the pegylated filgrastim have had a substance added that helps the drug to work for longer.~~

~~back to top~~

~~What it looks like~~

~~G-CSF is available as a white powder, which is then dissolved in sterile water, or a colourless fluid in a small glass bottle. It is also available as a pre-filled syringe.~~

~~back to too~~

~~How G-CSF is given~~

G-CSF is usually given as an injection under the skin (subcutaneously), most often in the thigh, arm or abdomen. You, or people caring for you, can be taught how to give these injections so that you can back to top

~~How often it is given~~

G-CSF is usually started a few days after the chemotherapy has been given, and is given daily for up to 14 days. Pegylated filgrastim is given once with each cycle of chemotherapy. Which of the types of G-CSF you get will depend upon the chemotherapy treatment you are having. Your doctor or nurse can give you more information.

~~back to top~~

~~Possible side effects~~

When G-CSF injections are given, the amount in the body increases greatly, becoming much higher than that which occurs naturally. For this reason, it causes side effects, even though it is a naturally occurring substance. The side effects are not usually severe, however. People react to drugs in different ways, so it is not possible to predict who is going to have side effects or which they will have. The most common side effects are listed below.

~~Bone pain Some people have a dull ache or discomfort in the bones of the back, pelvis, arms or legs. This is usually mild and goes away when the growth-factor injections stop.~~

~~Red, Itchy skin Your skin may become red and itchy around the area in which the injection is given. This will disappear once the course of injections is over.~~

~~Fever, chilis and fluid retention G-CSF may cause fever, chills and fluid retention. Fluid retention may lead to swelling of the ankles or breathlessness .~~

~~Nausea, vomiting and diarrhoea Occasionally you may experience nausea, vomiting and diarrhoea .~~

~~Let your doctor o r nurse know if you have any side effects. Your doctor may prescribe painkillers such as paracetamol to help reduce you temperature and prevent chills.~~

~~back to top~~

~~Additional information~~

~~You will need to have the levels of white blood cells in your blood checked regularly (possibly twice a week) while you are having growth factor injections.~~

~~Filgrastim and pegylated filgrastim need to be stored in the fridge. Lβnograstim can be stored at room temperature. Follow any storage instructions given by your pharmacist.~~

~~back to top~~

~~References~~

~~This section has been compiled using information from a number of reliable sources including:~~

Martindalβ : The Complete Drug Reference (33rd edition). Sweetman et al. Pharmaceutical Press, 2002. British National Formulary (50th edition). British Medical Association and Royal Pharmaceutical Society of Great Britain, September I Taking stem cells from the bone marrow

~~Although it is much more common for the stem cells to be collected from your, or your donor's, blood, in some situations the stem cells may be collected from the bone marrow.~~

~~How is it done?~~

~~About a week or two before the bone marrow is taken, you (or a donor) may have 1-2 pints of blood taken. This will be given back to you after the bone marrow is collected.~~

The collection of bone marrow is done under a general anaesthetic or an anaesthetic given into the spinal cord, so you will feel nothing. Some marrow is taken from inside the bones at the back and front of the pelvis (the hip bones). Rarely, marrow is taken from the breastbone (sternum). It is not usually possible to collect bone marrow from areas that have previously been treated with radiotherapy.

The doctor inserts a special needle through the skin and into the bone. The marrow is then sucked out into a syringe and put in a sterile container with various liquids. These liquids keep the cells healthy during processing and storage. The bone marrow is taken from a number of different areas in the pelvis. This is done through several small punctures, which quickly heal. You are likely to have some bruising for a few weeks afterwards.

For an adult, approximately one litre of bone marrow will be removed in this way - about 10-15% of the body's total. This leaves plenty for your or your donor's needs, and very quickly the body will replace the bone marrow that is removed.

You will have to stay in hospital overnight to recover from the general anaesthetic. You will feel very sore for a few days afterwards and will need to take painkillers. You will be given a further supply of painkillers to take home if you need them.

~~http ://www .cancerbackup .org .uk/Treatmenta/Stemcellbonemarro wtransplanta/Beingtreated/Gettingitback~~

~~I Giving back the stem cells~~

~~A day or more after the high-dose treatment has finished, your own, or the donated, stem cells will be given to you through your central line. This is similar to having a blood transfusion .~~

The stem cells will find their way back through the bloodstream to the bone marrow. In the bone marrow they will start to grow and develop into mature blood cells. It will be at least two or three weeks before some of the 'new' blood cells are released into the bloodstream. It may then be up to six weeks before you can leave hospital. This is because you will be very vulnerable to infection until your body is once again producing enough blood cells to protect you.

You may be given growth factors through your central line. PICC line or implantable port. These stimulate your bone marrow to start producing new white blood cells more quickly. Using growth factors can reduce the length of time you are at risk from some of the side effects .

If you are having an allogeneic transplant (from a donor) you will usually stay in a single room to help protect you from the risk of infection. This is especially important when your blood count is at its lowest, about 2-4 weeks after the high-dose treatment. I Newer treatments

~~Mini transplants~~

These are similar to allogeneic transplants (from a donor) except that standard-dose chemotherapy is given instead of high-dose. This treatment may be given to people who cannot have high-dose chemotherapy, possibly due to their age or because they are not fit enough. A course of standard-dose chemotherapy is given, usually over five days, before the patient is given the donor's stem cells or bone marrow. A drug which works on the immune system to reduce the risks of qraft-versus-host disease is also given. It is hoped that this will trigger an immune reaction which will encourage the donor's cells to take over in the patient's bone marrow.

~~This is a new and still experimental treatment, which is being carried out in clinical trials. It is sometimes called a mini-allo or a non-myeloablative transplant.~~

~~Tandem transplants~~

In a tandem transplant, high-dose chemotherapy with stem cell support is followed within 3-6 months by another high-dose chemotherapy treatment with more stem cell support. It is thought that this may make the treatment more effective in controlling the illness. However, tandem transplants are still in the early stages of research.

~~Cord blood~~

It is now possible to take stem cells from the umbilical cord of a newly born baby. These stem cells may be used if the baby's cells are a match for a brother or sister who needs a transplant. A major drawback is that usually not enough stem cells are collected. This treatment is also experimental and further research is going on to look at using stem cells from cord blood.

~~Glossary of Terms~~

~~adult stem cells: Undifferentiated cells that are found in various specialised (differentiated) tissues of the body (eg in bone marrow, skin, intestine).~~

blolnformatics: The use of computing technologies to discover and manage systematic biological information by translating genetic and protein data into manageable forms that can be analysed and interpreted, often in conjunction with rapidly growing databases.

~~biomass: All the living organisms of a particular region, considered collectively.~~

bloprospectlng: The practice of screening samples of plants, animals and microorganisms (often collected from the wild) for active chemical compounds or molecules that can be developed into patented and marketable commodities.

~~biotechnology: The understanding and use of biological processes and organisms for health, social, environmental and economic applications or outcomes.~~

biotechnology industry: A collection of industries - eg pharmaceutical, food processing, plant and animal agriculture, environmental management or minerals processing - which utilise some of the techniques of biotechnology.

~~blastocyst: A hollow ball of 50 - IOO cells reached after 4-5 days embryonic development just before implantation in the uterus.~~

cell: The smallest structural unit of living organisms that is able to grow and reproduce independently. g g y cytotechnology: 'diagnostic cytopathology* - Involves the interpretation of cells that spontaneously exfoliate or are removed from tissues by abrasion or fine needle aspiration, eg specimens from the cervix (Pap tests), breast, thyroid, lymph node, liver, etc

~~de-differentiation: The process of inducing a specialised cell to revert towards pluripotency~~

~~differentiation: The process by which less specialised cells develop into more specialised cell types.~~

~~embryo: A general term applied to the developing organism from the completion of fertilisation, until 8 weeks when the organism becomes known as a foetus~~

~~enucleated: A cell from which the nucleus has been removed (usually an egg).~~

~~foetus: The term used for a developing human after the eighth week of development until birth.~~

~~functional genomics: The study of the functional consequences for a ceil or organism o f the presence, absence or modification o f a gene.~~

~~gamete: A mature male or female germ cell, a sperm or egg.~~

~~gene: The earner of hereditary characteristics, a piece of DNA that codes for the production of a particular molecule (usually a protein) used to make a part of machinery or tissue of an organism~~

genetically modified organism (GMO): An organism in which characteπstιc/s have been altered by a modification of the genome (for example, by the introduction of a modified gene from another organism).

~~genome: The collection of all the genes in a cell in an organism~~

~~genomics: The study of the organisation, structure and control of genes~~

~~genotype: The entire genetic constitution o f an individual~~

~~gene therapy: Treating, diagnosing or preventing disease by introducing specific alterations in the genetic material of the human body~~

~~germ cell: A sexual reproductive cell (sperm or egg).~~

~~germ line: Cells from which the next generation of eggs or sperm will be derived~~

~~germplasm: The total genetic variability of an organism, represented by the total available pool of germ cells or seed~~

~~Intellectual Property: Can be defined as any product of the human intellect that is unique, novel and unobvious (has some value in the marketplace)~~

~~In vitro fertilisation (IVF): Technologies by which eggs and sperm are collected and united to achieve fertilisation outside the body~~

monoclonal antibody: A highly specific antibody that is derived from only one clone of cells. nanotechnology: Functioning devices with moving parts that are only molecules in size, such as a biosensor with a tiny molecular switch as its central component nuclear magnetic resonance (NMR): A technique that provides information on the structural behaviour of complex molecules in their environments nuclear replacement: see somatic cell nuclear transfer nucleus: The cell structure that houses the genetic information (chromosomes), nutraceutical: A plant or natural product that when consumed orally, confers a health benefit oocyte: The female germ cell. pluripotent: Cells with the capacity to develop into every cell type in the human body but not the placenta and umbilical cord. Pluripotent cells are not capable of developing into an entire organism.

~~primitive streak: A collection of cells which appears about 14 days after fertilisation from which the central nervous system eventually develops.~~

~~protein: A complex organic compound composed of numerous amino acids. Proteins occur in all living organisms and their production is coded for by genes~~

protco me: As the genome is the genetic complement of an organism, so the proteome is the complement o f all proteins in an organism Proteins may differ in the sequence of their amino acids or in chemical modifications which result in changed properties that can be identified

proteomlcj: The high-throughput separation, identification and characterisation of proteins from a biological sample - a complementary technology to genomics, but starting with the protein rather than the gene

~~SCNT: Somatic cell nuclear transfer is a technique that involves transferring the nucleus of a somatic cell into an enucleated egg~~

~~somatic cell: Any body cell apart from a sperm or egg.~~

~~somatic cell nuclear transfer (SCNT): A technique that involves transferring the nucleus of a somatic cell into an enucleated egg.~~

~~sperm: The mature male germ cell.~~

~~stem cell: A cell with the ability to divide indefinitely and to give πse to specialised cells as well as new stem cells with identical potential.~~

~~stem cell line: Stem cells that are cultured in the laboratory and divide to give πse to more stem cells.~~

~~Therapeutic Cloning: (same as SCNT)~~

~~totipotent: Cells that have the capacity to differentiate into the embryo and into extra embryonic membranes and tissues Totipotent cells contribute to every cell type of the adult organism.~~

~~transgenic: The introduction of a modified gene from one organism into another~~

~~vector: The agent used to carry new genes into cells zygote: The single cell formed when the male sperm fertilises the female egg. http ://www .medicalnews today .com/medicalnews .php?new3id=4677~~

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NASDAQ authonzed Stem Cell Autho πty Ltd. (OTC: SCAL), the company which possesses the exclusive rights for the collection and storage of human umbilical cord matrix stem cells, to be publicly traded on July 6, 2006.

The Kansas State University Research Foundation has filed an application for a patent pertaining to the methods for the collection, storage and biotechnology and therapeutic use of umbilical cord matrix stem cells, and has granted Stem Cell Authority Ltd. and its subsidiary company the exclusive license to collect and cryogenically store human umbilical cord matrix stem cells. Thus, the collection and storage of human pluripotent umbilical cord matrix stem cell must be authorized by Stem Cell Authority Ltd. The company also has the right to grant sublicenses.

Recent evidence indicates that the umbilical cord matrix stem cells can differentiate into neurons, glia, skeletal muscle cells, heart muscle cells, bone cells, cartilage cells and liver cells. Published work indicates that the human umbilical cord matrix stem cells are therapeutically useful in an animal model of Parkinson's disease. Therefore, the umbilical cord matrix stem cells may have the potential for treating neurological diseases. In contrast, umbilical cord blood stem cells are used clinically to treat conditions calling for bone marrow stem cell transplant. These diseases include certain cancers and in-borne errors in metabolism.

Stem Cell Autho πty Ltd. collects the matrix cells from the Wharton's jelly which is within the human umbilical cord. The matrix cells are collected safely and painlessly without risk to the child after birth. The collection of the matrix stem cell is compatible with collection of umbilical cord blood stem cells. Thus, Stem Ceil right o of er this service o s custome s, p ng t in a s ro g pos o b o ch m rk .

Stem Cell Authonty Ltd. is the only entity worldwide that is permitted to collect and store the umbilical cord's pluripotent or matrix stem cells, which have the potential to be one of the most important sources of stem cells available. The company is poised to become a market leader in this expanding biotech industry.

~~Stem Cell Authority Ltd. http7/www stemcellauthontv com~~

~~htto://www stemcellauthontv com/oaQe/pa αe/26091 87 htm Advantages of Cord Blood Stem Cells~~

Cord blood stem cells provide a distinct advantage over bone marrow stem cells because they are less likely to cause graft- versus host disease ("GVHD") with similarly matched specimens than bone marrow. The immune cells present in cord blood seem to have immunologic immaturity, which reduces the risk of rejection from the host system. In GVHD, donor lymphocytes attack the recipient's tissues leading to rash, diarrhea, jaundice, or a scieroderma-like disease; it is a major cause of morbidity and mortality following transplantation. In unrelated cord blood transplants, there is a 40% chance of GVHD versus 2% for related cord blood transplants, with a 20% chance of chronic GVHD versus 0%, respectively. Umbilical cord blood stem cells also seem to require less stringent HLA matching for unrelated donors '. Furthermore, the cord blood is a perfect match for the Infant from whom It is collected. Siblings have up to a 50% chance of being a suitable match for future transplantation in another sibling in need. Parents and grandparents are potential matches as well Umbilical cord blood is much easier and less traumatic to collect than bone marrow and the procedure is, by far, much less expensive.

~~http: //www.ncbi .nlm.nih.gov/entrez/query. fcgi?cmd=Retrieve& db=pubmed&dopt=Abstract&list uids=1719684 5~~

~~In vitro differentiation of cord blood unrestricted somatic stem cells expressing dopamlne-assoclated genes Into neuron-like cells.~~

~~Fallahi-Sichani M. Soleimanl M. Naiafi SM. Kianl J. Arefian E. Atashi A.~~

~~Department of Biotechnology, University College of Science, University of Tehran, Tehran 14155-6455, Iran.~~

An intensive study is underway to evaluate different potential candidates for cell therapy of neurodegenerative disorders such as Parkinson's disease (PD). Availability and lower immunogenicity compared to other sources for stem ceil therapy such as bone marrow have made human umbilical cord biood stem cells ( ), serum-withdrawal medium. Using reverse transcription polymerase chain reaction and lmmunocytochemlstry assays, we have shown the expression of neuron-specific genes following a 2week treatment of USSCs in serum- withdrawal induction medium. I n addition, we have found that USSCs and USSC- derlved neuron-like cells express transcripts of genes associated with development and/or survival of dopaminergic mesencephalic neurons including EnI, En2, Nurrl, Ptx3, Pax2, Wntl and Wnt3a. The expression of dopamlne- associated genes suggests that these cells may be potential candidates to be used for cell therapy of PD.

~~PMID: 17196845 [PubMed - in process]~~

~~PLURIPOTENT SCs FROM CORD BLOOD http: //www.google.com/search?hl=en&lr=&as qdr=all&q=%22plur ipotent+cord%2 2&btnG=Search~~

http: //www. the-scientist.com/blog/display/23 093/ Primer on the Science of Stem Cells: The definition of a stem cell is a cell that divides asynchronously, such that one daughter cell is a specialized cell and the other daughter cell is "self-renewing" - that is, it gives rise to another daughter cell which is self -renewing. There are loosely, three different type of stem cell:

~~• Pturipotent stem cell - can give rise t o any one of the approximately 200 cell types found within the body~~

~~• Multipotent stem cell - can give rise to a more restricted (and usually tissue-specific) set of cell types (e.g. hematopoetic, neural)~~

~~• Restricted stem cell - can give rise to one or only a few differentiated cell types (e.g. gut, germ cell)~~

The rule of thumb i s that the more different cell types that a stem cell can give rise to the earlier it comes in the lineage process - thus embryonic stem cells (those derived from a blastocyst) are pluripotent, cord blood stem cells (from the umbilical cord) are multipotent, giving rise to the entire blood cell lineages, both myeloid (erythrocytes, neutrophils, macrophages...) and lymphoid ( T and B cells, NK cells...), and adult stem cells are the most restricted of all - gut and skin stem cells only being able to give rise to a few cell types of those tissues. material, giving stability to the chromosomes. The enzyme is a reverse transcriptase that carries its own RNA molecule, which is used as a template when it elongates telomeres, which are shortened after each replication cycle. Telomerase was discovered by Carol W. Greige n 1984.

~~Contents~~

~~[hide]~~

• 1 Structure • 2 Function • 3 Clinical implications o 3.1 Aging o 3.2 Cancer o 3.3 Additional roles in cancer, heart disease, and a socioeconomic and quality of life aspect o 3.4 Role in other human diseases o 3.5 Telomerase as a potential drug target • 4 References • 5 See also • 6 External links

~~[edit] Structure~~

The composition of human telomerase was identified in 2007 by Dr Scott Cohen and his team at the Children's Medical Research Institute in Australia. It consists of two molecules each of human Telomerase Reverse Transcriptase (TERT), Telomerase RNA (hTR or TERC) and dyskerin. The two subunits of the enzyme are coded by two different genes in the genome. The coding region of the TERT gene is 3396bg, and translates to a protein of 1131 amino acids. The polypeptide folds with TERC (451 nucleotides long), which is not translated and remains as RNA. TERT has a 'mitten' structure that allows it to wrap around the chromosome to add single-stranded telomere repeats.

TERT is a reverse transcriptase , which is a class of enzyme that creates single stranded DNA using single stranded RNA as a template. Enzymes of this class (not TERT specifically, but the ones isolated from viruses) are utilized by scientists in the molecular biological process of Reverse Transcriptase PCR (RT-PCR), which allows the creation of several DNA copies of a target sequence using RNA as a template. As stated above TERT carries its own template around, TERC. [edit] Function The template region of TERC is 3'-CAAUCCCAAUC-S 1. This way, telomerase can bind the first few nucleotides of the template to the last telomere sequence on the chromosome, add a new telomere repeat (5'-GGTTAG-3') sequence, let go, realign the new 3'-end of telomere to the template, and repeat the process. (For an explanation on why this elongation is necessary see Telomere shortening .) [edit] Clinical implications

~~[edit] Aging~~

The enzyme telomerase has been proposed as the key to cellular immortality, as a veritable "fountain of youth". It is the reason most cancers are able to grow unchecked, as if they too are immortal. The enzyme allows for rapid growth of cells without the problems of senescence . Embryonic stem cells, for instance, express telomerase, which allows them to divide repeatedly and form the individual. In adults, telomerase is expressed in cells that need to divide regularly, such as the male gonads, although most somatic cells do not express it. One of the main obvious symptoms of old age is decreased skin vitality, and if telomerase therapy can improve this alone, it will be an advance for those who suffer from such problems. Both Revive Skincare and TaSciences.com have telomerase-based products on the market for cosmetic purposes. Revive's Dr. Brown has claimed that it costs that company $4 million to produce a gram of telomerase.

Geron Corporation has granted a license to TAsciences.com to sell TA-65, a telomerase activator agent derived from the Chinese Astragalus plant. TA Sciences will offer this product beginning in April of 2007 in New York.

A variety of premature aging syndromes are associated with short telomeres . These include Werner syndrome. Ataxia telangiectasia. Bloom syndrome. Fanconi anemia. Nijmegen breakage syndrome and ataxia telangiectasia-like disorder. The genes which have been mutated in these diseases all have roles in the repair of DNA damage, and their precise roles in maintaining telomere length are an active area of investigation. While it is currently unknown to what extent telomere erosion contributes to the normal aging process, maintenance of DNA in general, and telomeric DNA specifically, have emerged as major players. Dr. Michael Fossel has suggested in an interview that telomerase therapies may be used not only to combat cancer, but to actually get around human aging and extend lifespan significantly. He believes human trials of telomerase-based therapies for extending lifespan will occur within the next 10 years. This timeline is significant because it coincides with the retirement of Baby Boomers in the United States and Europe.

~~feditl Cancer~~

When cells are approaching the Hayflick limit in cell cultures, the time to senescence can be extended by the inactivation of the tumor suppressor proteins - TP53 and Retinoblastoma protein (pRb). Cells which have been thus altered will eventually division. Exposed chromosome ends are interpreted as a double stranded breaks (DSB) in DNA; such damage is usually repaired by reattaching (religating) the broken ends together. When the cell does this due to telomere shortening, the ends of different chromosomes can be attached together. This temporarily solves the problem of lacking telomeres, but during anaphase of cell division the fused chromosomes are randomly ripped apart causing many mutations and chromosomal abnormalities. As this process continues, the cell's genome becomes unstable. Eventually, either sufficient damage will be done to the cell's chromosomes such that cell dies (via programmed cell death, apoptosis) . or an additional mutation will take place that activates telomerase.

With the activation of telomerase, some types of cells and their offspring become immortal, that is, their chromosomes won't become unstable no matter how many cell divisions they undergo (they bypass the Hayflick limit) thus avoiding cell death as long as the conditions for their duplication are met. Many cancer cells are considered 'immortal' because telomerase activity allows them to divide virtually forever, which is why they can form tumors . A good example of cancer cells' immortality is HeLa cells , which were originally removed from the cervical cancer of Henrietta Lacks in 1951 and are still used in laboratories as a model cell line. They are indeed immortal - daily production of HeLa cells is estimated at several tons even up to this day - all from the few cells taken from Ms. Lacks' rumor.

While this method of modeling human cancer in cell culture is effective and has been used for many years by scientists, it is also very imprecise. The exact changes which allow for the formation of the tumorigenie clones in the above experiment are not clear. Scientists have subsequently been able to address this question by the serial introduction of several mutations present in a variety of human cancers. This has led to the elucidation of several combinations of mutations which are sufficient for the formation of rumorigenic cells, in a variety of cell types. While the combination varies depending on the cell type, a common theme is that the following alterations are required: activation of TERT, loss of p53 pathway function, loss of pRb pathway function, activation of the Ras or mvc proto-oncogenes. and aberration of the PP2A protein phosphatase . That is to say the cell has an activated telomerase eliminating the process of death by chromosome unstability or loss, absence of apoptosis-induction pathways and continued activation of mitosis .

This model of cancer in cell culture accurately describes the role of telomerase in actual human tumors. Telomerase activation has been observed in ~90% of all human tumors, suggesting that the immortality conferred by telomerase plays a key role in cancer development. Of the tumors which have not activated TERT, most have found a separate pathway to maintain telomere length termed ALT (Alternative Lengthening of Telomeres). The exact mechanism behind telomere maintenance in the ALT pathway has not been elucidated, but likely involves multiple recombination events at the telomere.

~~[edit] Additional roles in cancer, heart disease, and a socioeconomic and quality of life aspect facilitate their programmed growth rate.(roughly the growth rate of a fetus)~~

~~E.V. Gostjeva et al (MIT) recently imaged colon cancer stem cells and compared them to fetal colon stem cells trying to make a new colon, they were the same.~~

Dr. Elizabeth Blackburn et al UCSF has shown work that mothers caring for their very sick children have shorter telomeres when they report that their emotional stress is at the greatest point. She also found telomerase active at the site of blockages in coronary artery tissue. This could be why heart attacks can come on so suddenly, telomerase is driving the growth of the blockage.

Other work has shown that the poor have shorter telomeres than the rich. Short telomeres can lead to telomeric crisis and the initiation of cancer if many other conditions are also met, or so the discussion goes at this point. fW< i

Dr. Blackburn and the two other co-discoverers of telomerase won the Lasker Prize (2006) for the discovery of telomerase and subsequent work on telomerase. Dr. Blackburn also won this years Gruber Genetics Prize for same. Seventy winners of the Lasker have gone on to be awarded the Nobel.

~~[edit Role in other human diseases~~

~~Aplastic anemia, a disorder in which the bone marrow fails to produce blood cells. Mutations in TERT have been implicated in predisposing patients to aplastic anemia in a 2005. .~~

Cri du chat Syndrome (CdCS). is a complex disorder involving the loss of the distal portion of the short arm of chromosome 5. TERT is located in the deleted region, and loss of one copy of TERT has been suggested as a cause or contributing factor of this disease.

Dyskeratosis congenita (DC) is a disease of the bone marrow which can be caused by a mutation in the telomerase RNA subunit, TERC. Mutation of TERC only accounts for 5% of all cases, and when DC occurs by this mutation, it is inherited as an autosomal dominant disorder. Mutations in the gene Dyskerin (DKCl) account for about 35% of DC cases, and in this case the inheritance pattern is X-linked recessive.

Patients with DC have severe bone marrow failure manifesting as abnormal skin pigmentation, leucoplakia (a white thickening of the oral mucosa), and nail dvstophv. as well as a variety of other symptoms. Individuals with either TERC or DKCl mutations have shorter telomeres and defective telomerase activity in vitro than other individuals of the same age.

There has also been one family in which autosomal dominant DC has been linked to a heterozygous mutation in TERT. These patients also exhibited an increased rate of [edit! Telomerase as a potential drug target

Cancer is a very difficult disease to fight because the immune system cannot recognize it, and cancer cells are immortal; they will always continue dividing. Because telomerase is necessary for the immortality of so many cancer types, it is thought to be a potential drug target. If a drug can be used to turn off telomerase in cancer cells, the above process of telomere shortening will resume—telomere length will be lost as the cells continue to divide, mutations will occur and cell stability will decrease. Experimental drug therapies targeting active telomerase have been tested in mouse models, and some have now entered early clinical trials. Geron Corporation is currently conducting three human clinical trials involving telomerase inhibition using two different approaches. One is a vaccine (GRNVACl) and the other is a lipidated drug (GRNl 63L). Indeed, telomerase suppression in many types of cancer cells grown in culture has led to the massive death of the cell population. However, a variety of caveats, including the presence of the ALT pathway , complicate such therapies. Some have reported ALT methods of telomere maintenance and storage of DNA in cancer stem cells, however Geron claims to have killed cancer stem cells with their telomerase inhibitor GRNl 63L at Johns Hopkins. GRNl 63L binds directly to the RNA template of telomerase. Even a mutation of the RNA template of telomerase would render the telomerase unable to extend telomeres, and therefore not be able to grant replicative immortality to cancer, not allow glycolysis to be inititated, and not upregulate Blackburn's 70 cancer genes. (Blackburn et al) Since Blackburn has shown that most of the bad effects of telomerase are dependent on an intact RNA template, it seems a very worthwhile target. If indeed some cancer stem cells use an alternative method of telomere maintenance, it should be noted that they are still killed when the RNA template of telomerase is blocked. According to Blackburn's opinion at most of her lectures, it is a big mistake to think that telomerase is only involved with extending telomeres. Stopping glycolysis in cancer stem cells and preventing the upregulation of 70 bad genes is probably what is killing cancer stem cells if they are using ALT methods. [edit] References

1. Greider, CW. & Blackburn, E.H. (1985) "Identification of a specific telomere terminal transferase activity in Tetrahymena extracts." Cell v.43, (2 Pt. 1) pp. 405-413. 2. Cohen S, Graham M, Lovrecz G, Bache N, Robinson P, Reddel R (2007). "Protein composition of catalytically active human telomerase from immortal cells". Science 315 (5820): 1850-3. PMID 17395830. 3. A Blasco MA. Telomeres and human disease: ageing, cancer and beyond. Nat Rev Genet. 2005 Aug;6(8):611-22. PMID 16136653 4. fjYamaguchi H, Calado RT, Ly H, Kajigaya S, Baerlocher GM, Chanock SJ, Lansdorp PM, Young NS. Mutations in TERT, the gene for telomerase reverse transcriptase, in aplastic anemia. N EnglJ Med. 2005 Apr 7;352(14):1413-24. PMID 15814878 Free text after registration 5. fllZhang A, Zheng C, Hou M, Lindvall C, Li KJ, Erlandsson F, Bjorkholm M, Gruber A, Blennow E, Xu D.Deletion of the telomerase reverse transcriptase gene and haploinsufficiency of telomere maintenance in Cri du chat syndrome. Am J Hum Genet. 2003 Apr;72(4):940-8. Epub 2003 Mar 10. PMID 12629597 Cohen AR, Chakravarti A, Hamosh A, Greider CW. Haploinsufficiency of telomerase reverse transcriptase leads to anticipation in autosomal dominant dyskeratosis congenita. Proc Natl Acad Sci USA. 2005 Nov 1; 102(44): 15960-4. PMID 16247010 8. tferyan TM, Englezou A, Gupta J, Bacchetti S, Reddel RR. Telomere elongation in immortal human cells without detectable telomerase activity. EMBOJ. 1995 Sep l;14(17):4240-8. PMID 7556065

~~[edit] See also~~

~~DNA repair feditl External links~~

• Telomerase.org - A bloe-style discussion of telomerase and anti-aging therapies. • Three-dimensional model of telomerase at MUN • MeSH Telomerase CLAIMS autologous anti-aging method comprising: cc. Periodic collection of stem cells and/or bone marrow from a donor from after conception to an adult chronological age, dd. Providing for the long term storage of said donor-recipient stem cells and/or bone marrow in sterile conditions in containers, ee. Thawing a portion of said stored stem cells and/or bone marrow after a period of storage, and ff. Periodic and regular infusions or infusion of said stem cells and/or bone marrow into the donor starting after age 10 years, whereby said periodic infusions result in an average biological age of the new body comprised of replaced tissues from the donor DNA at least 5 years younger than the actual chronological age of said recipient. INTERNATIONAL SEARCH REPORT International application No PCT/US 08/04738

A CLASSIFICATION OF SUBJECT MATTER IPC(8) - A01 N 63/00; A01 N 65/00; C 12N 5/08 (2008.04) USPC - 424/93.7; 435/372 According to International Patent Classification (IPC) or to both national classification and IPC

~~B FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) 1PC(8) - A 01N 63/00, A01N 65/00, C12N 5/08 (2008 04) USPC - 424/93 7, 435/372~~

~~Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched USPC - 435/372, 374~~

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) PubWEST(USPT,PGPB,EPAB,JPAB), Google Patents, Google Scholar Search terms stem cell, anti-aging, collect or harvest, store, autologous, Infusion, bone marrow, periodic

~~C DOCUMENTS CONSIDERED TO BE RELEVANT~~

~~Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No~~

~~US 2004/0151706 A 1 (SHAKHOV et al ) 5 August 2004 (05 08 2004) para [0023], [0029]- [0031], [0036], [0038] and [0044]~~

~~I Further documents are listed in the continuation of Box C |~~

* Special categories of cited documents "T" later document published after the international filing date or priority "A" document defining the general state of the art which is not considered date and not in conflict with the application but cited to understand to be of particular relevance the principle or theory underlying the invention "E" earlier application or patent but published on or after the international "X" document of particular relevance, the claimed invention cannot be filing date considered novel or cannot be considered to involve an inventive "L" document which may throw doubts on priority claιm(s) or which is step when the document is taken alone cited to establish the publication date of another citation or other "Y" document of particular relevance, the claimed invention cannot be special reason (as specified) considered to involve an inventive step when the document is "O" document referring to an oral disclosure, use, exhibition or other combined with one or more other such documents, such combination means being obvious to a person skilled in the art "P" document published prior to the international filing date but later than "&" document member of the same patent family the priority date claimed Date of the actual completion of the international search Date of mailing of the international search report

~~29 June 2008 (29 06 2008) 1 8 AUG 2008~~