US 20060010509A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0010509 A1 Johnson et al. (43) Pub. Date: Jan. 12, 2006
(54) METHODS AND COMPOSITIONS FOR Related U.S. Application Data PRODUCING GERM CELLS FROM BONE MARROW DERVED GERMLINE STEM (60) Provisional application No. 60/572,222, filed on May CELLS 17, 2004. Provisional application No. 60/574,187, filed on May 24, 2004. Provisional application No. (76) Inventors: Joshua Johnson, New Haven, CT 60/586,641, filed on Jul. 9, 2004. (US); Jonathan L. Tilly, Windham, NH (US) Publication Classification (51) Int. Cl. Correspondence Address: A0IK 67/027 (2006.01) EDWARDS & ANGELL, LLP CI2N 5/06 (2006.01) P.O. BOX 55874 (52) U.S. Cl...... 800/18; 435/354 BOSTON, MA 02205 (US) (57) ABSTRACT (21) Appl. No.: 11/131,153 The present invention relates to the use of bone marrow derived germline Stem cells and their progenitors, methods (22) Filed: May 17, 2005 of isolation thereof, and methods of use thereof. Patent Application Publication Jan. 12, 2006 Sheet 1 of 13 US 2006/0010509 A1
F.G. 1A
F.G. 1B
F.G. 1C Patent Application Publication Jan. 12, 2006 Sheet 2 of 13 US 2006/0010509 A1
F.G. 1E Patent Application Publication Jan. 12, 2006 Sheet 3 of 13 US 2006/0010509 A1 BM Mock Ovary
Dazil
Stella
Fragilis
Nobox
FIG. 2A
FG. 2B Patent Application Publication Jan. 12, 2006 Sheet 4 of 13 US 2006/0010509 A1
FG. 2C
FIG. 2D Patent Application Publication Jan. 12, 2006 Sheet 5 of 13 US 2006/0010509 A1 Peripheral blood Mh levels
MM levels
O. 3
7.5 2
5.0
2.5
O.0 O Estrus(E) Metostrus:E Dio strus:E Proostrus:E
FIG. 2E
Number of non-atretic primordial follocles per ovary
Estrug to strus Diestrus Prootstrus
FIG. 2F Patent Application Publication Jan. 12, 2006 Sheet 6 of 13 US 2006/0010509 A1
MMh expression (fold difference versus BM) 12
BM i in Other Sca-1 C.Kitt FG. 3A
BM P3 Mock
MWh
Dazil
Stella
Fragilis
Nobox Patent Application Publication Jan. 12, 2006 Sheet 7 of 13 US 2006/0010509 A1
Nurnber of Or-atretic immature follotes per ovary
500 400 300
O I-8 X 2OO X - IS X Xay. &
100 es E.
e. O Prinordial Primary Small Preantral Total
FG. 4 Patent Application Publication Jan. 12, 2006 Sheet 8 of 13 US 2006/0010509 A1
FIG. 5A FIGSB FG, SC
Patent Application Publication Jan. 12, 2006 Sheet 9 of 13 US 2006/0010509 A1
F.G. 6A F.G. 6B Wild-type Atm -/-
Patent Application Publication Jan. 12, 2006 Sheet 10 of 13 US 2006/0010509 A1
F.G. 6E F.G. 6F
Patent Application Publication Jan. 12, 2006 Sheet 11 of 13 US 2006/0010509 A1
- a rare.' ' trail ......
Octa
V
DaZl
Stella
FG. 7 Patent Application Publication Jan. 12, 2006 Sheet 12 of 13 US 2006/0010509 A1
FG. 8 Patent Application Publication Jan. 12, 2006 Sheet 13 of 13 US 2006/0010509 A1 BM 1 2 3 4 Mock Ut 1 Ut 2
FG. 9 US 2006/0010509 A1 Jan. 12, 2006
METHODS AND COMPOSITIONS FOR upon injection of bone marrow cells obtained from the donor PRODUCING GERM CELLS FROM BONE rat. Therefore, it was not believed that bone marrow derived MARROW DERVED GERMLINE STEM CELLS cells could Successfully repopulate the germline of the mammalian gonads. RELATED APPLICATIONS/PATENTS & INCORPORATION BY REFERENCE SUMMARY OF THE INVENTION 0001. This application claims priority to U.S. Application 0006. It has now been shown that bone marrow derived Ser. No. 60/572,222, filed on May 17, 2004 as Attorney germline Stem cells can repopulate the germline of repro Docket No. 910000-3073, U.S. Application Ser. No. 60/574, ductive organs, and thus restore gonadal function. Methods 187, filed on May 24, 2004 as Attorney Docket No. 910000 of the invention relate to the use of bone marrow derived 3074, and U.S. Application Ser. No. 60/586,641, filed on Jul. germline Stem cells and their progenitors to, among other 9, 2004 as Attorney Docket No. 910000-3076, the contents things, replenish or expand germ cell reserves of the testes each of which are incorporated herein by reference. and ovary, to enhance or restore fertility, and in females, to ameliorate Symptoms and consequences of menopause. STATEMENT OF POTENTIAL GOVERNMENT INTEREST 0007. In one aspect, the present invention provides com positions comprising bone marrow derived female germline 0002 The United States government may have certain Stem cells. rights in this invention by virtue of grant numbers R01 AG12279 and RO1-AG24999 from the National Institute on 0008. In one embodiment, the present invention provides Aging of the National Institutes of Health. compositions comprising bone marrow derived female ger mline Stem cells, wherein the cells are mitotically competent 0003. Each of the applications and patents cited in this and express Oct 4, Vasa, Dazl, Stella, Fragilis, and option text, as well as each document or reference cited in each of ally Nobox, c-Kit and Sca-1. Consistent with their mitoti the applications and patents (including during the prosecu cally competent phenotype, bone marrow derived female tion of each issued patent; "application cited documents”), germline Stem cells of the invention do not express growth/ and each of the PCT and foreign applications or patents differentiation factor-9 (“GDF-9"), Zona pellucida proteins corresponding to and/or claiming priority from any of these (e.g., Zona pellucida protein-3, "ZP3'), histone deacety applications and patents, and each of the documents cited or lase-6 (“HDAC6') and synaptonemal complex protein-3 referenced in each of the application cited documents, are (“SCP3”). Upon transplantation into a host, bone marrow hereby expressly incorporated herein by reference, and may derived female germline Stem cells of the invention can be employed in the practice of the invention. More generally, produce oocytes after a duration of at least 1 week, more documents or references are cited in this text, either in a preferably 1 to about 2 weeks, about 2 to about 3 weeks, Reference List before the claims, or in the text itself; and, about 3 to about 4 weeks or more than about 5 weeks post each of these documents or references (“herein cited refer transplantation. ences”), as well as each document or reference cited in each of the herein cited references (including any manufacturers 0009. In another aspect, the present invention provides Specifications, instructions, etc.), is hereby expressly incor compositions comprising progenitor cells derived from bone porated herein by reference. marrow derived female germline Stem cells. In one embodi ment, the present invention provides compositions compris BACKGROUND OF THE INVENTION ing bone marrow derived female germline Stem cell pro genitors, wherein the cells express Oct 4, Vasa, Dazl, Stella, 0004. A basic doctrine of reproductive biology, which Fragilis, and optionally Nobox, c-Kit and Sca-1 and wherein States that mammalian females lose the capacity for germ the cells do not express GDF-9, Zona pellucida proteins, cell renewal during fetal life, has only recently been Suc HDAC6 and SCP3. Upon transplantation into a host, bone cessfully challenged by Johnson et al., (2004) Nature 428: marrow derived female germline Stem cell progenitors of the 145. Johnson et al. are the first to conclusively demonstrate invention can produce oocytes after a duration of less than that juvenile and adult mouse ovaries possess mitotically 1 week, preferably about 24 to about 48 hours post trans active germ cells that, based on rates of oocyte degeneration plantation. and clearance, Sustain oocyte and follicle production in the postnatal mammalian ovary. However, it remains unclear 0010. In another embodiment, the present invention pro whether the precursors of germ cells are confined exclu vides an isolated bone marrow cell, wherein the cell is Sively to the Ovaries or whether extra-ovarian Sites in the mitotically competent and expresses Oct 4, Vasa, Dazl, body, contain precursors having the ability to form germ Stella, Fragilis, and optionally Nobox, c-Kit and Sca-1. cells. Preferably, the cell is a bone marrow derived female germ line Stem cell, or its progenitor cell, having an XX karyo 0005 Previously, Green and Bernstein (1970) Int. J. type. Preferably, the bone marow derived female germline Radiat. Biol. Vol. 17(1): 87, had attempted to show that cells not derived from reproductive organs can repopulate the Stem cells, or their progenitor cells, are non-embryonic, testicular germinal epithelium in a Series of bone marrow mammalian, and even more preferably, human. inoculation experiments. In these experiments, a male test 0011. In another embodiment, the present invention pro rat, which was sterilized by whole-body irradiation, received vides purified populations of bone marrow derived female injections of bone marrow from a donor rat, which was germline Stem cells and/or their progenitor cells. In specific Sterilized by testes-specific irradiation. These experiments embodiments, the purified population of cells is about 50 to failed to provide evidence that germinal epithelium of the about 55%, about 55 to about 60%, about 65 to about 70%, test rat could be repopulated to reinitiate Spermatogenesis about 70 to about 75%, about 75 to about 80%, about 80 to US 2006/0010509 A1 Jan. 12, 2006
about 85%, about 85 to about 90%, about 90 to about 95% 0018. In yet another embodiment, the invention provides or about 95 to about 100% of the cells in the composition. a method for oocyte production, comprising culturing a bone marrow derived female germline Stem cell, or its progenitor 0012. In yet another embodiment, the present invention cell, in the presence of an agent that differentiates a bone provides pharmaceutical compositions comprising bone marrow derived female germline Stem cell, or its progenitor marrow derived female germline Stem cells, and/or their cell, into an oocyte, thereby producing an oocyte. In a progenitor cells, and a pharmaceutically acceptable carrier. preferred embodiment, the agent includes, but is not limited The pharmaceutical compositions can comprise purified to, a hormone or growth factor (e.g., a TGF, BMP or Wnt populations of bone marrow derived female germline Stem family protein, kit-ligand (“SCF) or leukemia inhibitory cells and/or their progenitor cells. factor ("LIF)), a signaling molecule (e.g., meiosis-activat 0013 Compositions comprising bone marrow derived ing Sterol, “FF-MAS”), or a pharmacologic or pharmaceu female germline Stem cells of the invention can be provided tical agent (e.g., a modulator of Id protein function or by direct administration to ovarian tissue, or indirect admin Snail/Slug transcription factor function). istration, for example, to the circulatory System of a Subject 0019. In yet another embodiment, the invention provides (e.g., to the extra-ovarian circulation). a method for in vitro fertilization of a female subject, said 0.014. In yet another aspect, the invention provides meth method comprising the Steps of ods for manipulating bone marrow derived germline Stem 0020) a) producing an oocyte by culturing a bone cells, or their progenitor cells, in Vivo, eX Vivo or in Vitro as marrow derived female germline Stem cell, or its pro described herein below. genitor, in the presence of an oocyte differentiation 0.015. In one embodiment, the invention provides a agent, method for expanding bone marrow derived female germ line Stem cells, or their progenitor cells, in Vivo, ex vivo or 0021 b) fertilizing the oocyte in vitro to form a zygote; in vitro, comprising contacting bone marrow derived female and germline Stem cells, or their progenitor cells, with an agent 0022 c) implanting the Zygote into the uterus of a that increases the amount of bone marrow derived female female Subject. germline Stem cells, or their progenitor cells, by promoting proliferation or Survival thereof, thereby expanding the bone 0023. In yet another embodiment, the invention provides marrow derived female germline stem cells, or their pro a method for in vitro fertilization of a female Subject, said genitor cells. In a preferred embodiment, the agent includes, method comprising the Steps of but is not limited to, a hormone or growth factor (e.g., insulin-like growth factor (“IGF'), transforming growth 0024 a) producing an oocyte by contacting a bone factor (“TGF'), bone morphogenic protein (“BMP”), Wnt marrow derived female germline Stem cell, or its pro protein, or fibroblast growth factor (“FGF)), a cell-signal genitor cell, with an agent that differentiates said cell(s) ing molecule (e.g., Sphingosine-1-phosphate ("S1P), or into an oocyte, retinoic acid (“RA)), or a pharmacological or pharmaceu tical compound (e.g., an inhibitor of glycogen Synthase 0025 b) fertilizing the oocyte in vitro to form a zygote; kinase-3 (“GSK-3”), an inhibitor of apoptosis such as a Bax and inhibitor or a caspase inhibitor, an inhibitor of nitric oxide 0026 c) implanting the Zygote into the uterus of a production, or an inhibitor of HDAC activity). female Subject. 0016. In another embodiment, the invention provides a 0027. In yet another embodiment, the invention provides method for identifying an agent that promoteS proliferation a method for identifying an agent that induces differentiation or Survival of a bone marrow derived female germline Stem of a bone marrow derived female germline Stem cell, or its cell, or its progenitor cell, comprising contacting the bone progenitor cell, into an oocyte comprising contacting bone marrow derived female germline Stem cells, or their pro marrow derived female germline Stem cells, or their pro genitor cells, with a test agent; and detecting an increase in genitor cells, with a test agent; and detecting an increase in the number of bone marrow derived female germline stem the number of oocytes, thereby identifying an agent that cells, or their progenitor cells, thereby identifying an agent induces differentiation of a bone marrow derived female that promoteS proliferation or Survival of a bone marrow germline Stem cell, or its progenitor. derived female germline Stem cell, or its progenitor cell. 0028. In yet another embodiment, the present invention 0.017. In yet another embodiment, the invention provides provides a method for oocyte production, comprising pro a method for using the female germline Stem cells, or their Viding a bone marrow derived female germline Stem cell, or progenitor cells, to characterize pharmacogenetic cellular its progenitor cell, to a tissue, preferably the ovary, wherein responses to biologic or pharmacologic agents, comprising the cell engrafts into the tissue and differentiates into an isolating bone marrow derived female germline Stem cells, oocyte, thereby producing an oocyte. or their progenitor cells, from a population of Subjects, expanding Said cells in culture to establish a plurality of cell 0029. In yet another embodiment, the present invention cultures, optionally differentiating Said cells into a desired provides a method for inducing folliculogenesis, comprising lineage, contacting the cell cultures with one or more providing a bone marrow derived female germline Stem cell, biologic or pharmacologic agents, identifying one or more or its progenitor cell, to a tissue, preferably the ovary, cellular responses to the one or more biologic or pharma wherein the cell engrafts into the tissue and differentiates cologic agents, and comparing the cellular responses of the into an oocyte within a follicle, thereby inducing folliculo cell cultures from different Subjects. genesis. US 2006/0010509 A1 Jan. 12, 2006
0.030. In yet another embodiment, the present invention progenitor cells, to an ovary of the Subject, wherein the cells provides a method for treating infertility in a female Subject engraft into the Ovary and differentiate into oocytes within in need thereof comprising administering a therapeutically the Ovary, thereby restoring ovarian function in the Subject. effective amount of a composition comprising bone marrow derived female germline Stem cells, or their progenitor cells, 0035) In yet another embodiment, the present invention to the Subject, wherein the cells engraft into a tissue, provides a method for restoring ovarian function in a meno preferably ovarian tissue, and differentiate into oocytes, pausal female Subject, comprising administering a therapeu thereby treating infertility. Except where expressly Stated tically effective amount of a composition comprising bone herein, the female Subject in need of fertility treatment is not marrow derived female germline Stem cells, or their pro a Subject who has undergone prior chemotherapy or radio genitor cells, to the Subject, wherein the cells engraft into the therapy. ovary and differentiate into oocytes, thereby restoring ova rian function. The menopausal female Subject can be in a 0031. In yet another embodiment, the present invention Stage of either peri- or post-menopause, with Said meno provides a method for restoring fertility to a female Subject pause caused by either normal (e.g., aging) or pathological having undergone chemotherapy or radiotherapy (or both (e.g., Surgery, disease, ovarian damage) processes. treatments) and who desires restored fertility, comprising 0036 Restoration of ovarian function can relieve adverse administering a therapeutically effective amount of bone Symptoms and complications associated with menopausal marrow derived female germline Stem cells, or their pro disorders, including, but not limited to, Somatic disorders genitor cells, to the Subject, wherein the cells engraft into a Such as Osteoporosis, cardiovascular disease, Somatic Sexual tissue, preferably ovarian tissue, and differentiate into dysfunction, hot flashes, vaginal drying, sleep disorders, oocytes, thereby restoring fertility in the subject. Preferably, depression, irritability, loSS of libido, hormone imbalances, the bone marrow derived female germline Stem cells com and the like, as well as cognitive disorders, Such as loSS of prise a purified Sub-population of cells obtained from the memory; emotional disorders, depression, and the like. bone marrow. Chemotherapeutic drugs include, but are not limited to, buSulfan, cyclophosphamide, 5-FU, vinblastine, 0037 Methods of the present invention can be used in the actinomycin D, etopoSide, cisplatin, methotrexate, doxoru production of other reproductive cell types. Accordingly, in bicin, among others. Radiotherapy includes, but is not yet another aspect, the present invention provides compo limited to, ionizing radiation, ultraViolet radiation, X-rayS, Sitions comprising bone marrow derived male germline Stem and the like. cells, wherein the bone marrow derived male germline Stem cells are mitotically competent and express Vasa and Dazl. 0032. In yet another embodiment, the present invention Bone marrow derived male germline Stem cells of the provides a method for protecting fertility in a female Subject invention carry an XY karyotype, whereas bone marrow undergoing or expected to undergo chemotherapy or radio derived female germline Stem cells of the invention carry an therapy (or both treatments), comprising providing an agent XX karyotype. Preferably, the bone marrow derived male that protects against reproductive injury prior to or concur germline Stem cells are non-embryonic, mammalian, and rently with chemotherapy or radiotherapy (or both treat even more preferably, human. ments) and providing a bone marrow derived female germ line Stem cell, or its progenitor cell, to the Subject, wherein 0038. In one embodiment, the invention provides an the cell engrafts into a tissue, preferably ovarian tissue, and isolated bone marrow cell that is mitotically competent, has differentiates into an oocyte, thereby protecting fertility in an XY kayrotype and expresses Vasa and Dazl. the Subject. The protective agent can be S1P, a Bax antago 0039. In another embodiment, the present invention pro nist, or any agent that increaseS SDF-1 activity. vides a method for restoring or enhancing Spermatogenesis, 0033. In yet another embodiment, the present invention comprising providing a bone marrow derived male germline provides a method for repairing damaged ovarian tissue, Stem cell, or its progenitor cell, to the testes of a male comprising providing a therapeutically effective amount of Subject, wherein the cell engrafts into the Seminiferous a composition comprising bone marrow derived female epithelium and differentiates into a Sperm cell, thereby germline Stem cells, or their progenitor cells, to the tissue, restoring or enhancing Spermatogenesis. wherein the cells engraft into the tissue and differentiate into 0040. In yet another embodiment, the present invention oocytes, thereby repairing the damaged tissue. Damage can provides a method for restoring fertility to a male Subject be caused, for example, by exposure to cytotoxic factors, having undergone chemotherapy or radiotherapy (or both) hormone deprivation, growth factor deprivation, cytokine and who desires restored fertility, comprising administering deprivation, cell receptor antibodies, and the like. Except a therapeutically effective amount of bone marrow derived where expressly Stated herein, the damage is not caused by male germline Stem cells, or their progenitor cells, to the prior chemotherapy or radiotherapy. Damage can also be Subject, wherein the cells engraft into the Seminiferous caused be diseases that affect ovarian function, including, epithelium and differentiate into Sperm cells, thereby restor but not limited to cancer, polycystic ovary disease, genetic ing fertility. disorders, immune disorders, metabolic disorders, and the like. 0041. In yet another embodiment, the invention provides a method for reducing the amount of bone marrow derived 0034. In yet another embodiment, the present invention germline Stem cells, or their progenitor cells, in Vivo, ex vivo provides a method for restoring ovarian function in a female or in Vitro, comprising contacting bone marrow derived Subject having undergone chemotherapy or radiotherapy (or germline Stem cells, or their progenitor cells, with an agent both treatments) and who desires restored ovarian function, that reduces cell proliferation, thereby reducing the amount comprising administering a therapeutically effective amount of bone marrow derived germline Stem cells, or their pro of bone marrow derived female germline Stem cells, or their genitor cells. In a preferred embodiment, the agent includes, US 2006/0010509 A1 Jan. 12, 2006 but is not limited to, a hormone or growth factor (e.g., differentiates a bone marrow derived female germline Stem TGF-3), a peptide antagonist of mitogenic hormones or cell, or its progenitor cell, into an oocyte and instructions for growth factors (e.g., the BMPantagonists, Protein Related to using the agent to differentiate a bone marrow derived DAN and Cerberus (“PRDC) and Gremlin), or a pharma female germline Stem cell, or its progenitor cell, into an cological or pharmaceutical compound (e.g., a cell cycle oocyte in accordance with the methods of the invention. inhibitor or an inhibitor of growth factor signaling). 0048. In yet another embodiment, the present invention 0042. In yet another embodiment, the invention provides provides a kit for oocyte production, comprising an agent a method for reducing the amount of bone marrow derived that increases the amount of bone marrow derived female germline Stem cells, or their progenitor cells, in Vivo, ex vivo germline Stem cells, or their progenitor cells, by promoting or in Vitro, comprising contacting bone marrow derived proliferation or Survival thereof, and instructions for using germline Stem cells, or their progenitor cells, with an agent the agent to increase the amount of bone marrow derived that inhibits cell Survival or promotes cell death, thereby female germline Stem cells or their progenitor cells, thereby reducing the amount of bone marrow derived germline Stem producing oocytes in accordance with the methods of the cells, or their progenitor cells. In a preferred embodiment, invention. the agent the that inhibits cell Survival includes, but is not limited to, a hormone, growth factor or cytokine (e.g., a 0049. In yet another embodiment, the present invention pro-apoptotic tumor necrosis factor (“TNF") Super family provides a kit for oocyte production comprising an agent member such as TNF-C., Fas-ligand (“FasL') and TRAIL), that differentiates bone marrow derived female germline an antagonist of pro-Survival Bcl-2 family member function, Stem cells, or their progenitor cells, into oocytes and instruc a signaling molecule (e.g., a ceramide), or a pharmacologi tions for using the agent to differentiate the bone marrow cal or pharmaceutical compound (e.g., an inhibitor of derived female germline Stem cells, or their progenitor cells, growth factor Signaling). In a preferred embodiment, the into oocytes, thereby producing oocytes in accordance with agent the that promotes cell death includes, but is not limited the methods of the invention. to, a pro-apoptotic tumor necrosis factor Superfamily mem 0050. In yet another embodiment, the present invention ber (e.g., TNF-C, FasL and TRAIL), agonist of pro-apop provides a kit for treating infertility in a female Subject in totic Bcl-2 family member function and ceramide. need thereof comprising an agent that increases the amount 0043. In yet another embodiment, the invention provides of bone marrow derived female germline Stem cells, or their a method for identifying an agent that reduces proliferation progenitor cells, by promoting proliferation or Survival or Survival, or promotes cell death, of a bone marrow thereof and instructions for using the agent to increase the derived germline Stem cell, or its progenitor cell, comprising amount of bone marrow derived female germline Stem cells contacting bone marrow derived germline Stem cells, or or their progenitor cells, thereby treating infertility in the their progenitor cells, with a test agent, and detecting a Subject in accordance with the methods of the invention. decrease in the number of bone marrow derived germline 0051. In yet another embodiment, the present invention Stem cells, or their progenitor cells, thereby identifying an provides a kit for treating infertility in a female Subject in agent that reduces proliferation or Survival, or promotes cell need thereof comprising an agent that differentiates bone death, of a female germline Stem cell, or its progenitor cell. marrow derived female germline Stem cells, or their pro 0044) In yet another embodiment, the present invention genitor cells, into oocytes, and instructions for using the provides a method for contraception in a male or female agent to differentiate bone marrow derived female germline Subject comprising contacting bone marrow derived germ Stem cells, or their progenitor cells, into oocytes, thereby line Stem cells, or their progenitor cells, of the Subject with treating infertility in the Subject in accordance with the an agent that decreases the proliferation, function or Survival methods of the invention. of bone marrow derived germline Stem cells, or their pro 0052. In yet another embodiment, the present invention genitor cells, or the ability of Said cells to produce new provides a kit for protecting fertility in a female Subject oocytes or Sperm cells or other Somatic cell types required undergoing or expected to undergo chemotherapy or radio for fertility, thereby providing contraception to the Subject. therapy (or both treatments), comprising an agent that that 0.045. In yet another aspect, the present invention pro protects bone marrow derived female germline Stem cells, or vides kits for use in employing various agents of the their progenitor cells, against reproductive injury and invention. instructions for using the agent to protect bone marrow derived female germline Stem cells, or their progenitor cells, 0046. In one embodiment, the present invention provides against reproductive injury thereby protecting fertility in the a kit for expanding a bone marrow derived female germline female Subject in accordance with the methods of the Stem cell, or its progenitor cell, in Vivo, eX Vivo or in vitro, invention. comprising an agent that promotes cell proliferation or survival of the bone marrow derived female germline stem 0053. In yet another embodiment, the present invention cell, or its progenitor cell, and instructions for using the provides a kit for restoring ovarian function in a post agent to promote cell proliferation or Survival of the bone menopausal female Subject comprising an agent that marrow derived female germline Stem cell, or its progenitor, increases the amount of bone marrow derived female ger thereby expanding a female germline Stem cell, or its mline Stem cells, or their progenitor cells, by promoting progenitor cell in accordance with the methods of the proliferation or Survival thereof and instructions for using the agent to increase the amount of bone marrow derived invention. female germline Stem cells or their progenitor cells, thereby 0047. In another embodiment, the present invention pro restoring ovarian function in the Subject in accordance with vides a kit for oocyte production, comprising an agent that the methods of the invention. US 2006/0010509 A1 Jan. 12, 2006
0054. In yet another embodiment, the present invention (E). The data shown represent the combined results from an provides a kit for restoring ovarian function in a post analysis of 3-4 mice per group, with mean levels at estruS Set menopausal female Subject comprising an agent that differ as the reference point for comparisons to other Stages of the entiates bone marrow derived female germline Stem cells, or cycle following normalization against beta-actin for Sample their progenitor cells, into oocytes, and instructions for using loading. For mice in estrus, Mvh expression in bone marrow the agent to differentiate bone marrow derived female ger was detected during linear amplification in only 1 of the 3 mline Stem cells, or their progenitor cells, into oocytes, Samples analyzed. Number of non-atretic primordial oocyte thereby restoring ovarian function in the Subject in accor containing follicles in adult female mice at the indicated dance with the methods of the invention. Stages of the estrous cycle (meantSEM, n=4 mice per group) (F). 0055. In another embodiment, the present invention pro vides a kit for reducing the amount of bone marrow derived 0059 FIG. 3 depicts properties of bone marrow-derived germline Stem cells, or their progenitor cells, in Vivo, ex vivo germ cells. Quantitative analysis of Mvh levels in crude or in vitro, comprising an agent that inhibits cell Survival or (Total) and lineage-depleted (lin-) bone marrow Samples promotes cell death and instructions for using the agent to without or with further fractionation by FACS based on inhibit cell Survival or promote cell death of the bone cell-Surface expression of Sca-1 or c-Kit (A). All remaining marrow derived germline Stem cells, or their progenitor lin- cells not represented in the Sca-1-/c-Kit-- cell fraction cells, thereby the reducing the amount of bone marrow were pooled and analyzed together. The data shown repre derived germline Stem cells, or their progenitor cells, in sent the combined results from an analysis of 3 adult female accordance with the methods of the invention. mice, with mean Mvh levels in the crude bone marrow Sample Set as the reference point for comparisons following 0056. In yet another embodiment, the present invention provides a kit for contraception in a male or female Subject normalization against beta-actin for Sample loading. Germ comprising an agent that decreases the proliferation, func line marker expression in adherent bone marrow-derived tion or Survival of bone marrow derived germline Stem cells, cells following a total of three serial passages (P3) over a or their progenitor cells, or the ability of Said cells to produce six-week period in-vitro. BM, freshly isolated bone marrow. new oocytes or other Somatic cell types required for fertility beta-actin, house-keeping gene. Mock, mock reverse-tran and instructions for using the agent to decrease the prolif scribed RNA samples (B). eration, function or Survival of bone marrow derived germ 0060 FIG. 4 presents results indicating that bone mar line stem cells, or their progenitor cells, or the ability of Said row transplantation (BMT) reverses chemotherapy-induced cells to produce new oocytes or Sperm cells or other Somatic ovarian failure. The number of non-atretic immature fol cell types required for fertility, thereby providing contracep licles present in the ovaries of wild-type female mice 60 tion to the Subject in accordance with the methods of the days after treatment with buSulfan and cyclophosphamide on invention. day 42 postpartum without or with BMT 1 or 7 days later (meantS. E., n=5 mice per group, with 4 of the 5 mice BRIEF DESCRIPTION OF THE DRAWINGS exposed to chemotherapy without subsequent BMT com pletely lacking immature oocytes). 0057 FIG. 1 depicts an analysis of germ cells/progeni tors in adult ovaries. Immunohistochemical analysis of 0061 FIG. 5 presents results indicating that BMT sus SSEA1 expression (red, with nuclei highlighted by pro tains both short and long-term oocyte production in adult pidium iodide in blue) in adult mouse ovaries (B, higher wild-type female mice Sterilized by chemotherapy. Repre magnification of Stage-Specific embryonic antigen 1+ Sentative ovarian histology in adult female mice 2 months (SSEA1) cells shown in A; A and C, ovaries from different after treatment with vehicle and no BMT (A, control), mice). Single SSEA1+ cell in an adult ovary, showing cell combination chemotherapy without BMT (B), or combina Surface expression of the antigen (D). Gene expression tion chemotherapy with BMT performed 7 days later (C). profile of isolated and residual cell fractions prepared from Corpora lutea are denoted by asterisks. Ovarian histology of adult mouse ovaries following SSEA1 antibody-based mag adult wild-type female mice 11.5 months after combination netic bead Sorting (E). The ribosomal gene, L7, was ampli chemotherapy (cyclophosphamide and buSulfan) followed fied as an internal loading control. No product was observed by BMT on day 42 postpartum. Follicles at various stages of in any mock reverse-transcribed (Mock) ovarian RNA maturational development are highlighted (insets) (D-E). Samples. 0062 FIG. 6 depicts the histology of postpartum day 4 0.058 FIG. 2 indicates that bone marrow contains germ wild-type (FIG. 6A, magnified in FIG. 6C) and Atm-null cells. Germline marker expression in bone marrow (BM) of (FIG. 6B, magnified in FIG. 6D) ovaries. Representative adult wild-type female mice (A). As a positive control for histology of adult wild-type (FIG. 6E) and Atm-null (FIG. expression of germline markers, adult mouse ovary RNA 6F) ovaries from adult mice is also shown. was analyzed in parallel. L7, house-keeping gene. Mock, 0063 FIG. 7 depicts expression of germline marker mock reverse-transcribed RNA samples (B-C). Analysis of genes in Atm deficient mouse ovaries by RT-PCR analysis of MVH immunoreactivity (red, with nuclei highlighted by Octá, Mvh (Vasa), Dazl and Stella expression in ovaries of propidium iodide in blue, Scale bar=5 mm) in bone marrow adult Atm-null (-/-) female mice. The ribosomal gene, L7, of adult wild-type female mice (D). Mouse ovary analyzed was amplified as an internal loading control; no product was in parallel as a positive control for the immunostaining observed in mock reverse-transcribed (Mock) ovarian RNA shown in B-C, demonstrating a restricted expression of Samples. MVH (red) to germ cells (oocytes). Real-time PCR analysis of Mvh levels in bone marrow or peripheral blood of adult 0064 FIG. 8 depicts ovaries in a chemotherapy condi female mice during the indicated Stages of the estrous cycle tioned (buSulfan, cyclophosphamide) Atm-null mouse that US 2006/0010509 A1 Jan. 12, 2006 received exogenous, wild-type bone marrow. Both the wild 0073. “Engraft” refers to the process of cellular contact type mouse and the Atm-null mouse that received eXog and incorporation into an existing tissue of interest (e.g., enous, wild-type bone marrow after Sterilizing doses of ovary) in vivo. chemotherapy exhibited normal oocytes within normal 0074) “Agents” refer to cellular (e.g., biologic) and phar appearing follicles. maceutical factors, preferably growth factors, cytokines, 0065 FIG. 9 depicts analysis of germline markers in hormones or Small molecules, or to genetically-encoded bone marrow of humans. Expression of DAZL and STELLA products that modulate cell function (e.g., induce lineage in bone marrow collected from 4 human female donors commitment, increase expansion, inhibit or promote cell between 24-36 years of age. As a negative control, germline growth and Survival). For example, "expansion agents' are markers were not detected in two different adult human agents that increase proliferation and/or Survival of bone uterine (Ut) endometrial Samples analyzed in parallel. Glyc marrow derived germline stem cells. “Differentiation eraldehyde 3 phosphate dehydrogenase (GAPDH), a house agents' are agents that induce bone marrow derived germ keeping gene, amplified as an internal loading control. line Stem cells to differentiate into committed cell lineages, Mock, mock reverse-transcribed RNA samples. Such as oocytes and Sperm cells. DETAILED DESCRIPTION OF THE 0075 A“follicle' refers to an ovarian structure consisting INVENTION of a single oocyte Surrounded by Somatic (granulosa without or with theca-interstitial) cells. Somatic cells of the gonad Definitions enclose individual oocytes to form follicles. Each fully 0.066 “Bone marrow derived germline stem cells” are formed follicle is enveloped in a complete basement mem any multipotent cells obtained from bone marrow that brane. Although some of these newly formed follicles start to grow almost immediately, most of them remain in the include a population of female or male germline Stem cells. resting stage until they either degenerate or Some signal(s) 0067) “Expansion” refers to the propagation of a cell or activate(s) them to enter the growth phase. For reviews on cells without terminal differentiation. “Isolation phenotype” ovarian Structure, function and physiology, See Gougeon, A., refers to the Structural and functional characteristics of the (1996) Endocr Rev. 17:121-55; Anderson, L. D., and Hir bone marrow derived germline Stem cells upon isolation. shfield, A. N. (1992) Md Med J. 41: 614-20; and Hirshfield, “Expansion phenotype' refers to the Structural and func A. N. (1991) Int Rev Cytol. 124: 43-101. tional characteristics of the bone marrow derived germline Stem cells during expansion. The expansion phenotype can 0076 A “sperm cell” refers to a male germ cell, in either be identical to the isolation phenotype, or alternatively, the a pre-meiotic (i.e., mitotically competent) or post-meiotic expansion phenotype can be more differentiated than the State of development, including a fully mature Spermato isolation phenotype. Zoan. “Spermatogenesis” is the developmental proceSS by which a Sperm cell is formed. 0068 “Differentiation” refers to the developmental pro ceSS of lineage commitment. A "lineage” refers to a pathway 0.077 “Mitotically competent” refers to a cell that is of cellular development, in which precursor or “progenitor' capable of mitosis, the proceSS by which a cell divides and cells undergo progressive physiological changes to become produces two daughter cells from a single parent cell. a specified cell type having a characteristic function (e.g., nerve cell, muscle cell or endothelial cell). Differentiation 0078. A “non-embryonic” cell refers to a cell that is occurs in Stages, whereby cells gradually become more obtained from a post-natal Source (e.g., infant, child or adult specified until they reach full maturity, which is also referred tissue). to as “terminal differentiation.” A “terminally differentiated 0079 A “subject' is a vertebrate, preferably a mammal, cell' is a cell that has committed to a Specific lineage, and more preferably a primate and Still more preferably a human. has reached the end stage of differentiation (i.e., a cell that Mammals include, but are not limited to, primates, humans, has fully matured). Oocytes are an example of a terminally farm animals, Sport animals, and pets. differentiated cell type. 0080. The term “obtaining” as in “obtaining the agent” is 0069. The term "isolated” as used herein refers to a bone intended to include purchasing, Synthesizing or otherwise marrow derived germline Stem cell or its progenitor cell, in a non-naturally occurring State (e.g., isolated from the body acquiring the agent (or indicated Substance or material). or a biological Sample, Such as bone marrow, from the 0081. The terms “comprises”, “comprising”, are intended body). to have the broad meaning ascribed to them in U.S. Patent 0070) “Progenitor cells” as used herein are germ lineage Law and can mean “includes”, “including” and the like. cells that are 1) derived from germline stem cells of the invention as the progeny thereof which contain a set of Embodiments of the Invention common marker genes; 2) are in an early stage of differen I. Bone Marrow Derived Germline Stem Cells tiation; and 3) retain mitotic capacity. 0082 Methods of the invention relate to the use of bone 0.071) “Progeny” as used herein are all cells derived from marrow derived germline Stem cells, or progenitors of bone bone marrow derived germline Stem cells of the invention, marrow derived germline Stem cells, to restore or increase including progenitor cells, differentiated cells, and termi germ cell production. Methods of the invention can be used nally differentiated cells. to, among other things, enhance or restore fertility, and in 0.072 “Derived from as used herein refers to the process females, to ameliorate Symptoms and consequences of of obtaining a daughter cell. menopause. US 2006/0010509 A1 Jan. 12, 2006
0083. Without wanting to be bound by theory, it is 0088 Stella is a gene expressed in bone marrow derived understood that one or more mechanisms can be involved female germline Stem cells and their progenitor cells. Stella with the ability of bone marrow derived germline stem cells is a novel gene Specifically expressed in primordial germ to repopulate reproductive organs. Female germline Stem cells and their descendants, including oocytes (Bortvin et al. cells have been detected in the bone marrow, which may (2004) BMC Developmental Biology 4(2):1-5). Stella therefore Serve as a reservoir for Stem cells having the encodes a protein with a SAP-like domain and a splicing capacity to repopulate and/or expand the germ cell Supply of factor motif-like structure. Embryos deficient in Stella reproductive organs. Male germline Stem cells also exist in expression are compromised in preimplantation develop the bone marrow of male subjects. Other sub-populations of ment and rarely reach the blastocyst Stage. Thus, Stella is a cells in the bone marrow, Such as hematopoietic Stem cells, may likewise have the ability to repopulate and/or expand maternal factor implicated in early embryogenesis. the germ cell Supply of reproductive organs (Herzog, E. L., 0089 Dazl is a gene expressed in bone marrow derived et al., (2004) Blood 102(10): 3483), for example, through female germline Stem cells and their progenitor cells. The de-differentiation into a multipotent progenitor cell (see U.S. autosomal gene Dazl is a member of a family of genes that Pat. No. 6,090,625) which in turn migrates through periph contain a consensus RNA binding domain and are expressed eral blood to the reproductive tract, engrafts into an organ in germ cells. LOSS of expression of an intact Dazl protein in (e.g., ovary or testes) as a germline stem cell or a progenitor mice is associated with failure of germ cells to complete of a germline Stem cell and differentiates into an oocyte meiotic prophase. Specifically, in female mice null for Dazl, (ovary) or sperm (testis). loSS of germ cells occurs during fetal life at a time coincident 0084. As described herein, germline stem cells have been with progression of germ cells through meiotic prophase. In detected in the bone marrow of male and female Subjects. male mice null for Dazl, germ cells were unable to progreSS Bone marrow derived female germline Stem cells express beyond the leptotene Stage of meiotic prophase I. Thus, in markers including Oct 4, Vasa, Dazl, Stella, Fragilis, and the absence of Dazl, progression through meiotic prophase optionally Nobox, c-Kit and Sca-1. Bone marrow derived is interrupted (Saunders et al. (2003), Reproduction, female germline stem cells are mitotically competent (i.e., 126:589-597). capable of mitosis) and accordingly, do not express GDF-9, 0090 Vasa is a gene expressed in bone marrow derived Zona pellucida proteins (e.g., ZP3), HDAC6 or SCP3. female germline Stem cells and their progenitor cells. Vasa 0085. The present invention also provides bone marrow is a component of the germplasm that encodes a DEAD derived female germline Stem cell progenitors. Bone mar family ATP-dependent RNA helicase (Liang et al. (1994) row derived female germline Stem cell progenitors of the Development, 120:1201-1211; Lasko et al. (1988) Nature, 335:611-167). The molecular function of Vasa is directed to invention can circulate throughout the body and most pref binding target mRNAS involved in germ cell establishment erably can be localized in bone marrow, peripheral blood (e.g., Oskar and NanoS), oogenesis, (e.g., Gruken), and and Ovary. Progenitor cells of the invention express Oct 4, translation onset (Gavis et al. (1996) Development, 110: Vasa, Dazl, Stella, Fragilis, and optionally Nobox, c-Kit and 521-528). Vasa is required for pole cell formation and is Sca-1 but do not express GDF-9, Zona pellucida proteins exclusively restricted to the germ cell lineage throughout the (e.g., ZP3), HDAC6 or SCP3. development. Thus, Vasa is a molecular marker for the germ 0.086 Bone marrow derived female germline stem cells cell lineage in most animal species (Toshiaki et al. (2001) and their progenitor cells have functional distinctions. Upon Cell Structure and Function 26:131-136). Because Vasa has transplantation into a host, bone marrow derived female been associated with inhibition of cell migration, expression germline Stem cells of the invention can produce oocytes of Vasa in progenitor cells of the invention may be differ after a duration of at least 1 week, more preferably 1 to about entially regulated, depending on the migratory State of the 2 weeks, about 2 to about 3 weeks, about 3 to about 4 weeks progenitor. For example, while in the bone marrow, the or more than about 5 weeks post transplantation. Bone progenitor may express Vasa, and while migrating to the marrow derived female germline Stem cell progenitors have reproductive tract, the progenitor may down regulate expres the capacity to generate oocytes more rapidly than bone SO. marrow derived female germline Stem cells. Upon trans plantation into a host, bone marrow derived female germline 0091 Fragilis is a gene expressed in bone marrow Stem cell progenitors of the invention can produce oocytes derived female germline Stem cells and their progenitor after a duration of less than 1 week, preferably about 24 to cells. Fragilis is a putative interferon-inducible gene that codes for a transmembrane protein associated with the about 48 hours post transplantation. acquisition of germ cell competence by epiblast cells (Sai 0.087 Oct-4 is a gene expressed in bone marrow derived tou, M. et al. (2002) Nature 418:293-300). Extraembryonic female germline Stem cells and their progenitor cells. The ectoderm is able to induce fragilis expression in epiblast Oct-4 gene encodes a transcription factor that is involved in tissue. Fragilis is expressed in proximal epiblast at a region the establishment of the mammalian germline and plays a in which primordial germ cell (PGC)-competent cells reside Significant role in early germ cell specification (reviewed in according to clonal analysis (Lawson, KA et al. (1994) In Scholer (1991), Trends Genet. 7(10): 323-329). In the devel Wiley, Chichester (Ciba Foundation Symposium 182): oping mammalian embryo, Oct-4 is downregulated during 68-91). As these proximal cells move to the posterior the differentiation of the epiblast, eventually becoming con proximal region during gastrulation, fragilis expression fined to the germ cell lineage. In the germline, Oct-4 increases within a community of cells at the base of the expression is regulated Separately from epiblast expression. incipient allantoic bud. Cells with the highest expression of Expression of Oct-4 is a phenotypic marker of totipotency fragilis initiate the germ cell-characteristic expression of (Yeom et al. (1996), Development 122: 881-888). TNAP and stella/PGC-7 (Ginsburg, M. et al. (1990) Devel US 2006/0010509 A1 Jan. 12, 2006 opment 110:521-528; Sato, M. et al. (2002) Mech Dev which are gene products that comprise the Zona pellucida of 113:91-94) and show repression of Hox genes. the oocyte. Their expression is regulated by a basic helix loop-helix (bHLH) transcription factor, FIGC. Mice null in 0092 Nobox is a gene that is optionally expressed in FIGC. do not express the Zp genes and do not form primor bone marrow derived female germline Stem cells and their dial follicles (Soyal, S. M., et al (2000) Development 127: progenitor cells Nobox (Newborn Ovary Homeobox) is a 4645-4654). Individual knockouts of the ZP genes result in gene active in ovaries and testes that regulates the transition abnormal or absent Zonae pellucidae and decreased fertility of a primordial germ cell into a primary follicle. Female (Zp 1; Rankin T, et al (1999) Development. 126: 3847-55) mice lacking the Nobox gene lose all of their follicles by 6 or sterility (Zp2, Rankin TL, et al. (2001) Development 128: weeks of life and are essentially menopausal by that time; 1119-26; ZP3, Rankin T et al (1996) Development 122: males have normal testes but are 30% less fertile (Rajkovic, 2903-10). The ZP protein products are glycosylated, and A. et al. (2004) Science 305 (5687): 1157-1159). Nobox Subsequently Secreted to form an extracellular matrix, which appears to govern the activity of genes crucial to the is important for in Vivo fertilization and pre-implantation development of follicles, which hold the immature eggs development. Expression of the ZP proteins is precisely cells or oocytes. These follicles are Supposed to thicken as regulated and restricted to a two-week growth phase of the mouse develops; without Nobox, the follicles do not Oogenesis. Zp mRNA transcripts are not expressed in resting develop, and the oocytes deteriorate. oocytes, however once the oocytes begin to grow, all three 0.093 c-Kit is a gene that is optionally expressed in bone Zp transcripts begin to accumulate. marrow derived female germline Stem cells and their pro genitor cells. c-Kit is a proto-oncogene that encodes a 0097 Bone marrow derived female germline stem cells transmembrane protein tyrosine kinase receptor that is struc and their progenitor cells do not express HDAC6. HDACs, turally similar to the receptors for colony-stimulating fac or histone deacetylases are involved in ovarian follicle tor-1 (CSF-1) and platelet derived growth factor. c-Kit has development. HDAC6 in particular can be detected in rest been found to play a pivotal role in the normal growth and ing germinal vesicle-stage (primordial) oocytes (Verdel, A., differentiation of embryonic melanoblasts. c-kit, and its et al. (2003) Zygote 11: 323-8; FIG. 16). HDAC6 is a class ligand have been demonstrated to be essential to the pro II histone deacetylase and has been implicated as a micro ceSSes of germ cell migration, proliferation and Survival in tubule-associated deactylase (Hubbert, C. et al., (2002) the rodent. The expression of c-kit mRNA and protein is Nature 417: 455-8). HDACs are the target of inhibitors germ cell Specific in human fetal gonads and are consistent including, but not limited to, trichoStatin A and trapoxin, with an important role for the c-kit/kit ligand Signalling both of which are microbial metabolites that induce cell System in germ cell proliferation and Survival in the devel differentiation, cell cycle arrest, and reversal of the trans oping human gonad (Robinson, L.L., et al. (2001) Mol Hum formed cell morphology. Reprod 7(9):845-52). 0098 Bone marrow derived female germline stem cells and their progenitor cells do not express SCP3, consistent 0094 Sca-1 is a gene that is optionally expressed in bone with observations that they are pre-meiotic stem cells (i.e., marrow derived female germline Stem cells and their pro diploid). The synaptonemal complex protein SCP3 is part of genitor cells. Sca-1 (stem cell antigen 1, Ly-6A/E) is an 18 the lateral element of the Synaptonemal complex, a meiosis kDa phosphatidylinositol-anchored protein and member of Specific protein Structure essential for Synapsis of homolo the Ly-6 antigen family. Sca-1 has been used in the isolation gous chromosomes. The Synaptonemal complex promotes of hematopoietic stem cells (purification to homogeneity) pairing and Segregation of homologous chromosomes, influ from mouse bone marrow (Van de Rijn, M. et al. (1989) ences the number and relative distribution of croSSOvers, and Proc. Natl. Acad. Sci. USA 86:4634; Spangrude, G. I. et al. converts croSSOverS into chiasmata. SCP3 is meiosis-specific (1988) Science 241:58). Sca-1"HSCs can be found in the and can form multi-Stranded, croSS-Striated fibers, forming adult bone marrow, fetal liver and mobilized peripheral an ordered, fibrous core in the lateral element (Yuan, L. et al., blood and spleen within the adult animal (Morrison, S.J. et (1998) J. Cell. Biol. 142:331-339). The absence of SCP3 in al. (1997) Proc. Natl. Acad. Sci. USA 94:1908). Addition mice can lead to female germ cell aneuploidy and embryo ally, Sca-1 may be involved in regulating both B and T cell death, possibly due to a defect in Structural integrity of activation (Codias, E. K. et al. (1990) J. Immunol. meiotic chromosomes (Yuan, L. et al., (2002) Science 296: 145:1407). 1115-8). 0.095 Bone marrow derived female germline stem cells and their progenitor cells do not express GDF-9, a gene 0099 Bone marrow derived female germline stem cells expressed in cells that have already Started to differentiate and their progenitor cells can be isolated by Standard means into oocytes. Growth/differentiation factor-9 (GDF-9) is a known in the art for the Separation of Stem cells from the member of the transforming growth factor-B Superfamily, marrow (e.g., cell Sorting). Preferably, the isolation protocol expressed specifically in ovaries. GDF-9 mRNA can be includes generation of a kit"/linfraction that is depleted of found in neonatal and adult oocytes from the primary hematopoietic cells. Additional Selection means based on the one-layer follicle stage until after ovulation (Dong, J. et al unique profile of gene expression (e.g., Vasa, Oct-4, Dazl, (1996) Nature 383: 531-5). Analysis of GDF-9 deficient Stella, Fragilis) can be employed to further purify popula mice reveals that only primordial and primary one-layer tions of cells comprising bone marrow derived female follicles can be formed, but a block beyond the primary germline Stem cells and their progenitor cells. Compositions one-layer follicle Stage in follicular development occurs, comprising bone marrow derived female germline Stem cells resulting in complete infertility. and their progenitor cells can be isolated and Subsequently purified to an extent where they become substantially free of 0.096 Bone marrow derived female germline stem cells the biological Sample from which they were obtained (e.g. and their progenitor cells do not express ZP1, ZP2, and ZP3, bone marrow). US 2006/0010509 A1 Jan. 12, 2006
0100 Bone marrow derived female germline stem cell Sitions can also be lyophilized. The compositions can con progenitors can be obtained from female germline Stem cells tain auxiliary Substances Such as wetting, dispersing, or by, for example, expansion in culture. Thus, the progenitor emulsifying agents (e.g., methylcellulose), pH buffering cells can be cells having an “expansion phenotype.” agents, gelling or Viscosity enhancing additives, preserva tives, flavoring agents, colors, and the like, depending upon II. Administration the route of administration and the preparation desired. 0101 Compositions comprising bone marrow derived Standard texts, such as “REMINGTON'S PHARMACEU germline Stem cells or their progenitor cells can be provided TICAL SCIENCE", 17th edition, 1985, incorporated herein directly to the reproductive organ of interest (e.g., ovary or by reference, may be consulted to prepare Suitable prepara testes). Alternatively, compositions comprising bone mar tions, without undue experimentation. row derived germline Stem cells or their progenitors can be 0106 Various additives which enhance the stability and provided indirectly to the reproductive organ of interest, for Sterility of the compositions, including antimicrobial preser example, by administration into the circulatory System (e.g., Vatives, antioxidants, chelating agents, and buffers, can be to the extra-ovarian circulation). Following transplantation added. Prevention of the action of microorganisms can be or implantation, the cells can engraft and differentiate into ensured by various antibacterial and antifungal agents, for germ cells (e.g., oocytes or Sperm cells). “Engraft refers to example, parabens, chlorobutanol, phenol, Sorbic acid, and the process of cellular contact and incorporation into an the like. Prolonged absorption of the injectable pharmaceu existing tissue of interest (e.g., ovary) in Vivo. Expansion tical form can be brought about by the use of agents delaying and differentiation agents can be provided prior to, during or absorption, for example, aluminum monoStearate and gela after administration to increase production of germ cells in tin. According to the present invention, however, any WVO. vehicle, diluent, or additive used would have to be compat 0102 Compositions of the invention include pharmaceu ible with the bone marrow derived germline stem cells or tical compositions comprising bone marrow derived germ their progenitors. line Stem cells or their progenitor cells and a pharmaceuti 0107 The compositions can be isotonic, i.e., they can cally acceptable carrier. Administration can be autologous or have the same osmotic pressure as blood and lacrimal fluid. heterologous. For example, bone marrow derived germline The desired isotonicity of the compositions of this invention Stem cells, or their progenitor cells, can be obtained from may be accomplished using Sodium chloride, or other phar one Subject, and administered to the same Subject or a maceutically acceptable agents. Such as dextrose, boric acid, different, compatible subject. Sodium tartrate, propylene glycol or other inorganic or 0103 Bone marrow derived germline stem cells of the organic Solutes. Sodium chloride is preferred particularly for invention or their progeny (e.g., progenitors, differentiated buffers containing Sodium ions. progeny and terminally differentiated progeny) can be administered via localized injection, including catheter 0.108 Viscosity of the compositions, if desired, can be administration, Systemic injection, localized injection, intra maintained at the Selected level using a pharmaceutically venous injection, intrauterine injection or parenteral admin acceptable thickening agent. Methylcellulose is preferred istration. When administering a therapeutic composition of because it is readily and economically available and is easy to work with. Other Suitable thickening agents include, for the present invention (e.g., a pharmaceutical composition), it example, Xanthan gum, carboxymethyl cellulose, hydrox will generally be formulated in a unit dosage injectable form ypropyl cellulose, carbomer, and the like. The preferred (Solution, Suspension, emulsion). concentration of the thickener will depend upon the agent 0104 Compositions of the invention can be conveniently Selected. The important point is to use an amount that will provided as Sterile liquid preparations, e.g., isotonic aqueous achieve the selected viscosity. Obviously, the choice of Solutions, Suspensions, emulsions, dispersions, or Viscous Suitable carriers and other additives will depend on the exact compositions, which may be buffered to a Selected pH. route of administration and the nature of the particular Liquid preparations are normally easier to prepare than gels, dosage form, e.g., liquid dosage form (e.g., whether the other Viscous compositions, and Solid compositions. Addi composition is to be formulated into a Solution, a Suspen tionally, liquid compositions are Somewhat more convenient Sion, gel or another liquid form, Such as a time release form to administer, especially by injection. Viscous compositions, or liquid-filled form). on the other hand, can be formulated within the appropriate Viscosity range to provide longer contact periods with Spe 0109 Amethod to potentially increase cell Survival when cific tissues. Liquid or Viscous compositions can comprise introducing the cells into a Subject in need thereof is to incorporate bone marrow derived germline Stem cells or carriers, which can be a Solvent or dispersing medium their progeny (e.g., in Vivo, ex vivo or in vitro derived) of containing, for example, water, Saline, phosphate buffered interest into a biopolymer or Synthetic polymer. Depending Saline, polyol (for example, glycerol, propylene glycol, on the Subject's condition, the Site of injection might prove liquid polyethylene glycol, and the like) and Suitable mix inhospitable for cell Seeding and growth because of Scarring tures thereof. or other impediments. Examples of biopolymer include, but 0105 Sterile injectable solutions can be prepared by are not limited to, cells mixed with fibronectin, fibrin, incorporating the cells utilized in practicing the present fibrinogen, thrombin, collagen, and proteoglycans. This invention in the required amount of the appropriate Solvent could be constructed with or without included expansion or with various amounts of the other ingredients, as desired. differentiation factors. Additionally, these could be in Sus Such compositions may be in admixture with a Suitable pension, but residence time at Sites Subjected to flow would carrier, diluent, or excipient Such as Sterile water, physi be nominal. Another alternative is a three-dimensional gel ological Saline, glucose, dextrose, or the like. The compo with cells entrapped within the interstices of the cell US 2006/0010509 A1 Jan. 12, 2006 biopolymer admixture. Again, expansion or differentiation micrograms to milligrams, such as about 0.0001 to about 5 factors could be included with the cells. These could be wt %, preferably about 0.0001 to about 1 wt %, still more deployed by injection via various routes described herein. preferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, 0110 Those skilled in the art will recognize that the and still more preferably about 0.05 to about 5 wt %. Of components of the compositions should be Selected to be course, for any composition to be administered to an animal chemically inert and will not affect the viability or efficacy or human, and for any particular method of administration, of the bone marrow derived germline stem cells or their it is preferred to determine therefore: toxicity, Such as by progenitors as described in the present invention. This will determining the lethal dose (LD) and LDs in a Suitable present no problem to those skilled in chemical and phar animal model e.g., rodent Such as mouse; and, the dosage of maceutical principles, or problems can be readily avoided by the composition(s), concentration of components therein and reference to standard texts or by simple experiments (not timing of administering the composition(s), which elicit a involving undue experimentation), from this disclosure and Suitable response. Such determinations do not require undue the documents cited herein. experimentation from the knowledge of the skilled artisan, 0111. One consideration concerning the therapeutic use this disclosure and the documents cited herein. And, the time of bone marrow derived germline Stem cells and their for Sequential administrations can be ascertained without progeny is the quantity of cells necessary to achieve an undue experimentation. optimal effect. In current human Studies of autologous mononuclear bone marrow cells, empirical doses ranging III. Oocyte Production from 1 to 4x10" cells have been used with encouraging 0.115. In one embodiment, the present invention provides results. However, different Scenarios may require optimiza a method for oocyte production, comprising providing a tion of the amount of cells injected into a tissue of interest. bone marrow derived female germline Stem cell, or its Thus, the quantity of cells to be administered will vary for progenitor, to a female Subject, and more preferably to the the Subject being treated. In a preferred embodiment, ovary of Said Subject, wherein the cell engrafts into the ovary between 10 to 10, more preferably 10 to 107, and still and differentiates into an oocyte. more preferably, 3x10" stem cells of the invention can be administered to a human Subject. 0116 Preferably, the engrafted cells undergo folliculo genesis, wherein the cells differentiate into an oocyte and 0112 Less cells can be administered directly to the ovary become enclosed within a follicle. Preferably, the engrafted or testes. Preferably, between 10° to 10, more preferably cells differentiate into an oocyte within a follicle of the 10 to 10, and still more preferably, 10" bone marrow ovary. Folliculogenesis is a proceSS in which an ovarian derived germline Stem cells can be administered to a human Structure consisting of a single oocyte is Surrounded by subject. However, the precise determination of what would Somatic (granulosa without or with theca-interstitial) cells. be considered an effective dose may be based on factors Somatic cells of the gonad enclose individual oocytes to individual to each patient, including their size, age, Sex, form follicles. Each fully formed follicle is enveloped in a weight, and condition of the particular patient. AS few as complete basement membrane. Although Some of these 100-1000 cells can be administered for certain desired newly formed follicles Start to grow almost immediately, applications among Selected patients. Therefore, dosages most of them remain in the resting Stage until they either can be readily ascertained by those skilled in the art from degenerate or Some signal(s) activate(s) them to enter the this disclosure and the knowledge in the art. growth phase. A method of the invention can induce folli 0113 Bone marrow derived germline stem cells of the culogenesis by providing a bone marrow derived female invention can comprise a purified population of germline germline stem cell, or its progenitor, to a tissue (e.g., ovarian Stem cells or their progenitors. Those skilled in the art can tissue) by any one of Several routes of administration. The readily determine the percentage of cells in a population bone marrow derived female germline Stem cell, or its using various well-known methods, Such as fluorescence progenitor, can engraft into the tissue and differentiate into activated cell sorting (FACS). Preferable ranges of purity in an oocyte within a follicle. populations comprising female germline Stem cells or their 0117 The number of bone marrow derived female ger progenitors are about 50 to about 55%, about 55 to about mline Stem cells, or their progenitor cells can be increased 60%, and about 65 to about 70%. More preferably the purity by increasing the Survival or proliferation of existing bone is about 70 to about 75%, about 75 to about 80%, about 80 marrow derived female germline Stem cells, or their pro to about 85%; and still more preferably the purity is about genitor cells. 85 to about 90%, about 90 to about 95%, and about 95 to about 100%. Purity of female germline stem cells or their 0118 Agents (e.g., expansion agents) which increase progenitors can be determined according to the cell Surface proliferation or Survival of bone marrow derived female marker profile within a population. Dosages can be readily germline Stem cells, or their progenitor cells include, but are adjusted by those skilled in the art (e.g., a decrease in purity not limited to, a hormone or growth factor (e.g., a IGF, TGF, may require an increase in dosage). BMP, Wnt protein or FGF), a cell-signaling molecule (e.g., S1P or RA), or a pharmacological or pharmaceutical com 0114. The skilled artisan can readily determine the amount of cells and optional additives, vehicles, and/or pound (e.g., an inhibitor of GSK-3, an inhibitor of apoptosis carrier in compositions and to be administered in methods, Such as a Bax inhibitor or caspase inhibitor, an inhibitor of of the invention. Typically, any additives (in addition to the nitric oxide production, or an inhibitor of HDAC activity). active stem cell(s) and/or agent(s)) are present in an amount 0119) Agents comprising growth factors are known in the of 0.001 to 50% (weight) solution in phosphate buffered art to increase proliferation or Survival of Stem cells. For Saline, and the active ingredient is present in the order of example, U.S. Pat. Nos. 5,750,376 and 5,851,832 describe US 2006/0010509 A1 Jan. 12, 2006
methods for the in vitro culture and proliferation of neural No. 6,395,546 describes methods for generating dopamin Stem cells using TGF. An active role in the expansion and ergic neurons in vitro from embryonic and adult central proliferation of stem cells has also been described for BMPs nervous System cells using LIF. The contents of each of (Zhu, G. et al., (1999) Dev. Biol. 215: 118-29 and Kawase, these references are specifically incorporated herein by E. et al., (2001) Development 131: 1365) and Wnt proteins reference for their description of differentiation agents (Pazianos, G. et al., (2003) Biotechniques 35: 1240 and known in the art. Constantinescu, S. (2003) J. Cell Mol. Med. 7: 103). U.S. 0.124 Agents comprising signaling molecules are also Pat. Nos. 5.453,357 and 5,851,832 describe proliferative known to induce differentiation of oocytes. FF-Mas is stem cell culture systems that utilize FGFs. The contents of known to promote oocyte maturation (Marin Bivens, C. L. each of these references are specifically incorporated herein et al., (2004) BOR papers in press). The contents of each of by reference for their description of expansion agents known these references are specifically incorporated herein by in the art. reference for their description of differentiation agents 0120 Agents comprising cell-signaling molecules are known in the art. also known in the art to increase proliferation or Survival of 0.125 Agents comprising pharmacological or pharma Stem cells. For example, Sphingosine-1-phosphate is known ceutical compounds are also known in the art to induce to induce proliferation of neural progenitor cells (Harada, J. differentiation of stem cells. For example, modulators of Id et al., (2004) J. Neurochem. 88: 1026). U.S. Patent Appli are involved in hematopoietic differentiation (Nogueria, M. cation No. 2003.0113913 describes the use of retinoic acid in M. et al., (2000) 276:803) and Modulators of Snail/Slug are stem cell self renewal in culture. The contents of each of known to induce stem cell differentiation (Le Douarin, N. M. these references are specifically incorporated herein by et al., (1994) Curr. Opin. Genet. Dev. 4: 685-695; Plescia, C. reference for their description of expansion agents known in et al., (2001) Differentiation 68: 254). The contents of each the art. of these references are specifically incorporated herein by 0121 Agents comprising pharmacological or pharma reference for their description of differentiation agents ceutical compounds are also known in the art to increase known in the art. proliferation or survival of stem cells. For example, inhibi 0.126 The present invention also provides methods for tors of glycogen Synthase kinase maintain pluripotency of reducing bone marrow derived female germline Stem cells, embryonic Stem cells through activation of Wnt signaling or their progenitor cells, in Vivo, ex vivo or in vitro, (Sato, N. et al., (2004) Nat. Med. 10: 55-63). Inhibitors of comprising contacting bone marrow derived female germ apoptosis (Wang, Y. et al., (2004) Mol. Cell. Endocrinol. 218: line Stem cells or their progenitor cells with an agent that 165), inhibitors of nitric oxide/nitric oxide synthase reduces cell proliferation, inhibits cell Survival or promotes (Matarredona, E. R. et al., (2004) Brain Res. 995: 274) and cell death. Unwanted proliferation of the cells of the inven inhibitors of histone deacetylases (Lee, J. H. et al., (2004) tion can give rise to cancerous and pre-cancerous pheno Genesis 38: 32-8) are also known to increase proliferation types (e.g., germ cell tumors, ovarian cancer). Such methods and/or pluripotency. For example, the peptide humanin is an can be used to control unwanted proliferation (e.g., cancer) inhibitor of Bax function that Suppresses apoptosis (Guo, B. or for contraceptive measures by reducing the numbers of et al., (2003) Nature 423: 456-461). The contents of each of germline Stem cells, and optionally their progenitors or these references are specifically incorporated herein by oocytes. reference for their description of expansion agents known in the art. 0127 Agents that reduce cell proliferation include, but are not limited to, a hormone or growth factor (e.g., TGF-B), 0.122 Oocyte production can be further increased by a peptide antagonist of mitogenic hormones or growth contacting bone marrow derived female germline Stem cells, factors (e.g., the BMP antagonists, PRDC and Gremlin), or or their progenitor cells, with an agent that differentiates a pharmacological or pharmaceutical compound (e.g., a cell bone marrow derived female germline Stem cells or their cycle inhibitor, or an inhibitor of growth factor signaling). progenitor cells into oocytes (e.g., differentiation agents). Such differentiation agents include, but are not limited to, a 0128 Agents that inhibit cell Survival include, but are not hormone or growth factor (e.g., TGF, BMP, Wnt protein, limited to, a hormone, growth factor or cytokine (e.g., a SCF or LIF), a signaling molecule (e.g., meiosis-activating pro-apoptotic TNF Super family member such as TNF-C., sterol, “FF-MAS”), or a pharmacologic or pharmaceutical FasL and TRAIL), an antagonist of pro-Survival Bcl-2 agent (e.g., a modulator of Id protein function or Snail/Slug family member function, a signaling molecule (e.g., a cera transcription factor function). mide), or a pharmacological or pharmaceutical compound (e.g., an inhibitor of growth factor signaling). Pro-Survival 0123 Agents comprising growth factors are known in the Bcl-2 family members include Bcl-2, Bcl-X1 (Cory, S. and art to induce differentiation of Stem cells. For example, Adams, J. M. (2000) Nat Rev Cancer 209):647-656; Lutz, R. TGF-B can induce differentiation of hematopoietic stem J. (2000) Cell Survival Apoptosis 28:51-56), Bcl-W (Gib cells (Ruscetti, F. W. et al., (2001) Int. J. Hematol. 74: 18). son, L., et al. (1996) Oncogene 13, 665-675; Cory, S. and U.S. Patent Application No. 2002142457 describes methods Adams, J. M. (2000) Nat Rev Cancer 2(9):647-656), Mc1-1 for differentiation of cardiomyocytes using BMPs. Pera et al (Kozopas, K. M., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. describe human embryonic Stem cell differentiation using 90:3516-3520; Reynolds, J. E., et al. (1994) Cancer Res. BMP-2 (Pera, M. F. etal,(2004).J. Cell Sci. 117: 1269). U.S. 54:6348-6352; Cory, S. and Adams, J. M. (2000) Nat Rev Patent Application No. 20040014210 and U.S. Pat. No. Cancer 209):647-656) and A1 (Cory, S. and Adams, J. M. 6,485,972 describe methods of using Wnt proteins to induce (2000) Nat Rev Cancer 2(9):647-656; Gonzales, J., et al. differentiation. U.S. Pat. No. 6,586,243 describes differen (2003) Blood 101(7):2679-2685; Reed, J. C. (1997) Nature tiation of dendritic cells in the presence of SCF. U.S. Pat. 387:773-776). US 2006/0010509 A1 Jan. 12, 2006
0129 Agents that promote cell death include, but are not in Otto, D. et al., J. Neurosci. Res. 22: 83 and in Otto, D. and limited to, a pro-apoptotic tumor necrosis factor Superfamily Unsicker, K. J. Neurosci. 10: 1912. member (e.g., TNF-C., FasL and TRAIL), agonist of pro apoptotic Bcl-2 family member function and ceramide. 0133. In vitro and ex vivo applications can involve cul Pro-apoptotic Bcl-2 family members include Bax (Oltvai, Z. ture of the bone marrow derived germline stem cells or their N, et al. (1993): Cell 74: 609-619), Bak (Chittenden, T, et al. progenitors with the Selected agent to achieve the desired (1995) Nature 374:733-736), Bid (Luo, X., et al. (1998) Cell result. Cultures of cells (from the same individual and from 94:481-490), Hrk (Inohara, N. et al. (1997) EMBO J. different individuals) can be treated with differentiation 16(7): 1686-1694), Bod (Hsu, et al. (1998) Mol Endocrinol. agents of interest to Stimulate, for example, the production 12(9): 1432-1440), Bim (O'Connor, L., et al. (1998) EMBO of oocytes or Sperm cells, which can then be used for a J. 17(2):385-395), Noxa (Oda, E., et al. (2000) Science 288, variety of therapeutic applications (e.g., in vitro fertiliza 1053-1058; Yakovlev, A. G., et al. (2004) J Biol Chem tion). 279(27):28367-28374), puma (Nakano, K. and Vousden, K. 0.134 Differentiated cells derived from cultures of the H. (2001) Mol Cell 7(3):683-694), Bok (Yakovlev, A. G., et invention can be implanted into a host. The transplantation al. (2004) J Biol Chem 279(27):28367-28374; Hsu, S Y, et can be autologous, Such that the donor of the Stem cells from al. (1997) Proc Natl Acad Sci USA. 94(23):12401-6) and which organ or organ units are derived is the recipient of the Bcl-Xs (Boise, L. H., et al. (1993) Cell 74:597-608). engineered tissue. The transplantation can be heterologous, 0130 Several agents are known in the art to inhibit cell such that the donor of the stem cells from which organ or proliferation or Survival or promote cell death, including organ units are derived is not that of the recipient of the PRDC (Sudo et al. (2004) J. Biol. Chem., advanced publi engineered-tissue. Once transferred into a host, the differ cation), TNF (Wong, G. et al., (2004) Exp. Neurol. 187: 171), entiated cells the function and architecture of the native host FasL (Sakata, S. et al., (2003) Cell Death Differ. 10: 676) and tissue. TRAIL (Pitti, RM, et al. (1996) J Biol Chem 271: 12687 0.135 Bone marrow derived germline stem cells and the 12690; Wiley, S R, et al. (1995) Immunity 3: 673-682). progeny thereof can be cultured, treated with agents and/or Ceramide mediates the action of tumor necrosis factor on administered in the presence of polymer Scaffolds. Polymer primitive human hematopoietic cells (Maguer-Satta, V. et al., Scaffolds are designed to optimize gas, nutrient, and waste (2000) Blood 96: 4118-23). Agonist/antagonist of Bcl-2 eXchange by diffusion. Polymer Scaffolds can comprise, for family members, such as Bcl-2, Bcl-XL, Bcl-W, Mcl-1, A1, example, a porous, non-woven array of fibers. The polymer Bax, Bak, Bid, Hrk, Bod, Bim, Noxa, Puma, Bok and Scaffold can be shaped to maximize Surface area, to allow Bcl-XS, are known to inhibit stem cell survival (Lindsten, T. adequate diffusion of nutrients and growth factors to the et al., (2003) J. Neurosci. 23: 11112-9). Agents comprising cells. Taking these parameters into consideration, one of pharmacological or pharmaceutical compounds are also skill in the art could configure a polymer Scaffold having known in the art to inhibit cell Survival. For example, sufficient surface area for the cells to be nourished by inhibitors of growth factor Signaling, Such as QSulfl, a diffusion until new blood vessels interdigitate the implanted heparan Sulfate 6-O-endosulfatase that inhibits fibroblast engineered-tissue using methods known in the art. Polymer growth factor Signaling, can inhibit stem cell Survival scaffolds can comprise a fibrillar structure. The fibers can be (Wang, S. et al., (2004) Proc. Natl. Acad. Sci. USA 101: round, Scalloped, flattened, Star-shaped, Solitary or entwined 4833). The contents of each of these references are specifi with other fibers. Branching fibers can be used, increasing cally incorporated herein by reference for their description Surface area proportionately to Volume. of agents known in the art to inhibit cell Survival. 0.136 Unless otherwise specified, the term “polymer” 0131 Agents can be provided directly to the reproductive includes polymers and monomers that can be polymerized or organ of interest. Alternatively, agents can be provided adhered to form an integral unit. The polymer can be indirectly to the reproductive organ of interest, for example, non-biodegradable or biodegradable, typically via hydroly by administration into the circulatory System. sis or enzymatic cleavage. The term “biodegradable' refers to materials that are bioresorbable and/or degrade and/or 0132) Agents can be administered to subjects in need break down by mechanical degradation upon interaction thereof by a variety of administration routes. Methods of with a physiological environment into components that are administration, generally Speaking, may be practiced using metabolizable or excretable, over a period of time from any mode of administration that is medically acceptable, minutes to three years, preferably less than one year, while meaning any mode that produces effective levels of the maintaining the requisite Structural integrity. AS used in active compounds without causing clinically unacceptable reference to polymers, the term “degrade” refers to cleavage adverse effects. Such modes of administration include oral, of the polymer chain, Such that the molecular weight stays rectal, topical, intraocular, buccal, intravaginal, intracister approximately constant at the oligomer level and particles of nal, intracerebroVentricular, intratracheal, nasal, transder polymer remain following degradation. mal, within?on implants, e.g., fiberS Such as collagen, oSmotic pumps, or grafts comprising appropriately trans 0.137 Materials suitable for polymer scaffold fabrication formed cells, etc., or parenteral routes. The term include polylactic acid (PLA), poly-L-lactic acid (PLLA), "parenteral' includes Subcutaneous, intravenous, intramus poly-D-lactic acid (PDLA), polyglycolide, polyglycolic acid cular, intraperitoneal, intragonadal or infusion. Intravenous (PGA), polylactide-co-glycolide (PLGA), polydioxanone, or intramuscular routes are not particularly Suitable for polygluconate, polylactic acid-polyethylene oxide copoly long-term therapy and prophylaxis. A particular method of mers, modified cellulose, collagen, polyhydroxybutyrate, administration involves coating, embedding or derivatizing polyhydroxpriopionic acid, polyphosphoester, poly(alpha fibers, Such as collagen fibers, protein polymers, etc. with hydroxy acid), polycaprolactone, polycarbonates, polya therapeutic proteins. Other useful approaches are described mides, polyanhydrides, polyamino acids, polyorthoesters, US 2006/0010509 A1 Jan. 12, 2006 polyacetals, polycyanoacrylates, degradable urethanes, ali and FLIP, can be used to regulate apoptotic events during phatic polyester polyacrylates, polymethacrylate, acyl Sub spermatogenesis (Weikert S., (2004) Int. J. Androl. 27: 161; Stituted cellulose acetates, non-degradable polyurethanes, Giampietri, C. et al., (2003) Cell Death Differ. 10: 175). polystyrenes, polyvinyl chloride, polyvinyl flouride, poly Similarly, Bax inhibitorS Such as humanin, are also impli Vinyl imidazole, chloroSulphonated polyolifins, polyethyl cated in Spermatogenic apoptosis (Guo, B. et al., (2003) ene oxide, polyvinyl alcohol, teflon RTM, nylon silicon, and Nature 423: 456). shape memory materials, Such as poly(styrene-block-buta diene), polynorbornene, hydrogels, metallic alloys, and 0.143 Growth factors, such as fibroblast growth factor-4 oligo(e-caprolactone)diol as Switching segment/oligo(p-di (Hirai, K. et al., (2004) Exp. Cell Res. 294: 77) can also influence Spermatogenesis. FGF-4 can play a critical role as oxyanone)diol as physical crosslink. Other Suitable poly a Survival factor for germ cells by protecting them from mers can be obtained by reference to The Polymer Hand apoptosis. Upon FGF-4 stimulation in Sertoli cells, lactate book, 3rd edition (Wiley, N.Y., 1989). production was induced, which is indispensable for germ 0138 Factors, including but not limited to nutrients, cell Survival. FGF-4 stimulation can also reduce DNA growth factors, inducers of differentiation or de-differentia fragmentation in Sertoli cells. tion, products of Secretion, immunomodulators, inhibitors of inflammation, regression factors, hormones, or other bio 0144 Bone morphogenetic protein (BMP) signaling logically active compounds can be incorporated into or can pathways have also been implicated in maintenance of germ line stem cells in Drosophila (Kawase, E. et al., (2004) be provided in conjunction with the polymer scaffold. Development 131: 1365-75; Pellegrini, M. et al., (2003) J. 0139 Agents of the invention may be supplied along with Cell Sci. 116:3363). BMP4 stimulation of cultured sper additional reagents in a kit. The kits can include instructions matogonia can induce Smad-mediated proliferation, as well for the treatment regime or assay, reagents, equipment (test as differentiation through the c-kit gene. Additionally, BMP tubes, reaction vessels, needles, Syringes, etc.) and Standards Signals from Somatic cells were shown to be essential for for calibrating or conducting the treatment or assay. The maintaining germline Stem cells through repression of the instructions provided in a kit according to the invention may bam expression, indicating that Bmp signals from the be directed to Suitable operational parameters in the form of Somatic cells maintain germline Stem cells at least in part, by a label or a separate insert. Optionally, the kit may further repressing bam expression in the testis. comprise a Standard or control information So that the test 0145 Transforming growth factor (TGF) can also repress Sample can be compared with the control information stan bam expression in testis. Maintenance and proliferation of dard to determine if whether a consistent result is achieved. germ line Stem cells and their progeny depends upon the IV. Spermatogenesis ability of these cells to transduce the activity of a Somatically expressed TGF-B ligand, known in Drosophila as the 0140 Methods of the present invention can be used in the BMP5/8 ortholog Glass Bottom Boat (Shivdasani, A. A. and production of other reproductive cell types. Accordingly, in Ingham, P. W. (2003) Curr. Biol. 13: 2065). TGF-3 signaling one embodiment, the present invention provides a method represses the expression of bam, which is necessary and for restoring or enhancing Spermatogenesis, comprising Sufficient for germ cell differentiation, thereby maintaining providing a bone marrow derived male germline Stem cell, germ line Stem cells and Spermatogonia in their proliferative or its progenitor, to the testes of a male Subject, wherein the cell engrafts into the Seminiferous epithelium and differen State. tiates into a Sperm cell. Administration of a bone marrow 0146) Sphingosine-1-phosphate (S1P) is also known to derived male germline Stem cell, or its progenitor, to the affect the Survival and proliferation of germ line Stem cells testes is preferably carried out by testicular injection. Direct and Spermatogonia. In a study where irradiated testicular injection into the testes advantageously circumvents the tissue was treated with S1P, the numbers of primary sper blood barrier, and provides cells to Suitable locations, Such matocytes and Spermatogonia were higher than untreated as the Seminiferous epithelium. tissues, indicating that S1P treatment can protect germ line 0141 Spermatogenesis can be further increased by con Stem cells against cell death induced by radiation (Otala, M. tacting compositions comprising bone marrow derived male et al., (2004) Biol Reprod. Mar; 70(3):759-67). germline Stem cells, or their progenitor cells, with an agent 0147 Glial-derived neurotrophic factor was found to that differentiates bone marrow male germline derived Stem markedly amplify germline Stem cells in murine testis cells or their progenitor cells into Sperm cells (e.g., differ (Kubota, H. et al., (2004) Biol. Reprod. 71(3):722-31). entiation agents). Such differentiation agents can be, but are Transplantation analysis demonstrated not only germline not limited to, those described herein. Stem cells enrichment, but also differentiation from Stem 0.142 Spermatogenesis, or the formation of Spermato cells into sperm (Yomogida, K. et al., (2003) Biol. Reprod. cytes from Spermatogonia, can be regulated by numerous 69: 1303). factors. Regulators of apoptosis, including Bax, Bcl-X fam 0.148. The present invention also provides methods for ily members, and caspase family members, can modulate reducing bone marrow derived male germline Stem cells, or Spermatogenesis and affect male fertility (Said, T. M., et al. their progenitor cells, in Vivo, ex vivo or in vitro, comprising (2004) Hum. Reprod. Update 10: 39-51; Yan, W. et al, contacting bone marrow derived male germline Stem cells or (2003) Mol. Endocrinol. 17: 1868). Caspases have been their progenitor cells with an agent that reduces cell prolif implicated in the pathogenesis of multiple andrological eration, inhibits cell Survival or promotes cell death. pathologies, Such as, inter alia, impaired Spermatogenesis, Unwanted proliferation of the cells of the invention can give decreased Sperm motility, and increased levels of Sperm rise to cancerous and pre-cancerous phenotypes (e.g., germ DNA fragmentation. Caspase inhibitors, Such as Survivin cell tumors, testicular cancer). Such methods can be used to US 2006/0010509 A1 Jan. 12, 2006 control unwanted proliferation (e.g., cancer) or for contra 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plas ceptive measures by reducing the numbers of germline Stem mids (Cullet al. (1992) Proc Natl Acad Sci USA 89:1865 cells, and optionally their progenitors or Sperm cells. 1869) or on phage (Scott and Smith (1990) Science 249:386 0149 Agents that reduce cell proliferation include, but 390; Devlin (1990) Science 249:404–406; Cwirla et al. are not limited to, a hormone or growth factor (e.g., TGF-B), (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991).J. a peptide antagonist of mitogenic hormones or growth Mol. Biol. 222:301-310; Ladner supra). factors (e.g., the BMP antagonists, PRDC and Gremlin), or 0156 Chemical compounds to be used as test agents (i.e., a pharmacological or pharmaceutical compound (e.g., a cell potential inhibitor, antagonist, agonist) can be obtained from cycle inhibitor, or an inhibitor of growth factor signaling). commercial Sources or can be Synthesized from readily available Starting materials using Standard Synthetic tech 0150 Agents that inhibit cell survival include, but are not niques and methodologies known to those of ordinary skill limited to, a hormone, growth factor or cytokine (e.g., a in the art. Synthetic chemistry transformations and protect pro-apoptotic TNF Super family member such as TNF-C., ing group methodologies (protection and deprotection) use FasL and TRAIL), an antagonist of pro-Survival Bcl-2 ful in Synthesizing the compounds identified by the methods family member function, a signaling molecule (e.g., a cera described herein are known in the art and include, for mide), or a pharmacological or pharmaceutical compound example, those such as described in R. Larock (1989) (e.g., an inhibitor of growth factor signaling). Comprehensive Organic Transformations, VCH Publishers; 0151. Agents that promote cell death include, but are not T. W. Greene and P. G. M. Wuts, Protective Groups in limited to, a pro-apoptotic tumor necrosis factor Superfamily Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. member (e.g., TNF-C., FasL and TRAIL), agonist of pro Fieser and M. Fieser, Fieser and Fieser’s Reagents for apoptotic Bcl-2 family member function and ceramide. Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Syn IV. Screening ASSayS thesis, John Wiley and Sons (1995), and Subsequent editions 0152 The invention provides methods for identifying thereof. modulators, i.e., candidate or test compounds or agents (e.g., O157. In one aspect the compounds are organic Small proteins, peptides, peptidomimetics, peptoids, Small mol molecules, that is, compounds having molecular weight leSS ecules or other drugs) which modulate bone marrow derived than 1,000 amu, alternatively between 350-750 amu. In germline Stem cells or the progenitors thereof. Agents thus other aspects, the compounds are: (i) those that are non identified can be used to modulate, for example, prolifera peptidic, (ii) those having between 1 and 5, inclusive, tion, Survival and differentiation of a bone marrow derived heterocyclyl, or heteroaryl ring groups, which may bear germline Stem cell or its progenitor e.g., in a therapeutic further Substituents; (iii) those in their respective pharma protocol. ceutically acceptable Salt forms, or (iv) those that are pep 0153. The test agents of the present invention can be tidic. obtained Singly or using any of the numerous approaches in 0158. The term "heterocyclyl” refers to a nonaromatic combinatorial library methods known in the art, including: 3-8 membered monocyclic, 8-12 membered bicyclic, or biological libraries; peptide libraries (libraries of molecules 11-14 membered tricyclic ring System having 1-3 heteroat having the functionalities of peptides, but with a novel, oms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 non-peptide backbone which are resistant to enzymatic heteroatoms if tricyclic, Said heteroatoms Selected from O, degradation but which nevertheless remain bioactive, See, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms e.g., Zuckermann, R. N. (1994) et al., J. Med. Chem. of N, O, or S if monocyclic, bicyclic, or tricyclic, respec 37:2678-85); spatially addressable parallel solid phase or tively), wherein 0, 1, 2 or 3 atoms of each ring can be Solution phase libraries, Synthetic library methods requiring substituted by a substituent. deconvolution; the 'one-bead one-compound library method; and Synthetic library methods using affinity chro 0159. The term “heteroaryl” refers to an aromatic 5-8 matography Selection. The biological library and peptoid membered monocyclic, 8-12 membered bicyclic, or 11-14 library approaches are limited to peptide libraries, while the membered tricyclic ring System having 1-3 heteroatoms if other four approaches are applicable to peptide, non-peptide monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms oligomer or Small molecule libraries of compounds (Lam if tricyclic, said heteroatoms Selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or (1997) Anticancer Drug Des. 12:145). S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0154) Examples of methods for the synthesis of molecu 0, 1, 2, 3, or 4 atoms of each ring can be Substituted by a lar libraries can be found in the art, for example in: DeWitt Substituent. et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et 0160 The term “substituents” refers to a group “substi al. (1994) Proc. Natl. Acad. Sci. USA 91:11422, Zucker tuted” on an alkyl, cycloalkyl, aryl, heterocyclyl, or het mann et al. (1994) J.Med. Chem. 37:2678; Cho et al. (1993) eroaryl group at any atom of that group. Suitable Substitu Science 261: 1303; Carrell et al. (1994) Angew. Chem. Int. ents include, without limitation, alkyl, alkenyl, alkynyl, Ed. Engl. 33:2059; Carelletal (1994) Angew. Chem. Int. Ed. alkoxy, halo, hydroxy, cyano, nitro, amino, SOH, perfluo Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. roalkyl, perfluoroalkoxy, methylenedioxy, ethylenedioxy, 37: 1233. carboxyl, Oxo, thioxo, imino (alkyl, aryl, aralkyl), S(O)- O155 Libraries of compounds may be presented in solu alkyl (where n is 0-2), S(O), aryl (where n is 0-2), S(O), tion (e.g., Houghten (1992), Biotechniques 13:412-421), or heteroaryl (where n is 0-2), S(O), heterocyclyl (where n is on beads (Lam (1991), Nature 354:82-84), chips (Fodor 0-2), amine (mono-, di-, alkyl, cycloalkyl, aralkyl, het (1993) Nature 364:555-556), bacteria (Ladner, U.S. Pat. No. eroaralkyl, and combinations thereof), ester (alkyl, aralkyl, US 2006/0010509 A1 Jan. 12, 2006
heteroaralkyl), amide (mono-, di-, alkyl, aralkyl, het formats. The isolated mRNA can be used in hybridization or eroaralkyl, and combinations thereof), Sulfonamide (mono-, amplification assays that include, but are not limited to, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), Southern or Northern analyses, polymerase chain reaction unsubstituted aryl, unsubstituted heteroaryl, unsubstituted analyses and probe arrayS. One diagnostic method for the heterocyclyl, and unsubstituted cycloalkyl. In one aspect, detection of mRNA levels involves contacting the isolated the Substituents on a group are independently any one Single, mRNA with a nucleic acid molecule (probe) that can hybrid or any Subset of the aforementioned Substituents. ize to the mRNA encoded by the gene being detected. The 0.161 Combinations of substituents and variables in com nucleic acid probe is Sufficient to Specifically hybridize pounds envisioned by this invention are only those that under stringent conditions to mRNA or genomic DNA. The result in the formation of stable compounds. The term probe can be disposed on an address of an array, e.g., an “stable', as used herein, refers to compounds which possess array described below. Other suitable probes for use in the stability sufficient to allow manufacture and which main diagnostic assays are described herein. tains the integrity of the compound for a Sufficient period of 0.168. In one format, mRNA (or cDNA) is immobilized time to be useful for the purposes detailed herein (e.g., on a Surface and contacted with the probes, for example by transport, Storage, assaying, therapeutic administration to a running the isolated mRNA on an agarose gel and transfer Subject). ring the mRNA from the gel to a membrane, Such as 0162 The compounds described herein can contain one nitrocellulose. In an alternative format, the probes are immo or more asymmetric centers and thus occur as racemates and bilized on a surface and the mRNA (or cDNA) is contacted racemic mixtures, Single enantiomers, individual diastere with the probes, for example, in a two-dimensional gene omers and diastereomeric mixtures. All Such isomeric forms chip array described below. A skilled artisan can adapt of these compounds are expressly included in the present known mRNA detection methods for use in detecting the invention. The compounds described herein can also be level of mRNA encoded by the genetic markers described represented in multiple tautomeric forms, all of which are herein. included herein. The compounds can also occur in cis-or 0169. The level of mRNA in a sample can be evaluated trans-or E-or Z-double bond isomeric forms. All Such iso with nucleic acid amplification, e.g., by rtPCR (Mullis meric forms of Such compounds are expressly included in (1987) U.S. Pat. No. 4,683.202), ligase chain reaction the present invention. (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self 0163 Test agents of the invention can also be peptides Sustained sequence replication (Guatelli et al. (1990) Proc. (e.g., growth factors, cytokines, receptor ligants). Natl. Acad. Sci. USA 87: 1874-1878), transcriptional ampli fication system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. 0164. Screening methods of the invention can involve the USA86:1173-1177), Q-Beta Replicase (Lizardiet al. (1988) identification of an agent that increases the proliferation or Bio/Technology 6:1197), rolling circle replication (Lizardi survival of bone marrow derived germline stem cells or the et al., U.S. Pat. No. 5,854,033) or any other nucleic acid progenitors thereof. Such methods will typically involve amplification method, followed by the detection of the contacting bone marrow derived Stem or progenitor cells amplified molecules using techniques known in the art. AS with a test agent in culture and quantitating the number of used herein, amplification primers are defined as being a pair new bone marrow derived Stem or progenitor cells produced of nucleic acid molecules that can anneal to 5' or 3' regions as a result. Comparison to an untreated control can be of a gene (plus and minus Strands, respectively, or Vice concurrently assessed. Where an increase in the number of versa) and contain a short region in between. In general, Stem or progenitor cells is detected relative to the control, amplification primers are from about 10 to 30 nucleotides in the test agent is determined to have the desired activity. length and flank a region from about 50 to 200 nucleotides 0.165. In practicing the methods of the invention, it may in length. Under appropriate conditions and with appropriate be desirable to employ a purified population of bone marrow reagents, Such primers permit the amplification of a nucleic derived germline Stem cells or the progenitors thereof. A acid molecule comprising the nucleotide Sequence flanked purified population of bone marrow derived germline Stem by the primerS. cells or the progenitors thereof can have about 50-55%, 0170 For in situ methods, a cell or tissue sample can be 55-60%, 60-65% and 65-70% purity. More preferably the prepared/processed and immobilized on a Support, typically purity is about 70-75%, 75-80%, 80-85%; and still more a glass Slide, and then contacted with a probe that can preferably the purity is about 85-90%, 90-95%, and hybridize to mRNA that encodes the genetic marker being 95-100%. analyzed. 0166 Increased amounts of bone marrow derived female germline Stem cells or the progenitors thereof can also be 0171 Screening methods of the invention can involve the detected by an increase in gene expression of genetic identification of an agent that differentiates bone marrow markers including an Oct-4, Dazl, Stella Vasa, Fragilis, derived germline Stem cells, or their progenitor cells, into Nobox and c-Kit. The level of expression can be measured oocytes or Sperm cells. Such methods will typically involve in a number of ways, including, but not limited to: measur contacting the bone marrow derived Stem or progenitor cells ing the mRNA encoded by the genetic markers, measuring with a test agent in culture and quantitating the number of the amount of protein encoded by the genetic markers, or new oocytes or Sperm cells produced as a result. Comparison measuring the activity of the protein encoded by the genetic to an untreated control can be concurrently assessed. Where markers. an increase in the number of oocytes is detected relative to the control, the test agent is determined to have the desired 0167 The level of mRNA corresponding to a genetic activity. The test agent can also be assayed using a biological marker can be determined both by in situ and by in vitro Sample (e.g., Ovarian tissue); Subsequent testing using a US 2006/0010509 A1 Jan. 12, 2006 population of Stem or progenitor cells may be conducted to of an agent that protects against reproductive injury prior to distinguish the functional activity of the agent (e.g., differ or concurrently with chemotherapy and/or radiotherapy can entiation rather then increase in proliferation or Survival) protect fertility and enhance the restoration methods where the result is ambiguous. described herein. The protective agent includes, but is not limited to, S1P, Bax, or any agent that increases SDF-1 0172 Increased amounts of oocytes be detected by a activity (i.e., SDF-1 mediated migration and homing of stem decrease in gene expression of bone marrow derived female cells). For a description of the use of S1P in protecting germline Stem or progenitor genetic markers including Oct reproductive systems, see U.S. application Ser. No. 10/217, 4, Dazl, Stella Vasa, Fragilis, Nobox or c-Kit or an increase 259, filed on Aug. 12, 2002 and published as 20030157086 in oocyte markers, such as HDAC6, GDF9 and ZP3. on Aug. 21, 2003, the contents of which are herein incor VI. Methods of Treatment porated by reference. 0173 Bone marrow derived germline stem cells of the 0179 The present invention also provides methods for invention or their progenitors can be used in a variety of restoring ovarian function in a menopausal female Subject, therapeutic applications (e.g., sperm/oocyte generation for comprising providing a bone marrow derived female germ in Vivo restoration or eX Vivo procedures including in Vitro line Stem cell, or its progenitor, to the Subject, wherein the fertilization). Accordingly, methods of the invention relate cell engrafts into the ovary and differentiates into an oocyte. to, among other things, the use of bone marrow derived The menopausal female Subject can be in a Stage of either germline Stem cells, or their progenitor cells, to provide peri- or post-menopause, with Said menopause caused by germ cells in the treatment of reproductive disorders. either normal (e.g., aging) or pathological (e.g., Surgery, 0.174 Compositions comprising bone marrow derived disease, ovarian damage) processes. germline Stem cells or their progenitor cells can be provided 0180 Ovarian function in a post-menopausal female can directly to the reproductive organ of interest (e.g., ovary or also be restored by administering an agent that increases the testes). Alternatively, compositions comprising bone mar amount of bone marrow derived female germline Stem cells row derived germline Stem cells or their progenitor cells can or their progenitors (e.g., by increasing the number or life be provided indirectly to the reproductive organ of interest, span of bone marrow derived female germline Stem cells, as for example, by administration into the circulatory System well as by increasing the differentiation of bone marrow (e.g., to extra-ovarian circulation). derived female germline stem cells into oocytes). 0.175 Thus, the present invention provides methods for 0181 Restoration of ovarian function can relieve adverse treating infertility in a female Subject comprising providing Symptoms and complications associated with menopause, a bone marrow derived female germline Stem cell, or its including, but not limited to, Somatic disorderS Such as progenitor, to a female Subject in need thereof, wherein the Osteoporosis, cardiovascular disease, Somatic Sexual dyS cell engrafts into a tissue (preferably ovarian tissue) and function, hot flashes, vaginal drying, Sleep disorders, depres differentiates into an oocyte, which can later be provided for Sion, irritability, loSS of libido, hormone imbalances, and the fertilization following ovulation in the Subject. Alterna like, as well as cognitive disorders, Such as loSS of memory; tively, the engrafted oocyte can be harvested from the emotional disorders, depression, and the like. subject and provided for in vitro fertilization or somatic cell nuclear transfer. Except where expressly Stated herein, the 0182 Bone marrow derived germline stem cells of the female Subject in need of fertility treatment is not a Subject invention, their progenitors or their progeny, can be admin who has undergone chemotherapy or radiotherapy. istered as previously described, and obtained by all methods known in the art. Bone marrow derived germline Stem cells 0176) The present invention also provides methods for of the invention can be autologous (obtained from the treating infertility comprising administering an agent that Subject) or heterologous (e.g., obtained from a donor). increases the amount of bone marrow derived female ger Heterologous cells can be provided together with immuno mline Stem cells, or their progenitor cells, by increasing the Suppressive therapies known in the art to prevent immune proliferation or survival of the bone marrow derived female rejection of the cells. germline Stem cells or their progenitor cells, thereby enhanc ing oocyte production. Agents can be provided directly to 0183 Bone marrow derived germline stem cells of the the reproductive organ of interest. Alternatively, agents can present invention can be isolated by bone marrow aspiration. be provided indirectly to the reproductive organ of interest, For increased yield from female donors, it may be desirable for example, by administration into the circulatory System. to coordinate isolation with appropriate Stages of the female reproductive cycle that exhibit higher levels of female 0177. The present invention also provides methods for germline Stem cells in the bone marrow, as described in repairing damaged OVarian tissue, comprising providing a Example 1. U.S. Pat. No. 4,481,946, incorporated herein bone marrow derived female germline Stem cell, or its expressly by reference, describes a bone marrow aspiration progenitor cell, to the Ovarian tissue, wherein the cell method and apparatus, wherein efficient recovery of bone engrafts into the Ovarian tissue and differentiates into an marrow from a donor can be achieved by inserting a pair of oocyte. Except where expressly Stated herein, the ovarian aspiration needles at the intended Site of removal. Through tissue was not damaged by chemotherapy or radiotherapy. connection with a pair of Syringes, the preSSure can be Damage can be caused, for example, by exposure to cyto regulated to Selectively remove bone marrow and Sinusoidal toxic factors, hormone deprivation, growth factor depriva blood through one of the aspiration needles, while positively tion, cytokine deprivation, cell receptor antibodies, and the forcing an intravenous Solution through the other of the like. aspiration needles to replace the bone marrow removed from 0.178 Where damage may be caused by an anticipated the site. The bone marrow and sinusoidal blood can be course of chemotherapy and/or radiotherapy, administration drawn into a chamber for mixing with another intravenous US 2006/0010509 A1 Jan. 12, 2006
Solution and thereafter forced into a collection bag. The known in the art (e.g., a BD Biosciences FACScalibur heterogeneous cell population can be further purified by cytometer) based on cell Surface expression of Sca-1 (van de identification of cell-Surface markers to obtain the bone Rijn et al., (1989) Proc. Natl. Acad. Sci. USA 86, 4634 marrow derived germline Stem cell compositions for admin 4638) and/or c-Kit (Okada et al., (1991) Blood 78, 1706 istration into the reproductive organ of interest. 1712); (Okada et al., (1992) Blood 80, 3044-3050) follow ing an initial immunomagnetic bead column-based 0184 U.S. Pat. No. 4486,188 describes methods of bone marrow aspiration and an apparatus in which a Series of lines fractionation step to obtain lineage-depleted (lin) cells are directed from a chamber Section to a Source of intrave (Spangrude et al., (1988) Science 241, 58-62); (Spangrude nous Solution, an aspiration needle, a Second Source of and Scollay, (1990) Exp. Hematol. 18, 920-926), as intravenous Solution and a Suitable Separating or collection described (Shen et al., (2001).J. Immunol. 166,5027-5033); Source. The chamber Section is capable of Simultaneously (Calvi et al., (2003) Nature 425, 841-846). applying negative preSSure to the Solution lines leading from 0190. For serial passage-based enrichment of bone mar the intravenous Solution Sources in order to prime the lines row derived germline Stem cells, and their progenitor cells, and to purge them of any air. The Solution lines are then in-vitro (Meirelles and Nardi, (2003) Br. J. Haematol. 123, closed and a positive pressure applied to redirect the intra 702-711); (Tropel et al., (2004) Exp. Cell Res. 295, 395 venous Solution into the donor while negative preSSure is 406), isolated bone marrow can be plated on plastic in applied to withdraw the bone marrow material into a cham Dulbecco's modified Eagle's medium (Fisher Scientific, ber for admixture with the intravenous solution, following Pittsburgh, Pa.) with 10% fetal bovine serum (Hyclone, which a positive pressure is applied to transfer the mixture Logan, Utah), penicillin, Streptomycin, L-glutamine and of the intravenous Solution and bone marrow material into amphotericin-B. About forty-eight hours after the initial the Separating or collection Source. plating, the Supernatants containing non-adherent cells can be removed and replaced with fresh culture medium after 0185. According to methods of the invention, bone mar gentle washing. The cultures can then be maintained and row can be harvested during the lifetime of the subject, but passed once confluence is reached (e.g., for a total of about a pre-menopausal harvest is recommended. Furthermore, three times over the span of about 6 weeks) at which time the harvest prior to illness (e.g., cancer) is desirable, and harvest cultures can be terminated to collect adherent cells for prior to treatment by cytotoxic means (e.g., radiation or analysis. chemotherapy) will improve yield and is therefore also desirable. 0191 Prior to administration, bone marrow derived ger mline Stem cells, their progenitors or their progeny, 0186 U.S. Pat. No. 5,806,529 describes a method for described herein can optionally be genetically modified, in bone marrow transplantation from an HLA-nonmatched vitro, in vivo or ex vivo, by introducing heterologous DNA donor to a patient which comprises conditioning the patient or RNA or protein into the cell by a variety of recombinant under a Suitable regimen followed by transplant of a very methods known to those of skill in the art. These methods large dose of stem cells (at least about 3-fold greater than the are generally grouped into four major categories: (1) viral conventional doses used in T cell-depleted bone marrow transfer, including the use of DNA or RNA viral vectors, transplantation). The patient is conditioned under lethal or Such as retroviruses (including lentiviruses), Simian virus 40 Supralethal conditions for the treatment of malignant or (SV40), adenovirus, Sindbis virus, and bovine papillomavi non-malignant diseases, or under Sublethal conditions for rus, for example; (2) chemical transfer, including calcium the treatment of non-malignant diseases. The transplant may phosphate transfection and DEAE dextran transfection consist of T cell-depleted bone marrow stem cells and T methods; (3) membrane fusion transfer, using DNA-loaded cell-depleted stem cell-enriched peripheral blood cells from membranous vesicles Such as liposomes, red blood cell the HLA-nonmatched donor. Preferably a relative of the ghosts, and protoplasts, for example; and (4) physical trans patient, which donor was previously treated with a drug, e.g. fer techniques, Such as microinjection, electroporation, or a cytokine Such as granulocyte colony-stimulating factor direct “naked' DNA transfer. (G-CSF). 0.192 The bone marrow derived germline stem cells of 0187 Where radiation or chemotherapy is conducted the invention, their progenitors or their progeny, can be prior to administration, transplantation of bone marrow genetically altered by insertion of pre-Selected isolated derived germline Stem cells of the invention, their progeni DNA, by Substitution of a segment of the cellular genome tors or their progeny should optimally be provided within with pre-selected isolated DNA, or by deletion of or inac about one month of the cessation of therapy. However, tivation of at least a portion of the cellular genome of the transplantation at later points after treatment has ceased can cell. Deletion or inactivation of at least a portion of the be done with derivable clinical outcomes. cellular genome can be accomplished by a variety of means, 0188 As described herein, germline stem cells have been including but not limited to genetic recombination, by detected in the bone marrow. Therefore, bone marrow antisense technology (which can include the use of peptide derived germline Stem cells, and their progenitor cells, that nucleic acids, or PNAS), or by ribozyme technology, for can be used in the methods of the invention can comprise a example. The altered genome may contain the genetic purified Sub-population of cells including, but not limited to Sequence of a Selectable or Screenable marker gene that is expressed So that the cell with altered genome, or its male and female germline Stem cells. progeny, can be differentiated from cells having an unaltered 0189 Purified bone marrow derived germline stem cells, genome. For example, the marker may be a green, red, and their progenitor cells, can be obtained by Standard yellow fluorescent protein, B-galactosidase, the neomycin methods known in the art, including cell Sorting by FACs. resistance gene, dihydrofolate reductase (DHFR), or hygro Isolated bone marrow can be Sorted using flow cytometers mycin, but are not limited to these examples. US 2006/0010509 A1 Jan. 12, 2006
0193 In some cases, the underlying defect of a patho animals. Cells of the present invention can also be geneti logical State is a mutation in DNA encoding a protein Such cally modified using electroporation. as a metabolic protein. Preferably, the polypeptide encoded by the heterologous DNA lacks a mutation associated with 0196) Liposomal delivery of DNA or RNA to genetically a pathological State. In other cases, a pathological State is modify the cells can be performed using cationic lipoSomes, asSociated with a decrease in expression of a protein. A which form a stable complex with the polynucleotide. For genetically altered bone marrow derived germline Stem cell, Stabilization of the liposome complex, dioleoyl phosphati or its progeny, may contain DNA encoding Such a protein dylethanolamine (DOPE) or dioleoyl phosphatidylcholine under the control of a promoter that directs Strong expres (DOPQ) can be added. Commercially available reagents for Sion of the recombinant protein. Alternatively, the cell may liposomal transfer include Lipofectin (Life Technologies). express a gene that can be regulated by an inducible pro Lipofectin, for example, is a mixture of the cationic lipid moter or other control mechanism where conditions neces N-1-(2,3-dioleyloxy)propyl-N-N-N-trimethyl ammonia sitate highly controlled regulation or timing of the expres chloride and DOPE. Liposomes can carry larger pieces of Sion of a protein, enzyme, or other cell product. Such stem DNA, can generally protect the polynucleotide from degra cells, when transplanted into a Subject Suffering from abnor dation, and can be targeted to specific cells or tissues. mally low expression of the protein, produce high levels of Cationic lipid-mediated gene transfer efficiency can be the protein to confer a therapeutic benefit. For example, the enhanced by incorporating purified viral or cellular envelope bone marrow derived germline Stem cell of the invention, its components, Such as the purified G glycoprotein of the progenitor or its progeny, can contain heterologous DNA vesicular stomatitis virus envelope (VSV-G). Gene transfer encoding genes to be expressed, for example, in gene techniques which have been shown effective for delivery of therapy. Bone marrow derived germline Stem cells of the DNA into primary and established mammalian cell lines invention, their progenitors or their progeny, can contain using lipopolyamine-coated DNA can be used to introduce heterologous DNA encoding Atm, the gene responsible for target DNA into the bone marrow derived germline stem the human disease Ataxia-telangiectasia in which fertility is cells described herein. disrupted. Providing Atm via bone marrow derived female 0.197 Naked plasmid DNA can be injected directly into a germline Stem cells, their progenitors or their progeny, can tissue mass formed of differentiated cells from the isolated further relieve defects in ovarian function. DNA encoding a bone marrow derived germline Stem cells or their progeni gene product that alters the functional properties of bone tors. This technique has been shown to be effective in marrow derived germline Stem cells in the absence of any transferring plasmid DNA to skeletal muscle tissue, where disease State is also envisioned. For example, delivery of a expression in mouse skeletal muscle has been observed for gene that inhibits apoptosis, or that prevents differentiation more than 19 months following a single intramuscular would be beneficial. injection. More rapidly dividing cells take up naked plasmid 0194 Insertion of one or more pre-selected DNA DNA more efficiently. Therefore, it is advantageous to Sequences can be accomplished by homologous recombina stimulate cell division prior to treatment with plasmid DNA. tion or by viral integration into the host cell genome. The Microprojectile gene transfer can also be used to transfer desired gene Sequence can also be incorporated into the cell, genes into Stem cells either in vitro or in Vivo. The basic particularly into its nucleus, using a plasmid expression procedure for microprojectile gene transfer was described by vector and a nuclear localization Sequence. Methods for J. Wolff in Gene Therapeutics (1994), page 195. Similarly, directing polynucleotides to the nucleus have been described microparticle injection techniques have been described pre in the art. The genetic material can be introduced using viously, and methods are known to those of skill in the art. promoters that will allow for the gene of interest to be Signal peptides can be also attached to plasmid DNA to positively or negatively induced using certain chemicals/ direct the DNA to the nucleus for more efficient expression. drugs, to be eliminated following administration of a given drug?chemical, or can be tagged to allow induction by 0198 Viral vectors are used to genetically alter bone chemicals (including but not limited to the tamoxifen marrow derived germline Stem cells of the present invention responsive mutated estrogen receptor) expression in specific and their progeny. Viral vectors are used, as are the physical methods previously described, to deliver one or more target cell compartments (including but not limited to the cell genes, polynucleotides, antisense molecules, or ribozyme membrane). Sequences, for example, into the cells. Viral vectors and 0.195 Calcium phosphate transfection can be used to methods for using them to deliver DNA to cells are well introduce plasmid DNA containing a target gene or poly known to those of skill in the art. Examples of viral vectors nucleotide into isolated or cultured bone marrow derived that can be used to genetically alter the cells of the present germline Stem cells or their progenitors and is a Standard invention include, but are not limited to, adenoviral vectors, method of DNA transfer to those of skill in the art. DEAE adeno-associated viral vectors, retroviral vectors (including dextran transfection, which is also known to those of skill in lentiviral vectors), alphaviral vectors (e.g., Sindbis vectors), the art, may be preferred over calcium phosphate transfec and herpes virus vectors. tion where transient transfection is desired, as it is often more efficient. Since the cells of the present invention are 0199 Peptide or protein transfection is another method isolated cells, microinjection can be particularly effective for that can be used to genetically alter bone marrow derived transferring genetic material into the cells. This method is germline Stem cells of the invention and their progeny. advantageous because it provides delivery of the desired Peptides including, but not limited to, Pep-1 (commercially genetic material directly to the nucleus, avoiding both cyto available as Chariot'TM) and MPG, can quickly and effi plasmic and lysosomal degradation of the injected poly ciently transport biologically active proteins, peptides, anti nucleotide. This technique has been used effectively to bodies, and nucleic acids directly into cells, with an effi accomplish bone marrow derived modification in transgenic ciency of about 60% to about 95% (Morris, M. C. et al, US 2006/0010509 A1 Jan. 12, 2006
(2001) Nat. Biotech. 19: 1173-1176). Without wishing to be invention can be genetically modified, either through bound by theory, the peptide forms a non-covalent bond with manipulation of the oocyte in vitro prior to fertilization or the macromolecule of interest (i.e., protein, nucleic acid). manipulation of donor DNA prior to nuclear transfer into the The binding reaction Stabilizes the protein and protects it enucleated oocyte, to produce embryos having a desired from degradation. Upon delivery into the cell of interest, genetic trait. Such as Stem cells of the invention, the peptide-macromol ecule complex dissociates, leaving the macromolecule bio VII. In Vitro Fertilization logically active and free to proceed to its target organelle. Delivery can occur in the presence of absence of Serum. 0203 Oocytes produced from bone marrow derived Uptake and delivery can occur at 4 C., which eliminates female germline Stem cells of the invention, or their pro endoSomal processing of incoming macromolecules. Move genitor cells, as described herein can also be used for ment of macromolecules through the endoSomal pathway methods of in Vitro fertilization. Accordingly, the invention can modify the macromolecule upon uptake. Peptides Such provides methods for in vitro fertilization of a female as Pep-1, by directly delivering a protein, antibody, or Subject, comprising the Steps of peptide of interest, bypass the transcription-translation pro 0204 a) producing an oocyte by culturing a bone CCSS. marrow derived female germline Stem cell, or its pro 0200 Methods of the invention can provide oocyte genitor, in the presence of an oocyte differentiation reserves for use in eX Vivo procedures, Such as Somatic cell agent, nuclear transfer. Employing recombinant techniques prior to 0205 b) fertilizing the oocyte in vitro to form a zygote; nuclear transfer will allow for the design of customized and oocytes and ultimately produce embryoS from which embry onic Stem cells can be derived. In addition, genetic manipu 0206 c) implanting the Zygote into the uterus of a lation of donor DNA prior to nuclear transfer will result in female Subject. embryos that possess the desired modification or genetic trait. 0207 Methods of in vitro fertilization are well known in the art, and are now rapidly becoming commonplace. 0201 Methods of somatic cell nuclear transfer are well Couples are generally first evaluated to diagnose their par known in the art. See U.S. application Ser. No. 10/494,074, ticular infertility problem(s). These may range from unex filed on Mar. 24, 2004 and published as 20050064586; plained infertility of both partners to severe problems of the Wilmut et al. (1997) Nature, 385,810-813; Wakayama, et al. female (e.g., endometriosis resulting in nonpatent Oviducts (1998) Nature 394; 369-374; and Teruhiko et al., (1999) with irregular menstrual cycles or polycystic ovarian dis PNAS 96:14984-14989. Nuclear transplantation involves ease) or the male (e.g., low sperm count with morphological the transplantation of donor cells or cell nuclei into enucle abnormalities, or an inability to ejaculate normally as with ated oocytes. Enucleation of the oocyte can be performed in Spinal cord lesions, retrograde ejaculation, or reversed a number of manners well known to those of ordinary skill vasectomy). The results of these evaluations also determine in the art. Insertion of the donor cell or nucleus into the the Specific procedure to be performed for each couple. enucleated oocyte to form a reconstituted cell is usually by microinjection of a donor cell under the Zona pellucida prior 0208 Procedures often begin with the administration of a to fusion. Fusion may be induced by application of a DC drug to down-regulate the hypothalamic/pituitary System electrical pulse across the contact/fusion plane (electrofu (LHRH agonist). This process decreases Serum concentra Sion), by exposure of the cells to fusion-promoting chemi tions of the gonadotropins, and developing ovarian follicles cals, Such as polyethylene glycol, or by way of an inacti degenerate, thereby providing a Set of new follicles at earlier Vated virus, Such as the Sendai Virus. A reconstituted cell is Stages of development. This permits more precise control of typically activated by electrical and/or non-electrical means the maturation of these new follicles by administration of before, during, and/or after fusion of the nuclear donor and exogenous gonadotropins in the absence of influences by the recipient oocyte. Activation methods include electric pulses, hypothalamic pituitary axis. The progreSS of maturation and chemically induced shock, penetration by Sperm, increasing the number of growing follicles (usually four to ten stimu levels of divalent cations in the oocyte, and reducing phos lated per ovary) are monitored by daily observations using phorylation of cellular proteins (as by way of kinase inhibi ultrasound and serum estradiol determinations. When the tors) in the oocyte. The activated reconstituted cells, or follicles attain preovulatory size (18-21 mm) and estradiol embryos, are typically cultured in medium well known to concentrations continue to rise linearly, the OVulatory those of ordinary skill in the art and then transferred to the response is initiated by exogenous administration of human womb of an animal. chorionic gonadotropins (hCG). 0202 Methods for the generation of embryonic stem cells 0209 Oocytes can be obtained from bone marrow from embryos are also well known in the art. See Evans, et derived female germline Stem cells, or their progenitor cells, al. (1981) Nature, 29:154-156; Martin, et al. (1981) PNAS, as previously described herein. Bone marrow derived female 78:7634-7638; Smith, et al. (1987) Development Biology, germline Stem cells, or the progenitor cells, can be cultured 121:1-9; Notarianni, et al. (1991) J. Reprod. Fert., Suppl. in the presence of an oocyte differentiation agent which 43:255-260; Chen RL, et al. (1997) Biology of Reproduc induces differentiation into oocytes. The differentiation tion, 57 (4):756-764; Wianny, et al. (1999) Theriogenology, agent can be Supplied exogenously (e.g., added to the culture 52 (2): 195-212; Stekelenburg-Hamers, et al. (1995) Mol. medium) or from endogenous Sources during co-culture with Reprod. 40:444-454; Thomson, et al. (1995) PNAS, 92 allogenic or heterogenic ovarian tissue. Bone marrow (17):7844-8 and Thomson (1998) Science, 282 (6):1145 derived female germline Stem cells, or their progenitors, can 1147. Accordingly, embryoS produced from oocytes of the also be cultured in a tissue-engineered Structure wherein the US 2006/0010509 A1 Jan. 12, 2006 differentiation agent is either exogenously or endogenously Application Ser. No. , filed on May 17, 2005 as Supplied and oocytes are obtained. Attorney Docket No. 51588-62054, the contents of which 0210 Individual oocytes can be evaluated morphologi are herein incorporated by reference. Accordingly, the pos cally and transferred to a petri dish containing culture media Sibility that a larger germline Stem cell reservoir exists and heat-inactivated Serum. A Semen Sample is provided by Somewhere outside of the ovaries was considered. The first the male partner and processed using a “Swim up' proce clue in this regard was provided by the location of SSEA1* dure, whereby the most active, motile sperm will be cells in the medullary region of the ovary, which is the obtained for insemination. If the female's oviducts are principal entry and exit point for major blood vessels that present, a procedure called GIFT (gamete intrafallopian Supply the female gonads. SSEA1" cells may represent ger transfer) can be performed at this time. By this approach, mline Stem cells/progenitors thereof en-route to, rather than oocyte-cumulus complexes Surrounded by Sperm are placed resident in, the ovary. directly into the Oviducts by laproscopy. This procedure best Simulates the normal Sequences of events and permits fer 0214. During embryogenesis, primordial germ cells tilization to occur within the Oviducts. Not Surprisingly, (PGCs) and hematopoietic stem cells (HSCs) are known to GIFT has the highest success rate with 22% of the 3,750 originate from the same region-the proximal epiblast patients undergoing ova retrieval in 1990 having a live (Lawson and Hage (1994), Ciba Found. Symp. 182,68-84, delivery. An alternative procedure ZIFT (zygote intrafallo 84-91. Early HSCs then colonize the aorta-gonad-meso pian transfer) permits the Selection of in vitro fertilized nephric region of the developing embryo prior to migration Zygotes to be transferred to Oviducts the day following ova into the fetal liver (Medvinsky and Dzierzak, (1996) Cell 86, retrieval. Extra Zygotes can be cryopreserved at this time for 897-906), at roughly the equivalent time that PGCs enter the future transfer or for donation to couples without female Same region of the embryo to colonize the fetal gonads gametes. Most patients having more Serious infertility prob (McLaren, (2003) Dev. Bio. 262, 1-15); Molyneaux and lems, however, will require an additional one to two days Wylie, (2004) Int. J. Dev. Biol. 48, 537-544). In postnatal incubation in culture So that preembryos in the early cleav life, the hematopoietic System is maintained by Stem cells age States can be Selected for transfer to the uterus. This that eventually home to and reside in the bone marrow IVF-UT (in vitro fertilization uterine transfer) procedure (Morrison et al., (1995) Annu. Rev. Cell Dev. Biol. 11, entails the transcervical transfer of several 2-6 cell (day 2) 35-71); Attar and Scadden, (2004) Leukemia 18, 1760 or 8-16 (day 3) preembryos to the fundus of the uterus (4-5 1768). This information, along with the reported ability of preembryos provides optimal Success). PGCs to generate primitive HSCs in-vitro (Rich, (1995) Blood 86, 463-472) and the increasing number of studies 0211 Procedures for in vitro fertilization are also demonstrating the multi-lineage potential of adult bone described in U.S. Pat. Nos., 6,610,543 6,585,982, 6,544,166, marrow-derived cells (Herzog et al., (2003) Blood 102, 6,352,997, 6,281,013, 6,196,965, 6,130,086, 6,110,741, 3483-3493); (Grove et al., (2004) Stem Cells 22, 487-500); 6,040,340, 6,011,015, 6,010,448, 5,961,444, 5,882,928, (Heike and Nakahata, (2004) Int. J. Hematol. 79, 7-14), 5,827,174, 5,760,024, 5,744,366, 5,635,366, 5,691,194, prompted an investigation as to whether a molecular Signa 5,627,066, 5,563,059, 5,541,081, 5,538,948, 5,532,155, ture consistent with the presence of germ cells could be 5,512,476, 5,360,389, 5,296,375, 5,160,312, 5,147,315, identified in adult female bone marrow. 5,084,004, 4,902,286, 4,865,589, 4,846,785, 4,845,077, 4,832,681, 4,790,814, 4,725,579, 4,701,161, 4,654,025, 0215 For PCR analysis, total RNA was extracted from 4,642,094, 4,589,402, 4,339.434, 4,326,505, 4,193,392, each Sample of bone marrow isolated from adult female 4,062,942, and 3,854,470, the contents of which are spe mice and 1 lug was reverse transcribed (SuperScript II RT, cifically incorporated by reference for their description of Invitrogen) using oligo-dT primers. Amplification via 28-35 these procedures. cycles of PCR was then performed using Taq polymerase and Buffer-D (Epicentre) with primer sets specific for each 0212. The following examples are put forth for illustra gene. For each Sample, RNA encoded by the ribosomal gene tive purposes only and are not intended to limit the Scope of L7 (mouse Studies) was amplified and used as a loading what the inventors regard as their invention. control (house-keeping gene). All PCR products were isolated, Subcloned and Sequenced for confirmation. EXAMPLES 0216) For immunohistochemical detection, ovaries, tes Example 1 tes and bones (femurs) were fixed in 4% neutral-buffered paraformaldehyde, and bones were then decalcified for 72 hr Extra-Ovarian Female Germline Progenitor Cell in formic acid-EDTA. The tissues were subsequently sec Reservoirs tioned for immunohistochemical analysis using antibodies specific for MVH (T. Noce; Fujiwara et al., (1994) Cell 0213 The restricted pattern of SSEA1 expression in the Struct. Funct. 26, 131-136), HDAC6 (2162; Cell Signaling adult mouse ovary (FIG. 1a, b) suggested that the number Technology, Beverly, Mass.), NOBOX (A. Rajkovic; of germline Stem cells/progenitors thereof is relatively Suzumori et al., (2002) Science 305, 1157-1159), or GDF-9 Small. However, this would be incongruous with recent (AF739; R&D Systems, Minneapolis, Minn.) after high Studies indicating that germline Stem cells must offset an temperature antigen unmasking, as recommended by each extremely robust rate of oocyte death for the gonads to Supplier. The Sections were mounted with propidium iodide remain functional throughout reproductive life (Johnson, J. (Vectashield; Vector Laboratories, Burlingame, Calif.) or et al (2004) Nature 428, 145-150) as well as with the ability TO-PRO-3 iodide (Molecular Probes, Eugene, Oreg.) to of adult mouse ovaries to rapidly generate hundreds of new Visualize nuclei, and images were captured using a Zeiss primordial oocyte-containing follicles. For details, See U.S. LSM 5 Pascal Confocal Microscope. US 2006/0010509 A1 Jan. 12, 2006
0217. These experiments confirmed expression of Octá, which in adult mice is restricted to the germ lineage (Schöler TABLE 1. et al., (1989) EMBO J. 8, 2543-2550); (Yoshimizu et al., (1999) Dev. Growth Differ. 41, 675-684), as well as Mvh, Quantitative analysis of Mvh expression in adult mice. Dazl, Stella and a fifth germline marker gene termed Fragilis Tissue Analyzed Fold Difference in Mwh Levels (Saitou et al., (2002) Nature 418, 293-300), in bone marrow Female Bone Marrow - Estrus (1.0) isolated from adult female mice (FIGS. 2A-2D). In addition, Female Bone Marrow - Metestrus 9.52 expression of the female germ cell-specific homeobox gene, Ovary 598.29 Nobox (Suzumori et al., (2002) Mech. Dev. 111, 137-141), Male Bone Marrow 1.72 which is critical for directing expression of Octá and Gdf) in primordial oocytes as well as for folliculogenesis (Rajk Levels of Mvh expression in bone marrow of adult female ovic et al., 2004) Science 305, 1157-1159), was also detected mice at estrus were used as a reference point for compari in bone marrow of adult females (FIG. 2A). Sons, and all data were normalized against the levels of B-actin mRNA in each Sample prior to analysis. 0218. In light of these results, several public microarray 0219 Interestingly, Mvh expression was also detected in databases were Searched to provide independent confirma bone marrow of adult male mice, with a level of expression tion of the findings that multiple germline markers are slightly less than 20% of that detected in bone marrow of expressed in mouse and human bone marrow. For example, adult females at metestrus (Table 1). In addition, male bone expression of Mvh, Dazl, Stella and Fragilis have been marrow was also positive for Dazl expression, whereas identified in mouse bone marrow (Benson et al., (2004) Stella expression was below detectable limits. Nucleic Acids Res. 32 Database Issue, D23-D26); (Su et al., 0220. Using established bone marrow fractionation pro (2004) Proc. Natl. Acad. Sci. USA 101, 6062-6067), and tocols, bone marrow Samples were Sorted based on cell expression of STELLA has been demonstrated in human Surface Stem cell markers and quantitatively analyzed the bone marrow (GenBank Accession CV414052 from Dias resultant cell fractions for Mvh levels. Briefly, bone marrow Neto et al., 2000). Given the large number of studies was isolated from adult female mice and Sorted using a BD documenting the germline-restricted nature of Vasa gene Biosciences FACScalibur cytometer based on cell surface expression throughout the animal kingdom (Roussell and expression of Sca-1 (van de Rijn et al., (1989) Proc. Natl. Bennett, (1993) Proc. Natl. Acad. Sci. USA90,9300-9304); Acad. Sci. USA 86, 4634-4638) and/or c-Kit (Okada et al., (Fujiwara et al., (1994) Proc. Natl. Acad. Sci. USA 91, (1991) Blood 78, 1706-1712); (Okada et al., (1992) Blood 80, 3044-3050) following an initial immunomagnetic bead 12258-12262); (Komiya et al., (1994) Dev. Biol. 162,354 column-based fractionation Step to obtain lineage-depleted 363); (Rongo et al., (1997) Cold Spring Harb. Symp. Quant. (lin) cells (Spangrude et al., (1988) Science 241, 58-62); Biol. 62, 1-11); (Ikenishi)(1998) Growth Differ. 40, 1-10); (Spangrude and Scollay, (1990) Exp. Hematol. 18, 920 (Braat et al., (1999) RNA. Dev. Dyn. 216, 153-167); (Cas 926), as described (Shen et al., (2001) J. Immunol. 166, trillon et al., (2000) Proc. Natl. Acad. Sci. USA97, 9585 5027-5033); (Calvi et al., (2003) Nature 425, 841-846). For 9590); (Noce et al., (2001) Cell Struct. Funct. 26, 131-136); Serial passage-based enrichment of bone marrow-derived (Dearden et al., (2003) Dev. Genes Evol. 212, 599–603); stem cells in-vitro (Meirelles and Nardi, (2003) Br. J. (Fabioux et al., (2004) Biochem. Biophys. Res. Commun. Haematol. 123, 702-711); (Tropel et al., (2004) Exp. Cell 320, 592-598) Mvh was selected as a representative end Res. 295,395-406), bone marrow isolated from adult female point to next quantitatively assess potential changes in the mice was plated on plastic in Dulbecco's modified Eagle's medium (Fisher Scientific, Pittsburgh, Pa.) with 10% fetal levels of germline marker expression in bone marrow during bovine Serum (Hyclone, Logan, Utah), penicillin, Strepto the female reproductive cycle. Using real-time PCR with mycin, L-glutamine and amphotericin-B. Forty-eight hr Standardization against the levels of B-actin mRNA in each after the initial plating, the Supernatants containing non Sample, marked estrous cycle-related changes in Mvh adherent cells were removed and replaced with fresh culture expression in bone marrow of adult female mice were medium after gentle Washing. The cultures were then main uncovered, with a 9.52-fold difference noted between estrus tained and passed once confluence was reached for a total of (nadir) and metestrus (peak) (FIG. 2E). A parallel evalua three times over the Span of 6 weeks, at which time the tion of ovarian germ cell dynamics in the same animals cultures were terminated to collect adherent cells for analy revealed a Striking positive correlation between the estrous SS. cycle-related changes in bone marrow MVh expression and 0221. After removal of differentiated cells committed to primordial follicle numbers, with ovaries at meteStruS con hematolymphoid lineages by negative Selection, Mvh taining over 800 more primordial follicles than ovaries at expression was retained in the lineage-depleted (lin) cell estrus (FIG.2F). In light of these findings, the levels of Mvh fraction with levels comparable to those observed in crude in bone marrow of adult females in meteStruS were com bone marrow (FIG. 3A). Subsequent separation of the pared to levels present in adult ovaries, which contain lin cells based on cell Surface expression of Sca-1 (van de thousands of Mvh-expressing oocytes (Fujiwara et al., 1994; Rijn et al., 1989) or c-Kit (Okada et al., 1991) further Noce et al., 2001; see also FIG. 2D), or in bone marrow of revealed that the majority of Mvh-expressing cells were adult male mice. These experiments demonstrated that Mvh negative for expression of Sca-1 but positive for c-Kit transcript levels in bone marrow of adult females at (Sca-1/c-Kit") (FIG. 3A). Moreover, expression of the metestrus were 1.6% of those detected in adult ovaries other germline markers co-Segregated with Mvh in the (Table 1). Sca-1/c-Kit"cell fraction. In parallel experiments, in-vitro US 2006/0010509 A1 Jan. 12, 2006 22 culture of adult female bone marrow-derived cells on plastic Example 3 under conditions shown previously to permit the progressive enrichment of stem cells from bone marrow (Meirelles and Bone Marrow Transplantation Rescues Oocyte Nardi, (2003) Br. J. Haematol. 123,702-711); (Tropel et al., Production in Atm Mutants (2004) Exp. Cell Res. 295, 395-406), demonstrated that all of the germline markers present in freshly isolated bone 0226 Atm(homozygous null) mice, created by tar marrow Samples were expressed by the adherent cell frac geted inactivation of the Atm gene, display many of the tion and remained So following multiple Serial passages over hallmarks of the Ataxia-telangiectasia Syndrome in humans, a 6-week period (FIG. 3B). including growth retardation, defects in T lymphocyte matu ration and infertility (Bagley et al. (2004) Blood 12: 1). Example 2 Atm-deficient male and female mice have been shown to be infertile due to the complete loSS of the production of mature gametes, i.e., Spermatozoa and oocytes (Barlow, C. et al. Bone Marrow Transplantation Reverses (1996) Cell 86: 159). These gametogenesis defects in mutant Pathological Ovarian Failure mice lacking Atm result from apoptosis and degeneration of 0222 To assess the functional capacity of bone marrow the developing gametes that exhibit aberrant early Stages of derived germ cells to produce new oocytes, bone marrow meiosis, detected as early as the leptotene stage (Barlow, C. was isolated from adult wild-type female mice and trans et al. (1998) Development 125: 4007). Ovaries from Atm planted using Standard procedures into recipient adult deficient females were shown to be completely barren of females Sterilized by treatment with a combination of cyclo oocytes and follicles by at least 11 days of age postpartum phosphamide and buSulphan to destroy the existing pre- and (Barlow, C. et al. (1998) Development 125: 4007). post-meiotic germ cell pools prior to BMT. 0227 To first confirm and extend these findings, the 0223 Bone marrow was harvested from adult (6-10 ovaries of wild-type mice were compared with ovaries from weeks of age) wild-type C57BL/6 female mice on the day of Atm gene-deficient mice. Representative histology of post transplantation, and 2-5x10" cells were injected intrave partum day 4 wild-type (FIG. 6A, magnified in C) and nously via the tail vein into recipients using Standard pro Atm-null (B, D) ovaries is shown in FIG. 6. Representative cedures. To prepare recipients, female mice received 0.5 mg histology of adult wild-type (E) and Atm-null (F) ovaries anti-CD4 antibody (GK1.5) (Dialynas, D. P. et al. (1984) J. from adult mice is also shown in FIG. 6. In keeping with Immunol. 131, 2445-2451) and 1 mg anti-CD8 antibody past reports, ovaries from Atm-null animals, irrespective of (2.43) (Sarmiento, M. et al. (1980) J. Immunol. 125, 2665 postnatal age, are barren of oocytes. However, in light of the 2672) one week prior to a second injection of each antibody recent detection of pre-meiotic germline Stem cells in the along with 120 mg kg cyclophosphamide (Cytoxan; Bris postnatal mouse ovary (Johnson et al., (2004) Nature 428: tol-Meyers Squibb) and 12 mg kg busulphan (Sigma) at 6 145), it was possible that pre-meiotic germline stem cells weeks of age. Mice were conditioned before BMT with were present and capable of Self-renewal, but ongoing cyclophosphamide and buSulphan, the latter of which Selec oocyte production was impossible due to failed meiotic tively removes the contribution of germline Stem cells to entry in the absence of Atm. adult gonadal function in both male and female mice. Bone 0228. The expression of germline lineage markers in the marrow transplantation was performed 1 or 7 days later. Atm-deficient ovary versus wild-type controls was per Animals were then euthanized for collection and analysis of formed by reverse-transcription followed by PCR (RT-PCR) ovaries at the indicated times following BMT. and representative data (n=3) are shown in FIG. 7. As 0224. Two months later, very few, if any, immature predicted, the pluripotency marker Oct-4 (Brehm et al., oocytes or follicles were detected in the ovaries of those (1998) APMIS 106: 114) and the germline markers Dazl females given cyclophosphamide and buSulphan alone (McNeilly et al., (2000) Endocrinology 141:4284); (Nishi et (FIG. 4). However, ovaries of mice receiving BMT after al., (1999) Mol Hum Reprod 5:495); Stella (Bortvin et al., combination chemotherapy possessed hundreds of oocyte (2004) BMC Dev Biol 23:2 and the mouse Vasa homologue, containing follicles at all Stages of development, including Mvh (Fujiwara, Y. et al. (1994) Proc. Natl. Acad. Sci. USA the resting primordial Stage that is most Susceptible to the 91, 12258-12262) are all expressed in the Atm-deficient cytotoxic actions of these drugs (FIGS. 4 and 5C). ovary at postnatal day 71. Semi-quantitative comparison of the relative levels of these genes by examination of the 0225. Histological evaluations further substantiated that loading control L7 shows that, as expected, these genes are the chemotherapy regimen essentially destroyed the ova expressed at much lower levels than in wild-type ovaries ries-which, after treatment, were composed of little more containing oocytes. The contralateral ovary in each animal than Stromal and interstitial cells with a random cystic follicle or old corpus luteum occasionally observed (FIG. used for RT-PCR analysis was prepared for histology, and 5B). By comparison, ovaries of mice receiving BMT, even the Sampling and examination of histological Sections from when the transplants were given a week after inflicting the Atm-null mice did not reveal any oocytes or structures damage to the tissue, possessed a spectrum of maturing resembling follicles, as expected. Thus, Atm-deficiency follicles as well as corpora lutea indicative of a resumption results in a pool of germline Stem cells that persist into adult of normal ovulatory cycles (FIG.5C). Furthermore, oocytes life (day 71) but these cells cannot, as reported, produce and follicles were found in ovaries of chemotherapy-Steril viable oocytes due to the meiotic defect that results in ized females more than 11 months after the initial transplan gamete death when Atm is absent. tation (FIGS. 5D-5E), indicating that bone marrow-derived 0229 Whether extra-ovarian cells have the ability to germ cells are capable of Sustaining long-term oocyte pro form germ cells was further investigated. Due to its pheno duction. type, the Atm-null mouse was Selected for evaluation as US 2006/0010509 A1 Jan. 12, 2006 23 these animals are genetically incapable of producing 0239 Borum, K. Oogenesis in the mouse. (1961). A study oocytes. If oocytes were detected in the ovaries of Atm-null of meiotic prophase. Exp. Cell Res. 24, 495-507. mice that received the transplants, they must be derived from the tissue transplanted (i.e., bone marrow) based on the 0240 Braat, A. K., Zandbergen, T., van de Water, S., nature of the Atm defect in the host animal. Bone marrow Goos, H. J., and Zivkovic, D. (1999). Charatcerization of transplantation as performed as described above. Zebrafish primordial germ cells: morphology and early distribution of vasa RNA. Dev. Dyn. 216, 153-167. 0230. Although the mutant females are genetically inca pable of generating oocytes from early germ cells, Atm-null 0241 Brinster, C. J., Ryu, B. Y., Avarbock, M. R., female mice were nonetheless conditioned with cyclophoS Karagenc, L., Brinster, R. L., and Orwig, K. E. (2003). phamide and buSulfan (See above) to eliminate the possibil Restoration of fertility by germ cell transplantation ity of host germ cell contribution to oocyte production requires effective recipient preparation. Biol. Reprod. 69, following BMT. In contrast to the complete absence of 412-420. oocytes in non-transplanted Atm mutants, both the wild-type 0242 Brinster, R. L. (2002). Germline stem cell trans mouse and the Atm-null mouse that received exogenous, planation and transgenesis. Science 296, 2174-2176. wild-type bone marrow exhibited normal oocytes within normal appearing follicles (FIG. 8). Oocyte containing 0243 Bucci, L. R., and Meistrich, M. L. (1987). Effects follicles were found in transplanted Atm-null females for at of buSulfan on murine Spermatogenesis: cytotoxicity, Ste least 11 months after the initial BMT. rility, Sperm abnormalities, and dominant lethal muta 0231. These results indicate that transplanted wild-type tions. Mutat. Res. 176, 259-268. bone marrow contains female germline Stem cells which can 0244 Calvi, L. M., Adams, G. B., Weilbrecht, K. W., then go on to Successfully differentiate into oocytes via Weber, J. M., Olson, D. P., Knicht, M. C., Martin, R. P., meiosis Since these transplanted cells contain functional Schipani, E., Divietti, P., Bringhurst, F. R., Milner, L.A., Atm. Kronenberg, H. M., and Scadden, D. T. (2003). 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(1996). Germline regulatory element of Oct-4 specific for 14. A method of oocyte production, comprising culturing the totipotent cycle of embryonal cells. Development 122, the isolated cell of claim 1 in the presence of an agent that 881-894. differentiates the cell into an oocyte, thereby producing an oocyte. 0340 Yoshimizu, T., Sugiyama, N., De Felice, M., Yeom, 15. The method of claim 14, wherein the agent is selected Y. I., Ohbo, K., Masuko, K., Obinata, M., Abe, K., from the group consisting of a transforming growth factor, Schöler, H. R., and Matsui, Y. (1999). Germline-specific bone morphogenic protein, Wnt family protein, kit-ligand, expression of the Oct-4/green fluorescent protein (GFP) leukemia inhibitory factor, meiosis-activating Sterol, modu transgene in mice. Dev. Growth Differ. 41, 675-684. lator of Id protein function and modulator of Snail/Slug 0341 Yuan, L., Liu, J. G., Hoja, M. R., Wilbertz, J., transcription factor function. Nordqvist, K., and Hoog, C. (2002). Female germ cell 16. A pharmaceutical composition comprising a purified aneuploidy and embryo death in mice lacking the meiosis population of cells that are mitotically competent, have an specific protein SCP3. Science 296,1115-1118. XXkaryotype and express Vasa, Oct-4, Dazl, Stella, Fragilis and optionally, Nobox, c-Kit and Sca-1 and a pharmaceuti 0342 Zhu, C. H., and Xie, T. (2003). Clonal expansion of cally acceptable carrier. Ovarian germline Stem cells during niche formation in 17. The pharmaceutical composition of claim 16, wherein Drosophila. Development 130, 2579-258. the cells are purified from the bone marrow. 0343 Zuckerman, S. (1951). The number of oocytes in 18. The pharmaceutical composition of claim 16, wherein the mature ovary. Recent Prog. Horm. Res. 6, 63-108. the cells are mammalian cells. 0344). Zuckerman, S., and Baker, T. G. (1977). The devel 19. The pharmaceutical composition of claim 16, wherein opment of the ovary and the process of oogenesis. In The the cells are human cells. 20. The pharmaceutical composition of claim 16, wherein Ovary, S. Zuckerman and B. J. Weir, eds. (New York, the purified population of cells is about 50 to about 55%, N.Y.: Academic Press), pp. 41-67. about 55 to about 60%, about 65 to about 760%, about 70 to 1. An isolated bone marrow cell that is mitotically com about 75%, about 75 to about 80%, about 80 to about 85%, petent, has an XX kayrotype and expresses Vasa, Oct-4, about 85 to about 90%, about 90 to about 95% or about 95 Dazl, Stella, Fragilis and optionally, Nobox, c-Kit and Sca-1. to about 100% of the cells in the composition. 2. The isolated cell of claim 1, wherein the cell can 21. A method of oocyte production in a Subject, compris produce oocytes after a duration of at least 1 week, 1 to ing providing the pharmaceutical composition of claim 16 to about 2 weeks, about 2 to about 3 weeks, about 3 to about a tissue of the Subject, wherein the cells engraft into the 4 weeks or more than about 5 weeks post transplantation into tissue and differentiate into oocytes, thereby producing a host. oocytes in the Subject. 3. The isolated cell of claim 1, wherein the cell can 22. The method of claim 21, wherein the tissue is ovarian produce oocytes after a duration of less than 1 week post tissue. transplantation into a host. 23. A method of inducing folliculogenesis in a Subject, 4. The isolated cell of claim 1, wherein the cell can comprising providing the pharmaceutical composition of produce oocytes after a duration of less than about 24 to claim 16 to the Subject, wherein the cells engraft into a tissue about 48 hours post transplantation into a host. of the subject and differentiate into oocytes within follicles, 5. The isolated cell of claim 2, wherein the cell is a bone thereby inducing folliculogenesis in the Subject. marrow derived female germline Stem cell. 24. The method of claim 23, wherein the tissue is ovarian 6. The isolated cell of claim 3, wherein the cell is a bone tissue. marrow derived female germline Stem cell progenitor. 25. A method of treating infertility in a female subject in 7. The isolated cell of claim 1, wherein the cell is a need thereof comprising administering a therapeutically mammalian cell. effective amount of the pharmaceutical composition of claim 8. The isolated cell of claim 1, wherein the cell is a human 16 to the Subject, wherein the cells engraft into the ovary and cell. differentiate into oocytes, thereby treating infertility. 9. The isolated cell of claim 1, wherein the cell is a 26. A method of repairing damaged ovarian tissue in a non-embryonic cell. Subject, comprising providing a therapeutically effective 10. The isolated cell of claim 1, wherein the cell expresses amount of the pharmaceutical composition of claim 16 to the Nobox. tissue, wherein the cells engraft into the tissue and differ 11. The isolated cell of claim 1, wherein the cell expresses entiate into oocytes, thereby repairing the damaged tissue in c-Kit. the Subject. 12. The isolated cell of claim 1, wherein the cell expresses 27. The method of claim 26, wherein the damage is a Sca-1. result of exposure to chemotherapeutic drugs or radiation. 28. The method of claim 27, wherein the chemotherapeu 13. A method of in vitro fertilization of a female subject, tic drug is Selected from the group consisting of buSulfan, Said method comprising the Steps of: cyclophosphamide, 5-FU, vinblastine, actinomycin D, eto a) producing an oocyte by culturing the isolated cell of poside, cisplatin, methotrexate, and doxorubicin. claim 1 in the presence of an agent that differentiates 29. The method of claim 26, wherein the damage is a the cell into an oocyte, result of a cancer, polycystic ovary disease, genetic disorder, immune disorder or metabolic disorder. b) fertilizing the oocyte in vitro to form a zygote; and 30. A method of restoring ovarian function in a meno c) implanting the Zygote into the uterus of a female pausal female Subject, comprising administering a therapeu Subject. tically effective amount of the pharmaceutical composition US 2006/0010509 A1 Jan. 12, 2006 28 of claim 16 to the Subject, wherein the cells engraft into the inhibitor, caspase inhibitor, inhibitor of nitric oxide produc ovary and differentiate into oocytes, thereby restoring ova tion and inhibitor of histone deacetylase activity. rian function in the Subject. 42. A method of restoring fertility to a female subject who 31. The method of claim 30, wherein the menopausal desires restored fertility, comprising administering a thera female Subject is in a stage of either peri- or post-meno peutically effective amount of bone marrow derived female pause. germline Stem cells, or their progenitor cells, to the Subject, 32. A method for oocyte production in a Subject, com wherein the cells engraft into a tissue and differentiate into prising contacting bone marrow derived female germline oocytes, thereby restoring fertility in the Subject. Stem cells, or their progenitor cells, of the Subject with an 43. The method of claim 42, wherein the tissue is ovarian agent that differentiates the bone marrow derived female tissue. germline Stem cells, or their progenitor cells, into oocytes, 44. A method of protecting fertility in a female subject thereby producing oocytes in the Subject. undergoing or expected to undergo chemotherapy, radio 33. The method of claim 32, wherein the agent is selected therapy or both treatments, comprising providing an agent from the group consisting of a transforming growth factor, that protects against reproductive injury prior to or concur bone morphogenic protein, Wnt family protein, kit-ligand, rently with chemotherapy, radiotherapy or both treatments leukemia inhibitory factor, meiosis-activating Sterol, modu and providing a bone marrow derived female germline Stem cell, or its progenitor cell, to the Subject, wherein the cell lator of Id protein function and modulator of Snail/Slug engrafts into a tissue and differentiates into an oocyte, transcription factor function. thereby protecting fertility in the Subject. 34. A kit for oocyte production comprising an agent of 45. The method of claim 44, wherein the agent is selected claim 33 and instructions for using the agent to differentiate from the group consisting of S1P, a Bax antagonist, or any the bone marrow derived female germline Stem cells, or their agent that increases SDF-1 activity. progenitor cells, into oocytes, thereby producing oocytes. 46. A kit for protecting fertility in a female subject 35. A method of expanding bone marrow derived female undergoing or expected to undergo chemotherapy, radio germline Stem cells, or their progenitor cells, in Vivo, ex vivo therapy or both treatments, comprising an agent of claim 45 or in vitro, comprising contacting the bone marrow derived and instructions for using the agent to protect bone marrow female germline Stem cells, or their progenitor cells, with an derived female germline Stem cells, or their progenitor cells, agent that increases the amount of bone marrow derived against reproductive injury thereby protecting fertility in the female germline Stem cells, or their progenitor cells, thereby female Subject. expanding the bone marrow derived female germline stem 47. A method for in vitro fertilization of a female Subject, cells, or their progenitor cells. Said method comprising the Steps of: 36. The method of claim 35, wherein the amount of bone marrow derived female germline Stem cells, or their pro a) producing an oocyte by contacting a bone marrow genitor cells, is increased with an agent that promotes cell derived female germline Stem cell, or its progenitor proliferation or survival. cell, with an agent that differentiates the bone marrow 37. The method of claim 36, wherein the agent is selected derived female germline Stem cell, or its progenitor from the group consisting of an insulin-like growth factor, cell, into an oocyte, transforming growth factor, bone morphogenic protein, Wnt b) fertilizing the oocyte in vitro to form a zygote; and protein, fibroblast growth factor, Sphingosine-1-phosphate, c) implanting the Zygote into the uterus of a female retinoic acid, inhibitor of glycogen Synthase kinase-3, Bax Subject. inhibitor, caspase inhibitor, inhibitor of nitric oxide produc 48. An isolated bone marrow cell that is mitotically tion and inhibitor of histone deacetylase activity. competent, has an XY kayrotype and expresses Vasa and 38. A kit for expanding bone marrow derived female Dazl. germline Stem cells, or their progenitor cells, comprising an 49. The isolated cell of claim 48, wherein the cell is a agent of claim 37, and instructions for using the agent to mammalian cell. increase the amount of bone marrow derived female germ 50. The isolated cell of claim 48, wherein the cell is a line Stem cells or their progenitor cells, thereby expanding human cell. the bone marrow derived female germline Stem cells, or their 51. The isolated cell of claim 48, wherein the cell is a progenitor cells. non-embryonic cell. 39. A method for oocyte production in a subject, com 52. A method of restoring or enhancing Spermatogenesis, prising contacting bone marrow derived female germline comprising providing a bone marrow derived male germline Stem cells, or their progenitor cells, of the Subject with an Stem cell, or its progenitor cell, to the testes of a male agent that increases the amount of bone marrow derived Subject, wherein the cell engrafts into the Seminiferous female germline Stem cells, or their progenitor cells, thereby epithelium and differentiates into a Sperm cell, thereby producing oocytes in the Subject. restoring or enhancing Spermatogenesis. 40. The method of claim 39, wherein the agent increases 53. A method of restoring fertility to a male subject having the Survival or proliferation of the cells, thereby increasing undergone chemotherapy or radiotherapy, or both and who the amount of the cells. desires restored fertility, comprising administering a thera 41. The method of claim 40, wherein the agent that peutically effective amount of bone marrow derived male increases Survival or proliferation of the cells is Selected germline Stem cells, or their progenitor cells, to the Subject, from the group consisting of an insulin-like growth factor, wherein the cells engraft into the Seminiferous epithelium transforming growth factor, bone morphogenic protein, Wnt and differentiate into Sperm cells, thereby restoring fertility. protein, fibroblast growth factor, Sphingosine-1-phosphate, 54. A method of reducing the amount of bone marrow retinoic acid, inhibitor of glycogen Synthase kinase-3, Bax derived germline Stem cells, or their progenitor cells, in a US 2006/0010509 A1 Jan. 12, 2006 29
Subject comprising contacting bone marrow derived germ an agent that promotes cell death, thereby reducing the line Stem cells, or their progenitor cells, in the Subject with amount of bone marrow derived germline Stem cells, or their an agent that reduces cell proliferation, thereby reducing the progenitor cells, in the Subject. amount of bone marrow derived germline Stem cells, or their 62. The method of claim 61, wherein the agent that progenitor cells, in the Subject. promotes cell death is Selected from the group consisting of 55. The method of claim 54, wherein the agent is selected a pro-apoptotic tumor necrosis factor Superfamily member, from the group consisting of a transforming growth factor-B, agonist of pro-apoptotic Bcl-2 family member function and bone morphogenic protein antagonist, Protein Related to ceramide. DAN and Cerberus and Gremlin. 63. The method of claim 62, wherein the pro-apoptotic 56. A method of reducing the amount of bone marrow tumor necrosis factor Superfamily member is Selected from derived germline Stem cells, or their progenitor cells, in a the group consisting of TNF alpha, Fas-ligand and TRAIL. Subject comprising contacting bone marrow derived germ 64. The method of claim 62, wherein the pro-apoptotic line Stem cells, or their progenitor cells, in the Subject with Bcl-2 family member is Selected from the group consisting an agent that inhibits cell Survival, thereby reducing the of BAX, BAK, BID, HRK, BOD, BIM, NOXA, PUMA, amount of bone marrow derived germline Stem cells, or their BOK and BCL-XS. progenitor cells, in the Subject. 65. Akit for reducing the amount of bone marrow derived 57. The method of claim 56, wherein the agent that germline Stem cells, or their progenitor cells, comprising an inhibits Survival is Selected from the group consisting of a agent of claim 62, and instructions for using the agent to pro-apoptotic tumor necrosis factor Super family member, promote cell death of bone marrow derived germline Stem antagonist of pro-Survival Bcl-2 family member function cells, or their progenitors, thereby reducing the amount of and ceramide. bone marrow derived germline Stem cells, or their progeni 58. The method of claim 57, wherein the pro-apoptotic tor cells. tumor necrosis factor Super family member is Selected from 66. The method of claims 54, 56 or 61, wherein the the group consisting of tumor necrosis factor-C, Fas-ligand Subject has a precancerous or cancerous condition. and TRAIL. 67. The method of claim 66, wherein the cancerous 59. The method of claim 57, wherein the pro-survival condition is a germ cell tumor, Ovarian cancer or teratoma. Bcl-2 family member is Selected from the group consisting 68. A method of providing contraception to a Subject of Bcl-2, Bcl-XL, Bcl-W. Mcl-1 and A1. comprising contacting bone marrow derived germline Stem 60. Akit for reducing the amount of bone marrow derived cells, or their progenitor cells, with an agent that decreases germline Stem cells, or their progenitor cells, comprising an the amount of bone marrow derived germline Stem cells, or agent of claim 57, and instructions for using the agent to their progenitor cells, thereby providing contraception to the inhibit cell survival of bone marrow derived germline stem Subject. cells, or their progenitor cells, thereby reducing the amount 69. A kit for contraception in a Subject comprising an of bone marrow derived germline Stem cells, or their pro agent of claim 68, and instructions for using the agent to genitor cells. decrease the amount of bone marrow derived germline Stem 61. A method of reducing the amount of bone marrow cells, or their progenitor cells, thereby providing contracep derived germline Stem cells, or their progenitor cells, in a tion to the Subject. Subject comprising contacting bone marrow derived germ line Stem cells, or their progenitor cells, in the Subject with