USOO898063 OB2

(12) United States Patent (10) Patent No.: US 8,980,630 B2 Woodbury et al. (45) Date of Patent: Mar. 17, 2015

(54) OBTAINING MULTIPOTENT (56) References Cited AMNION-DERIVED STEM CELL (ADSC) U.S. PATENT DOCUMENTS FROM AMNOTIC MEMBRANE TISSUE M WITHOUT ENZYMATIC DIGESTION 2005, 0118712 A1 6, 2005 Tsai et al. 2007/0243172 A1 10, 2007 Ra et al. (75) Inventors: Dale Woodbury, Middletown, NJ (US); Akiva J. Marcus, Teaneck, NJ (US) FOREIGN PATENT DOCUMENTS (73) Assignee: Rutgers, The State University of New WO WOOOf73421 * 12/2000 WO O2O64.755 A2 8, 2002 Jersey, Somerset, NJ (US) WO 2005042703 A2 5, 2005 WO 2006O19357 A1 2, 2006 (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 1293 days. Zhao et al. Human Amniotic Mesenchymal Cells Have Some Char acteristics of Cardiomyocytes Transplantation, Mar. 2005, vol. 79, (21) Appl. No.: 12/306,871 pp. 528-535.* Tsai et al. Isolation of human multipotent mesenchymal stem cells (22) PCT Filed: Jun. 28, 2007 from second-trimester amniotic fluid using a novel two-stage culture protocol Human Reproduction, 2004, vol. 19, pp. 1450-1456.*

(86). PCT No.: PCT/US2007/072356 notypessgyet of Neuroglial al. As Progenior style Uells. J Neuroscience Cells E.s: Kes. S371 (c)(1), vol. 78, pp. 208-214.* (2), (4) Date: May 6, 2009 Grueterichet al. Connexin 43 Expression and Proliferation of Human Limbal Epithelium on Intact and Denuded Amniotic Membrane. (87) PCT Pub. No.: WO2008/003042 Investigative Ophthalmology and VicSula Science, 2002, vol.43, pp. 63-71.* PCT Pub. Date: Jan. 3, 2008 Jietal. Cryopreservation of Adherent Human Embryonic StemCells Lin. Biotechnology and Bioengineering, 2004, vol.88, pp. 299-312.* (65) Prior Publication Data Zhao et al. Human Amniotic Mesenchymal Cells Have Some Char acteristics of Cardiomyocytes. Transplantation, Mar. 2005, vol. 79, US 2009/O2388O1 A1 Sep. 24, 2009 pp. 528-535.* Casey et al. Interstitial Collagen Synthesis and Processing in Human Amnion: A Property of the Mesenchymal Cells. Biology of Repro O O duction, 1996, vol. 55, pp. 1253-1260.* Related U.S. Application Data Soncini et al. Isolation and characterization of mesenchymal cells (60) Provisional application No. 60/816,987, filed on Jun. from human fetal membranes. J. Tissue Engeering Regenerative 28, 2006, provisional application No. 60/930,782, Medicine, 2007, vol. 1 pp. 296-305.* filed on May 18, 2007. Kim et al. Ex Vivo Characteristics of Human Amniotic Membrane Derived StemCells. Cloning and StemCells, 2007, vol. 9, pp. 581 594 * (51) y;'/02 (2006.01) Kim et al. Amniotic mesenchymal stem cells have robust angiogenic AOIN I/02 (200 6. 01) properties and are effective in treating hindlimb ischaemia. Cardio vascular Research, 2012, vol. 93, pp. 525-534.* CI2N 5/073 (2010.01) Hu et al. Progress in studies on the characteristics of human amnion CI2N 5/0793 (2010.01) mesenchymal cells. Progress in Natural Science, 2009, vol. 19, pp. CI2N 5/077 (2010.01) 1047-1052. CI2N 5/071 (2010.01) Roubelakis et al. Amniotic Fluid and AmnioticMembrane Stem C1 2N 5/0775 (2010.01) Cells: Marker Discovery. StemCells International, vol. 2012, pp. 1-9, (52) U.S. Cl doi:10.1155, 2012/107836. CPC CI2N5/0605 (2013.01); C12N5/0668 De Rosa et al. Amniotic Fluid-Derived Mesenchymal Stem Cells ------2013 O1): CI2N 2509500 2 03 O1): CI2N Lead to Bone Differentiation when Cocultured with Dental Pulp ( .01): ( .01): StemCells, Tissue Engineering; Part Amvol. 17, pp. 645-654, 2011.* 2506/1353 (2013.01); C12N5/0619 (2013.01); (Continued) CI2N5/0653 (2013.01); CI2N5/0654 (2013.01); C12N5/067 (2013.01); C12N 2501/115 (2013.01); C12N 250I/I 19 (2013.01); Primary Examiner – Deborah Crouch CI2N 2501/12 (2013.01); C12N 2501/23 (74) Attorney, Agent, or Firm — Miller, Matthias & Hull (2013.01); C12N 2506/02 (2013.01) LLP USPC ...... 435/378; 435/383; 435/13 (58) Field of Classification Search (57) ABSTRACT CPC. C12N5/0605; C12N5/0606; C12N 5/0668; The present invention relates to stem cells obtained from the C12N 2501/11: C12N 5/0623; C12N 5/0647; amnion and their methods of obtaining and culturing. The C12N5/0663; C12N5/0675; C12N 2500/99; present invention further relates to compositions comprising C12N5/0607; C12N 2501/119; C12N amnion-derived stems cells (ADSCs) and to methods of using 2501/12: C12N 2501/15; C12N 2501/148; ADSCS. C12N 25O1/235 See application file for complete search history. 5 Claims, 14 Drawing Sheets US 8,980,630 B2 Page 2

(56) References Cited Miki et al., “StemCell Characteristics of Amniotic Epithelial Cells.” Stem Cells (2005): vol. 23; pp. 1549-1559. OTHER PUBLICATIONS Marcus et al., “Isolation, characterization, and differentiation of stem Deng et al. nVitro Differentiation of Human Marrow Stromal Cells into Early Progenitors of Neural Cells by Conditions That Increase cells derived from the rat amniotic membrane. Differentiation Intracellular Cyclic AMP. Biochm. Biophys. Res. Comm., vol. 282, (2008): vol. 76; pp. 130-144. pp. 148-152, 2001.* Marcus et al., "Fetal stem cells from extra-embryonic tissues: do not Tamagawa et al., “Establishment and Characterization of a Pluripotent Stem Cell Line Derived from Human Amniotic mem discard.” J. Cell. Mol. Med. (2008): vol. 12, No. 3; pp. 730-742. branes and Initiation of Germ. Layers in vitro.” Human Cell (2004): vol. 17, No. 3; pp. 125-130. * cited by examiner U.S. Patent Mar. 17, 2015 Sheet 1 of 14 US 8,980,630 B2

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US 8,980,630 B2 1. 2 OBTAINING MULTIPOTENT perspective, ES cells often form tumors following transplan AMNION-DERIVED STEM CELL (ADSC) tation into rodents (Evans and Kaufman, 1983). Furthermore, FROM AMNOTIC MEMBRANE TISSUE ES cells might not be able to overcome the immunological WITHOUT ENZYMATC DGESTION incompatibility that exists between host and grafted cells (Keller, 2005). With all these unresolved issues many inves CROSS-REFERENCE TO RELATED tigators have turned to adult and fetal tissue in search of less APPLICATIONS controversial stem and precursor populations. The wide distribution and plasticity of adult stem cells has This application is a National Stage filing under 35 U.S.C. only recently been appreciated. In addition to the well known S371 (c) of International Application Serial No. PCT/US07/ 10 stem cells of the adult marrow lymphohematopoietic (Shi 072356 filed Jun. 28, 2007, which, in turn, claims the benefit Zuru et al., 2005; Krause et al., 2001) and stromal mesenchy under 35 U.S.C. S 119(e) of U.S. Provisional Patent Applica mal lineages (Prockop, 1997: Jiang et al., 2002), adult stem tion Ser. No. 60/816,987, filed on Jun. 28, 2006, and U.S. Provisional Application No. 60/930,782, filed May 16, 2007, cells have been identified in fat (Zuk et al., 2001), liver 15 (Theise et al., 1999), muscle (Lee et al., 2000), and the central which are both incorporated herein by reference. nervous system (Reynolds and Weiss, 1992; Morshead et al., FIELD OF THE INVENTION 1994: Doetsch et al., 1999) and skin (Toma et al., 2001). Recent reports suggest that the differentiation of adult stem The present invention relates to stem cells obtained from cells is not restricted to derivatives of the tissue in which they the amnion and their methods of obtaining and culturing. The reside. Landmark Studies have demonstrated that adult stem present invention further relates to compositions comprising cells can differentiate into progeny of other embryonic germ amnion-derived stems cells (ADSCs) and to methods of using layers, a process termed transgerminal differentiation. For ADSCS. example, ectodermal neural stem cells can differentiate into mesenchymal derivatives, including blood (Bjornson et al., BACKGROUND OF THE INVENTION 25 1999), muscle (Galli et al., 2000) and endothelial cells (Wurmser et al., 2004). The plasticity exhibited by adult stem Stem cells have been considered as potential treatments for cells has provided hope for the development of new autolo debilitating diseases of various etiologies, including diabetes, gous cellular therapies. Parkinson's disease and . Thus, a criti Adult stem cells may offer advantages over ES cells; how cal goal is to define the spectrum of stem cell types displaying 30 ever, their potential use in cell replacement therapies is not characteristics advantageous for the treatment of selected without obstacles. Many reports have questioned the plastic disorders. While the pluripotency and self-renewal of embry ity of adult stem cells. Several studies have suggested that in onic stem cells is well-recognized, the potential of adult and vitro and in vivo transgerminal plasticity is the result of cell fetal stem cells has been appreciated only recently. fusion rather than actual differentiation (Terada et al., 2002: Stem cells have the potential to develop into many different 35 Ying et al., 2002; Wang et al., 2003). In contrast, others have cell types in the body. Stem cells can theoretically divide confirmed in vivo transdifferentiation of marrow cells in the without limit to replenish other cells. When a stem cell absence of cell fusion(Tran et al., 2003; Pochampally et al., divides, each new cell has the potential to either remain a stem 2004; Sato et al., 2005). It has also recently been demon cell or become another type of cell with a more specialized strated that MSCs transplanted into the adult brain fail to function, such as a muscle cell, ared blood cell, or a brain cell. 40 survive. Stem cells are often classified as totipotent, pluripotent, and Even more disconcerting, these cells transferred their cel multipotent. Totipotent stem cells (e.g., a Zygote) give rise to lular labels (bromodeoxyuridine and bis benzamide) to both the fetus and the extraembryonic tissues. Pluripotent endogenous glia and neural cells, giving a false representa stem cells can give rise to any type of cell except for the tion of donor cell plasticity (Coyne et al., 2006: Burns et al., extraembryonic tissues (e.g., placenta). Multipotent stem 45 2006). These apparent contradictory results have raised cells can give rise to two or more different cell types but only important issues concerning the nature of adult stem cell within a given organ or tissue type. In contrast to stem cells, plasticity and the broader therapeutic potential they represent. progenitor cells are unable to self-renew and they give rise to Fetal stem cells may offer a number of therapeutic advan only a few cell types. tages over ES and adult stem cells, making them well Suited A central dogma in embryonic development is that cells 50 for cell replacement therapy. Fetal stem cells are easily acces undergo a process of fate restriction and commitment. This sible from extra-embryonic tissue that is normally discarded process begins with the development of the blastocyst; a at birth, including the umbilical cord (Nakahata and Ogawa, structure composed of an outer trophoblast layer and an 1982; Knudtzon, 1974) and placenta (Kaviani et al., 2002: undifferentiated inner cell mass (ICM). Yen et al., 2005), circumventing many of the ethical concerns The ICM is the source of embryonic stem cells (ES cells), 55 presented by ES cell research. Fetal stem cells grow rapidly in which are regarded as the quintessential stem cell population culture and exhibit plasticity similar to ES cells, but without (Evans and Kaufman, 1981; Martin, 1981). ES cells demon documented tumor formation in vivo (Miki et al., 2005). strate long-term self-renewal and differentiate into multiple Moreover, fetal stem cells might be more amenable to trans cell types in vitro and in vivo (Smith, 2001; Thomson et al., plantation due to their immunoprivileged characteristics(Liet 1998: Bradley et al., 1984; Amit et al., 2000). 60 al., 2005; Kubo et al., 2001). Due to their remarkable in vitro and in vivo plasticity, ES The present invention describes the identification and char cells have been regarded as the “gold standard for cell acterization of a fetal stem cell population isolated from replacement therapy and regenerative medicine. Although the explants of amniotic membrane. These amnion-derived stem therapeutic potential of ES cells is promising, a number of cells (ADSCs) fulfill all criteria of a stem cell population, issues must be addressed prior to clinical use. Ethical con 65 including clonality, which has proven difficult in previous cerns and in Some cases governmental policies restrict the studies of putative fetal stem cells (Woodbury et al., 2006; isolation and cultivation of human ES cells. From a safety Miki et al., 2005). US 8,980,630 B2 3 4 SUMMARY OF THE INVENTION NF-M, MAP2, APP GLUT, NCAM, NeuroD, Nurr1, GFAP, NG2, Olig1, Alkaline Phosphatase, Vimentin, Osteonectin, The present invention provides stem cells from the amnion. Osteoprotegrin, Osterix, Adipsin, Erythropoietin, SM22-C. One aspect of the invention provides a method for obtaining HGF, c-MET, C.-1.-Antriptrypsin, Ceruloplasmin, AFP. an amnion-derived stem cell (ADSC) comprising: a. separat PEPCK1, BDNF, NT-4%, TrkA, BMP2, BMP4, FGF2, FGF4, ing an amniotic membrane tissue sample from chorion of a PDGF, PGF, TGFo, TGFB, and VEGF. mammalian embryo; b. culturing the amniotic membrane In one aspect the invention provides a method for neuro tissue sample; c. preparing a single-cell culture of ADSC genic differentiation of ADSCs, comprising culturing the isolated from the amniotic membrane tissue sample; d. cul ADSCs in a medium comprising an effective amount DMEM turing the ADSC; and e. obtaining or isolating the ADSCs. In 10 pH 7.0 (low glucose) buffered with 2.75 g/liter sodium bicar one embodiment the amniotic membrane tissue sample is bonate and 5.96 g/liter HEPES (no serum), 2 mM Valproic washed and fragmented after step a. and before step b. In one Acid, 15 mM Betaine, 2.5 mM Taurine, 175 uM butylated embodiment amniotic membrane tissue sample is cultured in hydroxyanisole, 27 nM selenium, 20 nM progesterone, 10 Dulbecco's modified Eagle's medium (DMEM) supple uM forskolin, 10 nM K252a, 5 Units/ml Heparin, 5 lug/ml mented with 20% fetal bovine serum (FBS). 15 Insulin, 1 mM sodium pyruvate, 50 mM C-thioglycerol, and In one embodiment the mammal is mouse, rat, or human. 20 nM Bathocuproinedisulfonic acid, the medium supple In one embodiment the single-cell culture is prepared by mented with 10 ng/ml FGF2 every 48 hours, for about 2 to enzymatically digesting the amniotic membrane tissue about 4 weeks to obtain a population of cells having at least sample. one characteristic of a neural cell. In one embodiment the ADSC is multipotent. In one In one aspect the invention provides a method of treating a embodiment the ADSC is pluripotent. neurodegenerative disease or a brain or spinal cord injury in a In one aspect the invention provides a multipotent ADSC patient, wherein the method comprises administering athera obtained by the method of the present invention. peutically effective amount of an ADSC of the present inven In one aspect the invention provides a pluripotent ADSC tion or a population of neurogenically differentiated cell of obtained by the method of the present invention. 25 the present invention to a patient having a neurodegenerative In one aspect the invention provides an isolated ADSC disease or a brain or spinal cordinjury. In one embodiment the having at least one of the following characteristics: a. positive neurodegenerative disease is selected from the group consist for cell markers CD29 and CD90; and b. negative for cell ing of Parkinson's disease, Alzheimer's disease, Pick's dis markers CD45 and CD 11b. In one embodiment the ADSC ease, Huntington's disease, spingolipidoses, mucosacchari has the following characteristics: a. positive for cell markers 30 doses, and amyotrophic lateral Sclerosis. CD29 and CD90; and b. negative for cell markers CD45 and In one aspect the invention provides a method for osteo CD11b. genic differentiation of ADSCs, comprising a culturing the In one aspect the invention provides an isolated ADSC ADSCs in a medium comprising an effective amount of which expresses at least one of the genes selected from the DMEM pH 7.4 (High glucose) supplemented with 100 nM group consisting of Telomerase, Nanog, Sox2, B-III-Tubulin, 35 Dexamethasone, 10 mM f-glycerol phosphate, 50 LM NF-M, MAP2, APP, GLUT, NCAM, NeuroD, Nurr1, GFAP, L-ascorbic acid-2-phosphate for about 2 to about 4 weeks: NG2, Olig1, Alkaline Phosphatase, Vimentin, Osteonectin, and b. obtaining a population of cells having at least one Osteoprotegrin, Osterix, Adipsin, Erythropoietin, SM22-C. characteristic of a bone cell. HGF, c-MET, C.-1.-Antriptrypsin, Ceruloplasmin, AFP. In one aspect the invention provides a method of treating a PEPCK1, BDNF, NT-4/s, TrkA, BMP2, BMP4, FGF2, FGF4, 40 bone disease in a patient, wherein the method comprises PDGF, PGF, TGFoTGFB, and VEGF. administering a therapeutically effective amount of an ADSC In one embodiment the ADSC expresses Telomerase, of the present invention or the population of osteogenically Nanog, Sox2, B-III-Tubulin, NF-M, MAP2, APP, GLUT, differentiated cells of the present invention to a patient having NCAM, NeuroD, Nurr1, GFAP, NG2, Olig1, Alkaline Phos the bone disease. In one embodiment the bone disease is phatase, Vimentin, Osteonectin, Osteoprotegrin, Osterix, 45 selected from the group consisting of osteoporosis, Paget’s Adipsin, Erythropoietin, SM22-C, HGF, c-MET, C.-1.-Antrip disease, osteogenesis imperfecta, and osteoarthritis. trypsin, Ceruloplasmin, AFP, PEPCK1, BDNF, NT-3/5, TrkA, In one aspect the invention provides a method for adipose BMP2, BMP4, FGF2, FGF4, PDGF, PGF, TGFc., TGFB, and differentiation of ADSCs, comprising a culturing the ADSCs VEGF. in a medium comprising an effective amount of 10% FBS/ In one aspect the invention provides a composition com 50 DMEM, 500 uM IBMX, 1 uM dexamethazone stock, 5 prising an ADSC of the present invention. ug/mL insulin and 50 uM indomethacin for about 3 days; b. In one aspect the invention provides a cryopreserved culturing the ADSCs in a medium comprising an effective ADSC of the present invention. amount of 10% FBS/DMEM and 5 mg/mL insulin for about In one aspect the invention provides a method of treating a 3 days; c. repeating steps a. and b. in order 0 to about 5 times; patient comprising administering to the patient a therapeuti 55 and d. obtaining a population of cells having at least one cally effective amount of a ADSC of the present invention. characteristic of an adipocyte. In one aspect the invention provides a method for obtaining In one aspect the invention provides a method for hepatic an ADSC comprising isolating an ADSC which expresses at differentiation of ADSCs, comprising culturing the ADSCs in least one of the genes selected from the group consisting of a medium comprising an effective amount of 1% FBS/ Telomerase, Nanog, Sox2, B-III-Tubulin, NF-M, MAP2, 60 DMEM-LG, 20 ng/ml HGF, 10 ng/ml oncostatin M and 10 APP, GLUT, NCAM, NeuroD, Nurr1, GFAP, NG2, Olig1 ng/ml FGF-4 for about 2 to about 4 weeks to obtain a popu Alkaline Phosphatase, Vimentin, Osteonectin, Osteoprote lation of cells having at least one characteristic of a hepatic grin, Osterix, Adipsin, Erythropoietin, SM22-C, HGF, cell. c-MET, C-1-Antriptrypsin, Ceruloplasmin, AFP, PEPCK1, In one aspect the invention provides a method of treating a BDNF, NT 4/3, TrkA, BMP2, BMP4, FGF2, FGF4, PDGF, 65 hepatic disease in a patient, wherein the method comprises PGF, TGFC., TGFB, and VEGF. In one embodiment the administering a therapeutically effective amount of an ADSC ADSC expresses Telomerase, Nanog, Sox2, B-III-Tubulin, of the present invention or the population of hepatically dif US 8,980,630 B2 5 6 ferentiated cells of the present invention to a patient having ease, type V Glycogen storage disease, type VI Glycogen the hepatic disease. In one embodiment the hepatic disease is storage disease, type VII Glycogen storage disease, Fructose selected from the group consisting of amebic liver abscess, intolerance, Essential fructosuria, Galactosemia, PCD, autoimmune hepatitis, biliary atresia, cirrhosis, coccidioido PDHA, Pentosuria, Renal glycosuria, GM2 gangliosidoses, mycosis: disseminated, delta agent (Hepatitis D), drug-in Sandhoff disease, Tay-Sachs disease, GM1 gangliosidoses, duced cholestasis, hemochromatosis, hepatitis A, hepatitis B. Mucolipidosis type IV. Gaucher's disease, Niemann-Pick hepatitis C, hepatocellular carcinoma, liver cancer, liver dis disease, Farber disease, Fabry's disease, Metachromatic leu ease due to alcohol, primary biliary cirrhosis, pyogenic liver kodystrophy, Krabbe disease, Neuronal ceroid lipofuscino abscess, Reye's syndrome, Sclerosing cholangitis, and Wil sis, Batten disease, Cerebrotendineous Xanthomatosis, Cho son's disease. 10 lesteryl ester storage disease, Wolman disease, In one aspect the invention provides a method of treating a Hyperlipidemia, Hypercholesterolemia, Familial hypercho cardiovascular disease in a patient, wherein the method com lesterolemia, Xanthoma, Combined hyperlipidemia, Lecithin prises administering a therapeutically effective amount of an cholesterol acyltransferase deficiency, Tangier disease, Abe ADSC of the present invention to a patient having the cardio talipoproteinemia, Adrenoleukodystrophy, primary carnitine vascular disease. In one embodiment the cardiovascular dis 15 deficiency, carnitine palmitoyltransferase I deficiency, car ease is selected from the group consisting of congenital heart nitine palmitoyltransferase II deficiency, carnitine-acylcar defects, peripheral artery disease, arterio-arterial fistula, arte nitine translocase deficiency, Wilson's disease, Menkes dis riovenous fistula, cerebral arteriovenous malformations, aor ease, Haemochromatosis, Acrodermatitis enteropathica, tic coarctation, cortriatum, coronary vessel anomalies, patent Hypophosphatemia, Hypophosphatasia, Hypermagnesemia, ductus arteriosus, Ebstein's anomaly, hypoplastic left heart Hypomagnesemia, Hypercalcaemia, Hypocalcaemia, Disor syndrome, levocardia, transposition of great vessels, double ders of calcium metabolism, Hyperuricemia, Lesch-Nyhan outlet right ventricle, tricuspidatresia, persistent truncus arte syndrome, Xanthinuria, Gilbert's syndrome, Crigler-Najjar riosus, and heart septal defects, such as aortopulmonary sep syndrome, Dubin-Johnson syndrome, Rotor syndrome, tal defect, endocardial cushion defects, Lutembacher's Syn Mucopolysaccharidosis, Hurler Syndrome, Hunter Syn drome, Ventricular heart septal defects, , 25 drome, Sanfilippo Syndrome, Morquio Syndrome, Maro (including bacterial), heart aneurysm, cardiac teaux-Lamy Syndrome, Sly Syndrome, Mucolipidosis, I-cell arrest, congestive , congestive , disease, Pseudo-Hurler polydystrophy, Aspartylglu paroxysmal dyspnea, cardiac edema, post-infarction heart cosaminuria, Fucosidosis, Alpha-mannosidosis, Sialidosis, rupture, Ventricular septal rupture, heart valve diseases, myo Alpha 1-antitrypsin deficiency, Cystic fibrosis, Amyloidosis, cardial diseases, , (including 30 Familial Mediterranean fever, and Acatalasia constrictive and tuberculous), , postperi one aspect the invention provides a method of cryopreserv cardiotomy syndrome, , rheumatic ing ADSCs comprising: a. washing a population of ADSCs; b. heart disease, Ventricular dysfunction, hyperemia, cardiovas Suspending the population of ADSCs in a cryopreservation cular pregnancy complications, cardiovascular syphilis, car medium comprising 60% Dulbecco's Modified Eagles Media diovascular tuberculosis, Such as sinus arrhyth 35 (DMEM)/30% Fetal Bovine Serum (FBS)/10% Dimethyl mia, atrial , , , extrasystole, sulfoxide (DMSO); and c. storing the population at a tem Adams-Stokes Syndrome, bundle-branch block, sinoatrial perature below about -80° C. block, long QT syndrome, parasystole, sick sinus syndrome, In one aspect the invention provides a method of cryopre Ventricular , Such as paroxysmal serving an amniotic membrane tissue sample comprising: a. , Supraventricular tachycardia, accelerated idio 40 washing the amniotic membrane tissue sample; b. Suspending Ventricular rhythm, atrioventricular nodal reentry tachycar the amniotic membrane tissue sample in a cryopreservation dia, ectopic , ectopic junctional tachycardia, medium comprising 60% Dulbecco's Modified Eagles Media sinoatrial nodal reentry tachycardia, , Tor (DMEM)/30% Fetal Bovine Serum (FBS)/10% Dimethyl sades de Pointes, and and heart valve sulfoxide (DMSO); and c. storing the population at a tem diseases such as insufficiency, aortic valve Steno 45 perature below about -80° C. sis, heart murmurs, aortic valve prolapse, pro In one aspect the invention provides a method of cryopre lapse, prolapse, mitral valve insufficiency, serving ADSCs comprising: a. washing a population of mitral valve Stenosis, pulmonary atresia, ADSCs; b. suspending the population of ADSCs in a cryo insufficiency, pulmonary valve Stenosis, tricuspidatresia, tri preservation medium comprising 60% DMEM/30% Liforcel cuspid valve insufficiency, and . 50 serum substitute/10% DMSO; and c. storing the population at In one aspect the invention provides a method of treating a a temperature below about -80° C. metabolic disease in a patient, wherein the method comprises In one aspect the invention provides a method of cryopre administering a therapeutically effective amount of an ADSC serving an amniotic membrane tissue sample comprising: a. of the present invention to a patient having the metabolic washing the amniotic membrane tissue sample; b. Suspending disease. In one embodiment the metabolic disease is selected 55 the amniotic membrane tissue sample in a cryopreservation from the group consisting of Phenylketonuria, Alkaptonuria, medium comprising 60% DMEM/30% Liforcel serum sub Ochronosis, Tyrosinemia, Albinism, Histidinemia, Maple stitute/10% DMSO; and c. storing the population at a tem syrup urine disease, Propionic acidemia, Methylmalonic aci perature below about -80° C. demia, Isovaleric acidemia, 3-Methylcrotonyl-CoA carboxy lase deficiency, Cystinuria, Cystinosis, Hartnup disease, 60 BRIEF DESCRIPTION OF THE DRAWINGS Homocystinuria, CyStathioninuria, N-cetylglutamate Syn thase deficiency, Carbamoyl phosphate synthetase I defi FIG.1. Isolation and Propagation ADSCs: (A) Cells (>'s) ciency, Omithine transcarbamylase deficiency, Citrullinemia, migrate from amnion tissue explants (arrow) within 24 hours Argininosuccinic aciduria, Hyperammonemia, Glutaric aci of plating. (B) By 72 hours numerous ameboid-shaped cells, demia type 1, Sarcosinemia, Lactose intolerance, type I Gly 65 can be observed migrating from the tissue explants. In addi cogen storage disease, type II Glycogen storage disease, type tion, a number of doublets can be observed (>'s), indicative of III Glycogen storage disease, type IV Glycogen storage dis cell proliferation. Explants are subsequently removed and the US 8,980,630 B2 7 8 remaining Small, ameboid shaped cells can be cultured indefi and 3 weeks post-induction demonstrate the up-regulation of nitely. Scale bars: A=50 um; B=100 um. liver specific genes albumin and C-1-antitrypsin. The expres FIG. 2. Immunocytochemical Analysis of Primary ADSCs sion of HNF-3C. and GS is maintained throughout the experi Cultures: (A-C) At 72 hours post-explanation close to 100% ment. GAPDH is used as a loading control. Scale bars: a, c, e of the migrating cells migrating express the mesenchymal and g-100 um; b, h, I andj=50 um; f=10um. protein vimentin. (D-F) Further analysis reveals that these FIG. 7. Flow Cytometric Analysis of Parental and Clonal cells express fibronectin, consistent with a mesenchymal phe ADSC Cultures: (A, B) The overwhelming majority (greater notype. Scale bars: A-C=100 um; D-F-50 um. than 90%) of parental (P5) and clonal (C2, C4) ADSCs FIG. 3. Gene Expression Profile of ADSCs: RT-PCR express the mesenchymal cell surface markers CD29 and reveals that ADSCs express an array of genes, including: (A) 10 CD90. (C, D) Variable levels of the CD44 and CD31 are proteins normally associated with stem cells, (B) neuroecto detected within the ADSCs cultures. (E. F.) Parental and dermal structural proteins and transcription factors, (C) clonal ADSC populations do not express the lympho-hemato mesodermal genes normally expressed in bone, fat and poietic markers CD45 and CD 11b. P5 parental ADSC cul muscle cells, (D) endodermal genes representing the lung and tures, C2=ADSC clonal line #2, C4=ADSC clonal cell line liver, (E) neurotrophins and the nerve growth factor receptor 15 H4. TrkA and (F) various growth factors. FIG.8. RT-PCR Analysis and InVitro Multidifferentiation FIG. 4. ADSCs are Multidifferentiated: (A-D) A subpopu of ADSC Clonal Lines: (A) The genetic expression profile of lation of ADSCs co-express the mesenchymal protein vimen two distinct clonal populations (C1, C2) is identical to the tin and neuroectodermal neurofilament-M (NF-M) (arrow). parent population. RT-PCR analysis reveals that both clones Vimentin-positive (+)/NF-M-negative (-) cells are also evi expressed stem cells markers, growth factors and genes rep dent (>). (E-H) A subset of ADSCs co-express the neural resenting all three embryonic layers. (B) Row 1: ADSC clonal progenitor marker nestin and the mesenchymal protein cell lines maintain the ability to differentiate into neural cells. fibronectin (arrows). Fibronectin--cells that do not express Two clonal ADSC cultures exposed to NIM for 1 week attain nestin are also present (>). Scale bars: A-D=50 um: E-H=100 typical neural morphologies: Small, round refractile cell bod lm. 25 ies (arrows) elaborating long processes (>) that form net FIG. 5. Neuroectodermal differentiation of ADSCs: (A-C) works. Row 2: Clonal ADSC lines accumulate fat droplets, as Time-lapse images of a single ADSC (arrow) at 0, 1 hour and indicated by positive Oil Red O staining, after culture for 3 5 days following exposure to NIM. ADSCs attain a neural weeks in AIM. Row 3: Clonal ADSC populations lay down morphology, exhibiting round, compact, refractile cell bodies mineralized matrix, as observed by Alizarin red staining, (arrow), while elaborating processes (>'s) greater than 500 30 following 6 weeks of OIM culture. Row 4: Clonal ADSC lines um in length. (D) Cellular extensions of differentiated differentiate into presumptive hepatocytes and took up Dil ADSCs make connections with neighboring cells and form Ac-LDL after exposure to HIM for 3 weeks. Scalebars: Rows complex neural networks similar to primary neural cultures. 1 3=100 um; Row 4-50 um. (E-H) Immunocytochemical analysis reveals the co-expres FIG. 9. Characterization of GFP-expressing ADSCs prior sion of tau and NFM throughout the cell body (arrow) and 35 to transplantation. (AC) Examination of the same field with processes (>'s) of differentiated ADSCs. (I) PCR analysis of phase contrast and fluorescent microscopy reveals uniform ADSCs exposed to NIM for one week up-regulate neural expression of GFP in ADSCs. Regardless of the passage specific genes, including GAP-43 and tau (three splice vari number close to 100% of the cells express GFP. (DG) GFP ants, >'s). Differentiated ADSCs down-regulate the neural expressing ADSCs express the mesodermal marker vimentin, stem cell marker Sox2, and the mesenchymal gene SM22O. 40 while a subset of cells (HK, arrowheads) express the neural GAPDH is utilized as a loading control. C-7 day serum-free progenitor marker nestin. (HK) The majority of ADSCs (ar control, N=7 day NIM treated ADSC cultures. Scale bars: rows) are negative for nestin. (L.) After culture in a defined A-H=100 um. neural induction media GFP+ ADSCs attain a neural mor FIG. 6. Mesodermal Differentiation of ADSCs: Osteo phology, exhibiting round, compact, refractile cell bodies genic Differentiation: (A) ADSCs maintained in OIM for 6 45 (arrow), while elaborating long processes extending more weeks reveal extensive areas of mineralized matrix deposi than 600 um in length (>'s). Scale Bars: AC=50 um; DG=100 tion (Alizarin red staining). (B) ADSCs exposed to osteo um; HL-100 um. genic conditions attain cuboidal morphologies and form FIG. 10. Infused ADSCs disperse throughout the embry Alizarin red-nodules (>). (C) Alizarin red staining of control onic ventricular system and form discrete spheres. (A) Sag cultures demonstrates no areas of mineralized matrix or 50 ittal section reveal extensive diffusion of donor ADSCs (ar nodular formation. (D) RNA extracted at 3 and 6 weeks row) throughout the ventricular system at E16.5 (24 hours post-induction reveals an up-regulation of the bone-specific post-transplantation). (A, inset, arrow) Some donor cells are gene osteopontin, and maintained expression of mesenchy observed within the parenchyma of the brain even at this early mal osteonectin. Expression of ectodermal NF-M, endoder time point. (B) By E17.5 donor cells form discrete GFP+ mal C-1-antitrypsin and the stem cell marker nanog decrease 55 spheres or clusters (arrow) within the lateral ventricles. (C) concomitantly. GAPDH is used as a loading control. Adipo Some spheres appear to have fused with the walls of the genic Differentiation: (E) Following 3 weeks of culture in ventricular cavity. Individual cells (arrows) can be observed AIM, ADSCs accumulate fat droplets (red) as indicated by migrating into the parenchyma. LV—lateral ventricle. *-cor Oil Red Ostaining. (F) High power magnification (>) reveals tex. Scale bars: A, B=100 um; A inset=20 um; C=50 um. Oil Red-O+ lipid droplets filling most of the cytoplasm. (G) 60 FIG. 11: Distribution of ADSCs at E20.5. (AC) At E20.5 Control cultures stained with Oil Red O display no lipid GFP+ ADSCs (arrow) are observed in a number of brain accumulation. Endodermal Differentiation of ADSCs: (H) areas, including the cortex. (D) Higher magnification of the ADSCs exposed to HIM for 3 weeks demonstrate the ability box in (C) reveals that a subset of donor cells (arrow) in the to take up Dil-Ac-LDL from culture media (red). (I) Phase brain associate with vascular structures. These cells have contrast image depicts cobblestone appearance of HIM 65 crescent morphologies, similar to endogenous endothelial treated ADSCs. (J) Control cultures do not show any Dil-Ac cells. (EG) Donor ADSCs (arrow) are also evident in other LDL uptake. (K) PCR analysis of HIM-treated ADSCs at 2 brain areas, including the midbrain. Scale bars: AG-20 lum. US 8,980,630 B2 9 10 FIG. 12. Morphology and Phenotypic characterization of brain cells. However, as discussed above, recent evidence transplanted ADSCs at one week postnatal. (AD) The major Suggests that hematopoietic stem cells may be pluripotent ity of transplanted GFP+ ADSCs (arrows) within the cortex because they may differentiate into other types of cells, attain elongated morphologies and expressed vimentin. (EH) including brain cells Some donor cells (arrows) are nestin positive and assumed 5 As used herein, the term “progenitor cell refers to a cell ameboid morphologies. (IJ) A Subpopulation of donor cells that is committed to differentiate into a specific type of cellor (arrow) around blood vessels attain crescent morphologies to form a specific type of tissue. and expressed von Willebrand factor (vWF). *=blood vessel. As used herein, the term "amnion” refers to a membranous Scale bar: AL-20 lum. sac which surrounds and protects the embryo. It is developed FIG. 13. A subpopulation of transplanted ADSCs assume 10 in reptiles, birds, and mammals. The primary function of this neuronal morphologies. (AC) At P7 a subset of GFP+ cells in the hippocampal formation assume typical neural morpholo is the protection of the embryo for its future development into gies: Small cell bodies (arrow) and the elaboration of long a fetus and eventually an animal. The amnion is the inner of processes (arrowheads). (DG) Donor cells (arrows) within the two fetal membranes surrounding the fetus (the chorion is the cortex do not express the neuronal protein, B-III tubule, in 15 the outer one). The terms "amnion”, “amniotic membrane'. contrast to neighboring endogenous cells (arrowheads). (HK) and "amniotic tissue' are all used interchangeably in the GFP+ ADSCs do not express the atrocity structural protein present application. The amnion may be obtained from any GFAP, DG=Dentate Gyros. Scale bar: AK=20 um. reptilian, avian or mammalian species, including rodents, FIG. 14. Long-term survival of transplanted ADSCs in the human, non-human primates, equines, canines, felines, neocortex. (AC) In utero transplanted ADSCs (arrows) sur bovines, porcines, Ovines, lagomorphs, and the like. In one vive 2.5 months in the neocortex of recipient animals. (D) embodiment, the amnion is obtained from mouse, rat, or Higher magnification of the box in (C) reveals that a subset of human. donor cells (arrows) in the adult brain associate with vascular In one embodiment the single-cell culture is prepared by structures. (EH) GFP+ ADSCs in the P7 rathippocampus do enzymatically digesting the amniotic membrane tissue not initiate a host immunological response as indicated by the 25 sample. absence of ED1+reactive microglia within the proximity of In one embodiment the ADSC is multipotent. In one donor cells (arrow). ED1+(EH, arrowhead) are present, but embodiment the ADSC is pluripotent. not closely associated with donor GFP ADSCs. Scale bar: In one aspect the invention provides a multipotent ADSC AH-20 Lum. obtained by the method of the present invention. 30 In one aspect the invention provides a pluripotent ADSC DETAILED DESCRIPTION OF THE INVENTION obtained by the method of the present invention. An ADSC may be characterized by its cell markers. A The present invention provides stem cells from the amnion. variety of cell markers are known. See e.g., Stem Cells: Sci One aspect of the invention provides a method for obtaining entific Progress and Future Research Directions. Department an amnion-derived stem cell (ADSC) comprising: a. separat 35 of Health and Human Services. June 2001. http://www. ing an amniotic membrane tissue sample from chorion of a nih.gov/news/stemcell/scireport.htm. Cell markers may be mammalian embryo; b. culturing the amniotic membrane detected by methods known in the art, Such as by immu tissue sample; c. preparing a single-cell culture of ADSC nochemistry or flow cytometry. Flow cytometry allows the isolated from the amniotic membrane tissue sample; d. cul rapid measurement of light scatter and fluorescence emission turing the ADSC; and e. obtaining or isolating the ADSCs. In 40 produced by suitably illuminated cells or particles. The cells one embodiment the amniotic membrane tissue sample is or particles produce signals when they pass individually washed and fragmented after step a. and before step b. In one through a beam of light. Each particle or cell is measured embodiment amniotic membrane tissue sample is cultured in separately and the output represents cumulative individual Dulbecco's modified Eagle's medium (DMEM) supple cytometric characteristics. Antibodies specific to a cell mented with 20% fetal bovine serum (FBS). 45 marker may be labeled with a fluorochrome so that it may be The present invention relates to stem cells from the detected by the flow cytometer. See, eg. Bonner et al., Rev. amnion, preferably the amniotic membrane. As used herein, Sci. Instrum 43: 404–409, 1972; Herzenberg et al., Immunol. the term “stem cell refers to a master cell that can reproduce Today 21: 383-390, 2000; Julius et al., PNAS 69; 1934-1938, indefinitely to form the specialized cells of tissues and organs. 1972: Ormerod (ed.), Flow Cytometry: A Practical Approach, A stem cell can divide to produce two daughter stem cells, or 50 Oxford Univ. Press, 1997: Jaroszeski et al. (eds.), Flow one daughter stem cell and one progenitor (“transit’) cell, Cytometry Protocols in Methods in Molecular Biology No. which then proliferates into the tissues mature, fully formed 91, Humana Press, 1997; Practical Flow Cytometry, 3. Sup.rd cells. As used herein, the term “stem cell includes multipo ed., Wiley-Liss, 1995. tent and pluripotent stem cells. In one aspect the invention provides an isolated ADSC As used herein, the term “pluripotent cell” refers to a cell 55 having at least one of the following characteristics: a. positive that has complete differentiation versatility, i.e., the capacity for cell markers CD29 and CD90; and b. negative for cell to grow into any of the mammalian body's cell types, except markers CD45 and CD 11b. In one embodiment the ADSC for the extraembryonic tissues. A pluripotent cell can be self has the following characteristics: a. positive for cell markers renewing, and can remain dormant or quiescent within a CD29 and CD90; and b. negative for cell markers CD45 and tissue. 60 CD11b. As used herein, the term “multipotent cell refers to a cell In one aspect the invention provides an isolated ADSC that has the capacity to grow into two or more different cell which expresses at least one of the genes selected from the types of the mammalian body within a given tissue or organ. group consisting of Telomerase, Nanog, Sox2, B-III-Tubulin, However, a multipotent cell may have the capacity to be NF-M, MAP2, APP GLUT, NCAM, NeuroD, Nurr1, GFAP, pluripotent. For example, hematopoietic stem cells were 65 NG2, Olig1, Alkaline Phosphatase, Vimentin, Osteonectin, originally believed to be multipotent cells, i.e., stem cells that Osteoprotegrin, Osterix, Adipsin, Erythropoietin, SM22-C. could develop into several types of blood cells, but not into HGF, c-MET, C.-1.-Antriptrypsin, Ceruloplasmin, AFP. US 8,980,630 B2 11 12 PEPCK1, BDNF, NT-4/s, TrkA, BMP2, BMP4, FGF2, FGF4, with Scissors. Before culturing the amniotic membrane, the PDGF, PGF, TGFo, TGFB, and VEGF. membrane may be enzymatically digested with, for example, In one embodiment the ADSC expresses Telomerase, trypsin, chymotrypsin, lysozyme, amylase, or protease K. Nanog, Sox2, B-III-Tubulin, NF-M, MAP2, APP, GLUT, The amniotic cells thus obtained may be cultured in culture NCAM, NeuroD, Nurr1, GFAP, NG2, Olig1, Alkaline Phos 5 medium comprising standard medium, Such as DMEM phatase, Vimentin, Osteonectin, Osteoprotegrin, Osterix, (Gibco) and 20% fetal bovine serum, and may be supple Adipsin, Erythropoietin, SM22-C, HGF, c-MET, C.-1.-Antrip mented with glucose and/or antibiotics, as appropriate. trypsin, Ceruloplasmin, AFP, PEPCK1, BDNF, NT-3/5, TrkA, ADSCs may be obtained by continued culture of the amniotic BMP2, BMP4, FGF2, FGF4, PDGF, PGF, TGFc., TGFB, and membrane in the culture medium. VEGF. 10 In one aspect the invention provides a composition com The ADSCs of the present invention may also be induced prising an ADSC of the present invention. into other cell types by methods known in the art. The present invention also embodies a homogeneous popu The presence of ADSCs in culture may be detected by their lation of ADSCs. As used herein, “homogeneous population' ability to differentiate into different cell types. For example, refers to a population of cells exhibiting substantially the 15 the cultured cells may be tested for their ability to undergo same phenotype, such as that determined by cell markers. A neuronal, adipogenic, hepatic, and/or osteogenic differentia homogeneous population may comprise at least about 70% of tion. substantially the same cells, or at least about 80%, 90%, 92%, In one aspect the invention provides a method for neuro 96%, or 99% of substantially the same cells. genic differentiation of ADSCs, comprising culturing the The present invention therefore provides a method of ADSCs in a medium comprising an effective amount DMEM obtaining an ADSC by isolating amniotic cells having certain pH 7.0 (low glucose) buffered with 2.75 g/liter sodium bicar cell characteristics. The amniotic cells having these cell char bonate and 5.96 g/liter HEPES (no serum), 2 mM Valproic acteristics may be isolated from a single-cell culture of amni Acid, 15 mM Betaine, 2.5 mM Taurine, 175 uM butylated otic cells obtained from amniotic membrane as described hydroxyanisole, 27 nM selenium, 20 nM progesterone, 10 above or from amniotic cells that have been cultured after 25 uM forskolin, 10 nM K252a, 5 Units/ml Heparin, 5 lug/ml isolating from the amniotic membrane. Cells may be isolated Insulin, 1 mM sodium pyruvate, 50 mM C-thioglycerol, and according to cell characteristics by, for example, flow cytom 20 nM Bathocuproinedisulfonic acid, the medium supple etry, as described above. In an embodiment of the present mented with 10 ng/ml FGF2 every 48 hours, for about 2 to invention, ADSCs are isolated by isolating amniotic cells about 4 weeks to obtain a population of cells having at least having at least one of the following characteristics: a. positive 30 one characteristic of a neural cell. for cell markers CD29 and CD90; and b. negative for cell In one aspect the invention provides a method for osteo markers CD45 and CD11b. In one embodiment ADSCs are genic differentiation of ADSCs, comprising a culturing the isolated by isolating amniotic cells having the following char ADSCs in a medium comprising an effective amount of acteristics: a. positive for cell markers CD29 and CD90; and DMEM pH 7.4 (High glucose) supplemented with 100 nM b. negative for cell markers CD45 and CD11b. 35 Dexamethasone, 10 mM f-glycerol phosphate, 50 LM In one embodiment ADSCs are isolated by isolating amni L-ascorbic acid-2-phosphate for about 2 to about 4 weeks: otic cells which express at least one of the genes selected from and b. obtaining a population of cells having at least one the group consisting of Telomerase, Nanog, Sox2, B-III-Tu characteristic of a bone cell. Osteogenic differentiation may bulin, NF-M, MAP2, APP, GLUT, NCAM, NeuroD, Nurr1 be induced by other methods known in the art. GFAP, NG2. Olig1, Alkaline Phosphatase, Vimentin, 40 Osteogenic differentiation may be detected by testing for Osteonectin, Osteoprotegrin, Osterix, Adipsin, Erythropoi the presence of osteogenic markers, which include, but are etin, SM22-C, HGF, c-MET, C.-1.-Antriptrypsin, Ceruloplas not limited to, osteopontin (OP), osteocalcin (OC), osteonec min, AFP, PEPCK1, BDNF, NT 4%, TrkA, BMP2, BMP4, tin (ON), and bone sialoprotein. Osteogenesis may also be FGF2, FGF4, PDGF, PGF, TGFC, TGFB, and VEGF. In one detected by using Von Kossa stain (Jaiswal et al., J. Cell embodiment ADSCs are isolated by isolating amniotic cells 45 Biochem. 64; 295-312, 1997)and/oralizarin red stain (Wanet which express Telomerase, Nanog, Sox2, B-III-Tubulin, al., Chin. J. Traumatol. 5: 374-379, 2002), which detect the NF-M, MAP2, APP, GLUT, NCAM, NeuroD, Nurr1, GFAP, presence of calcium deposit activity. Neuronal differentiation NG2, Olig1, Alkaline Phosphatase, Vimentin, Osteonectin, may be induced by other methods known in the art. Osteoprotegrin, Osterix, Adipsin, Erythropoietin, SM22-C. Neuronal differentiation may be detected by testing for the HGF, c-MET, C.-1.-Antriptrypsin, Ceruloplasmin, AFP. 50 presence of markers of neuronal and glial differentiation PEPCK1, BDNF, NT-4/s, TrkA, BMP2, BMP4, FGF2, FGF4, would include Nestin, Neurogenin-2 (Ngn-2), Musashi-1, PDGF, PGF, TGFo, TGFB, and VEGF. Microtubule Dendrite Associated Protein-2 (MAP-2), Neu The present invention also provides a method for obtaining rofilament-3 (NF-3), Synaptophysin (SYP), Tyrosine an ADSC. The method comprises a. separating an amniotic Hydroxylase (TH). Tryptophan Hydroxylase 2 (TPH2). membrane tissue sample from chorion of a mammalian 55 Myelin Basic Protein (MBP), and Glial Fibrillary Acidic embryo; b. culturing the amniotic membrane tissue sample; c. Protein (GFAP). preparing a single-cell culture of ADSC isolated from the In one aspect the invention provides a method for adipose amniotic membrane tissue sample; d. culturing the ADSC: differentiation of ADSCs, comprising a culturing the ADSCs and e. obtaining or isolating the ADSCs. In one embodiment in a medium comprising an effective amount of 10% FBS/ the amniotic membrane tissue sample is washed and frag 60 DMEM, 500 uM IBMX, 1 uM dexamethazone stock, 5 mented after step a. and before step b. In one embodiment ug/mL insulin and 50 uM indomethacin for about 3 days; b. amniotic membrane tissue sample is cultured in Dulbecco's culturing the ADSCs in a medium comprising an effective modified Eagle's medium (DMEM) supplemented with 20% amount of 10% FBS/DMEM and 5 mg/mL insulin for about fetal bovine serum (FBS). Post-partum amnion may be 3 days; c. repeating steps a. and b. in order 0 to about 5 times; obtained, for example, with informed consent from a cae 65 and d. obtaining a population of cells having at least one sarian procedure or normal birth. The amnion may be characteristic of an adipocyte. Adipogenic differentiation mechanically cut into Smaller pieces of tissue, for example, may be induced by other methods known in the art. US 8,980,630 B2 13 14 Adipogenic differentiation may be detected by testing for A patient is hereby defined as any person or non-human the presence of adipogenic transcription factors PPARy2 (per animal in need of treatment with an ADSC, or to any subject oxisome proliferator activated receptor gamma) and/or for whom treatment may be beneficial, including humans and CEBPO. (CCAAT/enhancer binding protein alpha), by meth non-human animals. Such non-human animals to be treated ods such as immunohistochemistry and reverse-transcriptase include all domesticated and feral mammals. In an embodi polymerase chain reaction. Alternatively, adipogenic differ ment of the present invention, the ADSC to be administered is entiation may be detected by lipid accumulation as demon obtained from the same species as the species receiving treat strated by Oil Red O staining after culture in an adipocyte ment. Examples of mammalian species include rodents, inducing medium (Conget and Minguell, J. Cellular human, non-human primates, equines, canines, felines, 10 bovines, porcines, Ovines, lagomorphs, and the like. Physiology 181: 67-73, 1999). Other methods of inducing The ADSCs of the invention may be used in the treatment and detecting adipogenic differentiation may be used (see, of any kind of injury due to trauma where tissues need to be e.g., Pittenger et al., Science 284: 143-147, 1999; replaced or regenerated. Examples of Such trauma-related Tchoukalova et al., Obesity Research 8: 664-672, 2000). conditions include central nervous system (CNS) injuries, In one aspect the invention provides a method for hepatic 15 including injuries to the brain, spinal cord, or tissue Surround differentiation of ADSCs, comprising culturing the ADSCs in ing the CNS injuries to the peripheral nervous system (PNS), a medium comprising an effective amount of 1% FBS/ or injuries to any other part of the body. Such trauma may be DMEM-LG, 20 ng/ml HGF, 10 ng/ml oncostatin M and 10 caused by accident, or may be a normal or abnormal outcome ng/ml FGF-4 for about 2 to about 4 weeks to obtain a popu of a medical procedure Such as Surgery or angioplasty. The lation of cells having at least one characteristic of a hepatic trauma may be related to a rupture or occlusion of a blood cell. Hepatic differentiation may be induced by other methods vessel, for example, in stroke orphlebitis. In specific embodi known in the art. ments, the cells may be used in autologous or heterologous Hepatic differentiation may be detected by testing for the tissue replacement or regeneration therapies or protocols, presence of hepatocyte genes such as albumin (Alb) and including, but not limited to treatment of corneal epithelial alpha-1-antitrypsin (A1AT), and the transcription factor, 25 defects, cartilage repair, facial dermabrasion, mucosal mem C/EBPO. To confirm expression of prototypical liver genes, branes, tympanic membranes, intestinal linings, neurological hepatically differentiated ADSCs can be immunostained with structures (e.g., retina, auditory neurons in basilar membrane, anti-serum albumin antibody and anti-hepatocyte nuclear olfactory neurons in olfactory epithelium), burn and wound factor (HNF)-4a antibodies. repair for traumatic injuries of the skin, or for reconstruction In one aspect the invention provides a method for endot 30 of other damaged or diseased organs or tissues. Injuries may helial differentiation of ADSCs, comprising culturing the be due to specific conditions and disorders including, but not ADSCs in a medium comprising an effective amount of limited to, , seizure disorder, multiple EGMTM-MV (catalog #CC-3125, from Cambrex) containing Sclerosis, stroke, hypotension, , , heparin, bovine brain extract, epithelial growth factor (e.g., inflammation, age-related loss of cognitive function, radia human recombinant epithelial growth factor), and hydrocor 35 tion damage, cerebral palsy, neurodegenerative disease, tisone for about 2 to about 4 weeks to obtain a population of Alzheimer's disease, Parkinson's disease, Leigh disease, cells having at least one characteristic of an endothelial cell. AIDS dementia, memory loss, amyotrophic lateral sclerosis Endothelial differentiation may be induced by other methods (ALS), ischemic renal disease, brain or spinal cord trauma, known in the art. heart-lung bypass, glaucoma, retinal ischemia, retinal Endothelial differentiation may be detected by testing for 40 trauma, inborn errors of metabolism, adrenoleukodystrophy, the presence of endothelial genes Such as E-selectin cystic fibrosis, glycogen storage disease, hypothyroidism, (CD62E), ICAM-2 (CD102), CD34, and STRO-1. sickle cell anemia, Pearson syndrome, Pompe's disease, phe In one aspect the invention provides a method of treating a nylketonuria (PKU), porphyrias, maple syrup urine disease, patient comprising administering to the patient a therapeuti homocystinuria, mucoplysaccharide nosis, chronic granulo cally effective amount of an ADSC of the present invention 45 matous disease and tyrosinemia, Tay-Sachs disease, cancer, and/or an ADSC which has differentiated into a particular tumors or other pathological or neoplastic conditions. progenitor cell or fully differentiated cell type. In the method The ADSC used in the treatment may also contain a nucleic of the present invention, the ADSCs and differentiated cells acid vector or biological vector in an amount Sufficient to are transplanted into a subject in need of treatment in an direct the expression of a desired gene(s) in a patient. The amount effective to treat the nervous tissue degeneration. As 50 construction and expression of conventional recombinant used herein, the phrase “therapeutically effective amount nucleic acid vectors is well known in the art and includes means effective to ameliorate or minimize the clinical impair those techniques contained in Sambrook et al., Molecular ment or symptoms of the disease or injury. For example, in the Cloning: A Laboratory Manual, Vols 1-3 (2d ed. 1989), Cold case of a neurodegenerative disease, the clinical impairment Spring Harbor Laboratory Press. Such nucleic acid vectors or symptoms of the neurodegenerative disease may be ame 55 may be contained in a biological vector Such as viruses and liorated or minimized by alleviating vasomotor symptoms, bacteria, preferably in a non-pathogenic or attenuated micro increasing deep-tendon reflexes, reducing muscle atrophy, organism, including attenuated viruses, bacteria, parasites, restoring sensory function, and strengthening muscles. The and virus-like particles. amount of ADSCs and/or differentiated neural cells effective The nucleic acid vector or biological vector may be intro to treat disease or injury in a Subject in need of treatment will 60 duced into the cells by an ex vivo gene therapy protocol, vary depending upon the particular factors of each case, which comprises excising cells or tissues from a patient, including the type of tissue, the stage of the disease or injury, introducing the nucleic acid vector or biological vector into the subjects weight, the severity of the subject’s condition, the excised cells or tissues, and reimplanting the cells or the type of differentiated cells, and the method of transplan tissues into the patient (see, for example, Knoellet al., Am. J. tation. This amount may be readily determined by the skilled 65 Health Syst. Pharm. 55: 899-904, 1998; Raymon et al., Exp. artisan, based upon known procedures, including clinical tri Neurol. 144: 82-91, 1997: Culver et al., Hum. Gene Ther. 1: als, and methods disclosed herein. 399-410, 1990; Kasid et al., Proc. Natl. Acad. Sci. U.S.A. 87: US 8,980,630 B2 15 16 473–477, 1990). The nucleic acid vector or biological vector heart disease, Ventricular dysfunction, hyperemia, cardiovas may be introduced into excised cells or tissues by, for cular pregnancy complications, cardiovascular syphilis, car example, calcium phosphate-mediated transfection (Wigler diovascular tuberculosis, arrhythmias Such as sinus arrhyth et al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell mia, , atrial flutter, bradycardia, extrasystole, Genetics 7: 603, 1981; Graham and Vander Eb, Virology 52: Adams-Stokes Syndrome, bundle-branch block, sinoatrial 456, 1973). Other techniques for introducing nucleic acid block, long QT syndrome, parasystole, sick sinus syndrome, vectors into host cells, such as electroporation (Neumann et Ventricular fibrillations, tachycardias Such as paroxysmal al., EMBO J. 1: 841-845, 1982), may also be used. tachycardia, Supraventricular tachycardia, accelerated idio The cells of the invention may also be co-administered Ventricular rhythm, atrioventricular nodal reentry tachycar with other agents, such as other cell types, growth factors, and 10 dia, ectopic atrial tachycardia, ectopic junctional tachycardia, antibiotics. Other agents may be determined by those of ordi sinoatrial nodal reentry tachycardia, sinus tachycardia, Tor nary skill in the art. sades de Pointes, and ventricular tachycardia and heart valve In one aspect the invention provides a method of treating a diseases such as aortic valve insufficiency, aortic valve Steno neurodegenerative disease or a brain or spinal cord injury in a sis, heart murmurs, aortic valve prolapse, mitral valve pro patient, wherein the method comprises administering athera 15 lapse, tricuspid valve prolapse, mitral valve insufficiency, peutically effective amount of an ADSC of the present inven mitral valve Stenosis, pulmonary atresia, pulmonary valve tion or a population of neurogenically differentiated cell of insufficiency, pulmonary valve Stenosis, tricuspidatresia, tri the present invention to a patient having a neurodegenerative cuspid valve insufficiency, and tricuspid valve Stenosis. disease or a brain or spinal cord injury. In one embodiment the In one aspect the invention provides a method of treating a neurodegenerative disease is selected from the group consist metabolic disease in a patient, wherein the method comprises ing of Parkinson's disease, Alzheimer's disease, Pick's dis administering a therapeutically effective amount of an ADSC ease, Huntington's disease, spingolipidoses, mucosacchari of the present invention to a patient having the metabolic doses, and amyotrophic lateral Sclerosis. disease. In one embodiment the metabolic disease is selected In one aspect the invention provides a method of treating a from the group consisting of Phenylketonuria, Alkaptonuria, bone disease in a patient, wherein the method comprises 25 Ochronosis, Tyrosinemia, Albinism, Histidinemia, Maple administering a therapeutically effective amount of an ADSC syrup urine disease, Propionic acidemia, Methylmalonic aci of the present invention or the population of osteogenically demia, Isovaleric acidemia, 3-Methylcrotonyl-CoA carboxy differentiated cells of the present invention to a patient having lase deficiency, Cystinuria, Cystinosis, Hartnup disease, the bone disease. In one embodiment the bone disease is Homocystinuria, CyStathioninuria, N-cetylglutamate Syn selected from the group consisting of osteoporosis, Paget’s 30 thase deficiency, Carbamoyl phosphate synthase I deficiency, disease, osteogenesis imperfecta, and osteoarthritis. Omithine transcarbamylase deficiency, Citrullinemia, In one aspect the invention provides a method of treating a Argininosuccinic aciduria, Hyperammonemia, Glutaric aci hepatic disease in a patient, wherein the method comprises demia type 1, Sarcosinemia, Lactose intolerance, type I Gly administering a therapeutically effective amount of an ADSC cogen storage disease, type II Glycogen storage disease, type of the present invention or the population of hepatically dif 35 III Glycogen storage disease, type IV Glycogen storage dis ferentiated cells of the present invention to a patient having ease, type V Glycogen storage disease, type VI Glycogen the hepatic disease. In one embodiment the hepatic disease is storage disease, type VII Glycogen storage disease, Fructose selected from the group consisting of amebic liver abscess, intolerance, Essential fructosuria, Galactosemia, PCD, autoimmune hepatitis, biliary atresia, cirrhosis, coccidioido PDHA, Pentosuria, Renal glycosuria, GM2 gangliosidoses, mycosis: disseminated, delta agent (Hepatitis D), drug-in 40 Sandhoff disease, Tay-Sachs disease, GM1 gangliosidoses, duced cholestasis, hemochromatosis, hepatitis A, hepatitis B. Mucolipidosis type IV. Gaucher's disease, Niemann-Pick hepatitis C, hepatocellular carcinoma, liver cancer, liver dis disease, Farber disease, Fabry's disease, Metachromatic leu ease due to alcohol, primary biliary cirrhosis, pyogenic liver kodystrophy, Krabbe disease, Neuronal ceroid lipofuscino abscess, Reye's syndrome, Sclerosing cholangitis, and Wil sis, Batten disease, Cerebrotendineous Xanthomatosis, Cho son's disease. 45 lesteryl ester storage disease, Wolman disease, In one aspect the invention provides a method of treating a Hyperlipidemia, Hypercholesterolemia, Familial hypercho cardiovascular disease in a patient, wherein the method com lesterolemia, Xanthoma, Combined hyperlipidemia, Lecithin prises administering a therapeutically effective amount of an cholesterol acyltransferase deficiency, Tangier disease, Abe ADSC of the present invention to a patient having the cardio talipoproteinemia, Adrenoleukodystrophy, primary carnitine vascular disease. In one embodiment the cardiovascular dis 50 deficiency, carnitine palmitoyltransferase I deficiency, car ease is selected from the group consisting of congenital heart nitine palmitoyltransferase II deficiency, carnitine-acylcar defects, peripheral artery disease, arterio-arterial fistula, arte nitine translocase deficiency, Wilson's disease, Menkes dis riovenous fistula, cerebral arteriovenous malformations, aor ease, Haemochromatosis, Acrodermatitis enteropathica, tic coarctation, cortriatum, coronary vessel anomalies, patent Hypophosphatemia, Hypophosphatasia, Hypermagnesemia, ductus arteriosus, Ebstein's anomaly, hypoplastic left heart 55 Hypomagnesemia, Hypercalcaemia, Hypocalcaemia, Disor syndrome, levocardia, transposition of great vessels, double ders of calcium metabolism, Hyperuricemia, Lesch-Nyhan outlet right ventricle, tricuspidatresia, persistent truncus arte syndrome, Xanthinuria, Gilbert's syndrome, Crigler-Najjar riosus, and heart septal defects, such as aortopulmonary sep syndrome, Dubin-Johnson syndrome, Rotor syndrome, tal defect, endocardial cushion defects, Lutembacher's Syn Mucopolysaccharidosis, Hurler Syndrome, Hunter Syn drome, Ventricular heart septal defects, cardiac tamponade, 60 drome, Sanfilippo Syndrome, Morquio Syndrome, Maro endocarditis (including bacterial), heart aneurysm, cardiac teaux-Lamy Syndrome, Sly Syndrome, Mucolipidosis, I-cell arrest, congestive heart failure, congestive cardiomyopathy, disease, Pseudo-Hurler polydystrophy, Aspartylglu paroxysmal dyspnea, cardiac edema, post-infarction heart cosaminuria, Fucosidosis, Alpha-mannosidosis, Sialidosis, rupture, Ventricular septal rupture, heart valve diseases, myo Alpha 1-antitrypsin deficiency, Cystic fibrosis, Amyloidosis, cardial diseases, pericardial effusion, pericarditis (including 65 Familial Mediterranean fever, and Acatalasia constrictive and tuberculous), pneumopericardium, postperi In one embodiment of the present invention, the ADSCs are cardiotomy syndrome, pulmonary heart disease, rheumatic useful for inducing new blood vessel formation in a patient. US 8,980,630 B2 17 18 New blood vessels can be formed by vasculogenesis (forma cryopreserved intact (without the need for cell dissociation) tion of blood vessels from embryonic precursors), angiogen in liquid nitrogen or in a standard-80° C. laboratory freezer esis (in-growth of blood vessels from the Surrounding tissue) indefinitely. When retrieved from cryopreservation and or the formation of neovascularization (formation of new placed into culture, viable stem cell populations are readily blood vessels where they had not been previously) including established. This methodology 1) minimizes handling forming blood vessels from endothelial progenitor cells link requirements at the time of amnion isolation, 2) decreases ing to existing blood vessels. There are numerous conditions costs associated with long-term cryopreservation (freezer in which a mammal may be in need of forming new blood storage) and 3) allows for simple and reliable establishment vessels such as injury due to trauma, Surgery or acute or of stem cell cultures for propagation and manipulation. This chronic diseases. For example, the mammal may have a 10 methodology may be adaptable to other solid tissues and wound that requires healing. In another non-limiting associated stem cells. example, the patient may have undergone cardiovascular Sur In one embodiment the invention provides a method of gery, cardiovascular angioplasty, carotid angioplasty, or coro cryopreserving an amniotic membrane tissue sample com nary angioplasty, which are all conditions requiring new prising: a. washing the amniotic membrane tissue sample; b. blood vessel formation. In another non-limiting example, 15 Suspending the amniotic membrane tissue sample in a cryo patients who have had a myocardial infarction, Such as an a preservation medium comprising 60% Dulbecco's Modified MI, are in need of new blood vessel formation. Other condi Eagles Media (DMEM)/30% Fetal Bovine Serum (FBS)/ tions which may require new blood vessel formation include 10% Dimethylsulfoxide (DMSO); and c. storing the popula sickle cell anemia and thalassemia. tion at a temperature below about -80° C. In another embodiment of the present invention, the In one embodiment the invention provides a method of ADSCs can be administered to the mammal in need of form cryopreserving an amniotic membrane tissue sample com ing new blood vessels by any route or method that allows the prising: a. washing the amniotic membrane tissue sample; b. preferential migration of the cells to the site in need of new Suspending the amniotic membrane tissue sample in a cryo blood vessel formation. Exemplary routes of administration preservation medium comprising 60% DMEM/30% Liforcel include, but are not limited to, systemic administration Such 25 serum substitute/10% DMSO; and c. storing the population at as intravenous injection, localized implantation Such as local a temperature below about -80° C. ized intramuscular or Subcutaneous injection of the progeni Other than in the operating examples, or where otherwise tor cells in biocompatible solutions or biodegradable biocom indicated, all numbers expressing quantities of ingredients, patible matrices. Biocompatible solutions are known to those reaction conditions, and so forth used in this application are to skilled in the art. Examples of biodegradable biocompatible 30 be understood as being modified in all instances by the term matrices include, but are not limited to, solubilized basement "about. Accordingly, unless the contrary is indicated, the membrane, autologous platelet gel, collagen gels or collag numerical parameters set forth in this application are approxi enous Substrates based on elastin, fibronectin, laminin, extra mations that may vary depending upon the desired properties cellular matrix and fibrillar proteins. sought to be obtained by the present invention. At the very The present invention further provides a composition com 35 least, and not as an attempt to limit the application of the prising an ADSC of the invention. The present invention also doctrine of equivalents to the scope of the claims, each provides a pharmaceutical composition comprising an ADSC numerical parameter should be construed in light of the num of the invention. The ADSC of the invention or formulations ber of reported significant digits and by applying ordinary thereof may be administered by any conventional method rounding techniques. including parenteral (e.g. Subcutaneous or intramuscular) 40 Notwithstanding that the numerical ranges and parameters injection or intravenous infusion. The treatment may consist setting forth the broad scope of the invention are approxima of a single dose or a plurality of doses over a period of time. tions, the numerical values set forth in the specific examples The pharmaceutical composition may comprise one or more are reported as precisely as possible. Any numerical value, acceptable carriers. The carrier(s) must be “acceptable' in the however, inherently contains certain errors necessarily result sense of being compatible with the ADSC and not deleterious 45 ing from the standard deviation found in the respective testing to the recipients thereof. Typically, the carriers may be water measurementS. or saline which will be sterile and pyrogen free. It must be noted that as used herein and in the appended In one aspect the invention provides a cryopreserved claims, the singular forms “a”, “and”, and “the include plural ADSC of the present invention. referents unless the context clearly dictates otherwise. In one embodiment the invention provides a method of 50 Unless defined otherwise, all technical and scientific terms cryopreserving ADSCs comprising: a. washing a population used herein have the same meaning as commonly understood of ADSCs; b. suspending the population of ADSCs in a cryo by one of ordinary skill in the art to which this invention preservation medium comprising 60% Dulbecco's Modified belongs. Although any methods and materials similar or Eagles Media (DMEM)/30% Fetal Bovine Serum (FBS)/ equivalent to those described herein can also be used in the 10% Dimethylsulfoxide (DMSO); and c. storing the popula 55 practice of the present invention, exemplary methods and tion at a temperature below about -80° C. materials are described for illustrative purposes. In one embodiment the invention provides a method of All publications mentioned in this application are incorpo cryopreserving ADSCs comprising: a. washing a population rated by reference to disclose and describe the methods and/ of ADSCs; b. suspending the population of ADSCs in a cryo or materials in connection with which the publications are preservation medium comprising 60% DMEM/30% Liforcel 60 cited. Additionally, the publications discussed herein are pro serum substitute/10% DMSO; and c. storing the population at vided solely for their disclosure prior to the filing date of the a temperature below about -80° C. present application. Nothing herein is to be construed as an In one aspect the invention provides a cryopreserved amni admission that the present invention is not entitled to antedate otic membrane tissue sample. In one aspect the invention such publication by virtue of prior invention. Further, the provides a method of cryopreserving an amniotic membrane 65 dates of publication provided may be different from the actual explant, which allows for the future isolation of ADSC popu publication dates, which may need to be independently con lations. Freshly isolated amniotic membrane tissue can be firmed. US 8,980,630 B2 19 20 Methods, techniques, and/or protocols (collectively dishes and incubated for 2 hours to allow cell attachment. “methods”) that can be used in the practice of the invention Supernatant is removed and dishes are washed with PBS to are not limited to the particular examples of these procedures eliminate unattached cells. Dishes are examined microscopi cited throughout the specification but embrace any procedure cally and isolated single cells are identified and marked. known in the art for the same purpose. Furthermore, although 5 Colonies arising from these single cells are then expanded Some methods may be described in a particular context in the into clonal lines. specification, their use in the instant invention is not limited to Flow Cytometry that context. ADSC parent and clonal cultures are trypsinized and Sus The specification is most thoroughly understood in light of pended in 20% FBS/DMEM. Samples are centrifuged and the teachings of the references cited within the specification, 10 suspended in PBS. 1x10° cells are placed into separate 1.5 ml all of which are hereby incorporated by reference in their tubes, washed 2x with PBS and incubated for 1 hour at room entirety. The embodiments within the specification provide temperature with the following FITC-conjugated primary an illustration of embodiments of the invention and should antibodies Becton Dickinson, Franklin Lakes, N.J.: CD11b not be construed to limit the scope of the invention. The (rat, 1:500), CD44 (rat, 1:500), CD45 (rat, 1:500), CD29 skilled artisan recognizes that many other embodiments are 15 (guinea pig, 1:500), CD90 (rat, 1:500) and PE-conjugated encompassed by the claimed invention and that it is intended CD31 (rat, 1:200). Control samples are incubated in PBS that the specification and examples be considered as exem without primary antibody. Cell suspensions are fixed with 4% plary only, with the true scope and spirit of the invention being paraformaldehyde (PFA) and analyzed by the Coulter indicated by the following claims. Cytomics FC500 Flow Cytometer. The present invention is illustrated by the following RNA Isolation Examples, which are not intended to be limiting in any way. RNA is isolated from induced and control ADSCs using Trizol reagent according to manufacturers recommendations EXAMPLES Life Technologies, Carlsbad, Calif.). The resulting RNA pel let is subjected to a chloroform extraction and two ethanol Example 1 25 precipitations. cDNA Synthesis Methods 2 ug of RNA are reverse-transcribed using Superscript II reverse transcriptase Life Technologies in a 20 Jul Volume ADSC Isolation, Culture and Cloning containing 1 lug of oligo-dT primer, 200 uM dNTPs, and Ratamnion membrane is mechanically separated from the 30 buffers supplied by the manufacturer. The reaction is carried chorion of embryonic day 18.5 (E18.5) Sprague-Dawley rat out in a Perkin-Elmer 9600 polymerase chain reaction (PCR) embryos. The tissue is washed extensively with phosphate machine with the following parameters: 25°C.5 minutes, 37° buffered saline (PBS) and subsequently cut into small pieces. C. 5 minutes, 42°C. 60 minutes, and 48°C. 10 minutes. A 5 Membrane fragments are placed in 6-well plastic tissue cul minute ramp time is employed between each temperature. In ture dishes in a minimal volume (0.5 ml) of Dulbecco's modi 35 control reactions the SuperScript II reverse transcriptase is fied Eagle's medium DMEM: Invitrogen, Carlsbad, Calif. omitted. supplemented with 20%. Fetal Bovine Serum FBS; Atlanta Polymerase Chain Reaction Biologicals, Atlanta, Ga. to encourage attachment. Cells cDNA target (1 Jul) is amplified by PCR using specific begin to emerge from the explanted tissues within 24 hours of primer pairs (see Table 1). Sequences for telomerase primers plating. After one week the tissue explants are removed and 40 are obtained from a previously published report (Ginis et al., the remaining adherent cells are trypsinized and re-plated into 2004). PCR reactions employed Tfl polymerase and Master 100 mm plastic culture dishes. Cultures are passaged at con Amp PCR Optimization Buffer Epicentre, Madison, Wis. fluency. All cultures are maintained under a humidified atmo according to manufacturer's recommendations. PCRs are sphere of 5% CO, at 37°C. ADSCs used in these studies are performed in a Rapid Cycler 2 Idaho Technologies, Salt Lake extensively propagated up to 50 passages in vitro. 45 City, Utah as follows: initial 2 minute denaturing step at 94° Generation of Clonal Lines C. followed by 35-40 cycles of 94° C. 1 second, 52-60° C. 11 Clonal lines are generated from single cells as previously seconds, 72°C.30-70 seconds. All reactions are performed in described (Black and Woodbury, 2001). Briefly, ADSCs are 10 ul volumes. Brain and liver tissue extracts are used as plated at a density of 1 cell/cm in 150 mm plastic culture positive PCR controls. TABLE 1.

Primer Sequences

Alolore - Forward Forward Rewerse Rewerse viated Primer Primer primer Primer Full Name Name s" ->3' SEO ID NO 5'-->3' SEO ID NO

Telomerase CTGCGT 1. CACCTC 2 GTGCGT AGCAAA. GCTCTG CAGCTT GAC GTTCTC

Nanog TGCTCT 3 TCTGGT 4. GCACAG TGCTCC AGACTG AGGTTG GTAAGG GGTTGG US 8,980,630 B2 21 TABLE 1 - continued Primer Sequences

Alolore- Forward Forward Rewerse Reverse viated Primer Primer primer Primer Full Name Name 5'-->3' SEO ID NO 5'-->3' SEC ID NO

SRY-related Sox2 ACATGA 5 GCTGCT 6 HMG-box gene TGGAGA CCTGCA 2 CGGAGC TCATGC TGAAGC TGTAGC

Neurogenic NeuroD TGACCA. 7 AGAAGT 8 differen- AATCAT TGCCAT tiation ACAGCG TGATGC factor AGAGC TGAGCG

Orphan Nurr - 1 CTCCCG 9 GTGTCT 10 nuclear GAGGA TCCTCT hormone ACTGCA GCTCGA receptor-1 CTTCGG TCATAT C

Neural cell NCAM TGCTCA 11 CTTCTC 12 adhesion AGTCCC GGGCTC molecule TAGACT TGTCAG GGAACG TGGTGT GG

NMDA GLUT AGTTTC 13 AACTGA. 14 glutamate TTGGTC TGGTCA binding TCTGGG GGATCG subunit GACAGC ACAGGG

B-III-Tubulin TGCTCA 1.5 ATGGCC 16 TCAGCA AGCATC AAGTGC TGCTCA GTGAGG TCCACC

Neuro- NF-M CTCGAC 17 TCTTTG 18 filament- TTCAGC CGCTCT M CAGTCC ACGGTG TCTTCG ATGTGC

Microtubule MAP-2 TAACCA. 19 TACCTC 2 O associated ACACTA TGGCAC protein-2 GCGGAA TGAACT CGATGG GAGACC

Tau GGCTTT 21 GGCCTG 22 GAAGCA ATCACA GCATGG AACCCT CTGAAC GCTTGG

Growth GAP 43 ATGCTG 23 TCAGGC 24 associated TGCTGT ATGTTC protein 43 ATGAGA TTGGTC AGAACC AGCCTC

Amyloid APP AGTTTC 25 AACTGA. 26 precursor TTGGTC TGGTCA protein TCTGGG GGATCG GACAGC ACAGGG

Glial fibrillary GFAP AGCTGA, 27 GGAAGC 28 acidic protein ACCAGC AACGTC TTCGAG TGTGAG CCAAGG GTCTGC

Memorane- NG-2 TGGTAG 29 TGTCCT 3 O spanning CCCAGA GCAGTC proteoglycan AGCAGG AGCTCA NG2 TACTCC GATTGC

Tyrosine TH GACGGC 31 CAGCAG 32 hydroxylase GACAGA TCCGGC GTCTCA TCAGGT TCGAGG GAATGC US 8,980,630 B2 23 24 TABLE 1 - continued Primer Sequences

Alolore Forward Forward Rewerse Reverse viated Primer Primer primer Primer Full Name Name s" ->3' SEO ID NO s" ->3' SEO ID NO

Alkaline AP AAGACC 33 CCTTTC 34 Phosphatase CCAGTT CGATGG ACTGGC CCTCAT GACAGC CCATCT

Erythropoietin CACCTC 35 CAGAGT 36 AGCAAA. GACGGT CAGCTT GAGCGA GTTCTC GTTGGC

Fibronectin F 37 R- 38 GGAATG GAATTC GACCTG CCACCT CAAGCC CGAGTC AATAGC TGAACC

Wimentin CCAACG 39 GCCATC 4 O AGAAG TTTACA GTGGAA TTGAGC TTGCAG AGGTCC G

Osteonectin CGGAAG 41 TGTCCT. 42 CTGCAG GCTCCT AAGAG TGATGC ATGGTG CAAAGC G

Osteopontin TCGGAG 43 TCCTCA 44 GAGAA TGGCTG GGCGCA TGAAAC TTACAG TCGTGG C

Osteoprotegrin ACGAGT 45 TCTCGT 46 GATGAA TCTCTC TGCGTG AATCTC TACTGC GTCTGG

Osterix CCTTCT 47 ACTGCC 48 CAAGCA TGCATA CCAATG TCCACC GTCTCC ACTGCC

Adipsin CAGTGC 49 TCAATC SO AAGTGA CACGGC ATGGCA ACGTAG CGCACG GTTGCC

Smooth muscle SM22 - O TCTCCT 51 CTTCCC 52 protein 22-C. TCCAGT TTTCTA CCACAA ACTGAT ACGACC GATCTG

Albumin TCGTGA 53 TGTTCT 54 CAACTA GTCTCA CGGTGA GCGAGA ACTGGC CACTGG

Glutamine GS CCCACT 55 GGTCCT 56 synthetase TGAACA ATCTGG AAGGCA AATTCC TCAAGC CACTGG

Hepatocyte HNF-3C GCAGGC GCTGAA 58 nuclear factor TCCATG CCTGAG AACATG AAGCCT TCATCC GTGTCC

Hepatocyte HGF CAAGCA 59 GCACAG 60 growth factor ATCCAG GATATT AGGTAC ACAGGA GCTACG TGGTCC US 8,980,630 B2 25 26 TABLE 1 - continued Primer Sequences

Alolore- Forward Forward Rewerse Reverse viated Primer Primer primer Primer Full Name Name s" ->3' SEO ID NO 5'-->3' SEO ID NO

Hepatocyte C-MET CCCTAG 61 GGAATC 62 growth factor TGAAGT TCGGAA receptor CTGAGA TTCTGG TGAACG CAGGAC C.-1-antitrypsin C.-1.-AT GACAAG 63 GCTGGC 64 GCAGTT AGCACG CCAGCA TTGAGG TGCTGC CAATGC

Ceruloplasmin CTACAG 65 AGTAAC 66 TTGCTC CAGCTT CAACGT CCAGGC TGCCAG GTTTGG G

C.-fetoprotein AFP GACAAG 67 GCTGGC 68 GCAGTT AGCACG CCAGCA TTGAGG TGCTGC CAATGC

Phosphoenolpy PEP-CK1 CTCCTC 69 TGGGGT 7 O ruvate AGCTGC TCATCA carboxykinase- ATAATG TGGCCA 1. GTCTGG GGTTGG

Nerve growth NGF CCAAAC 71 CAAAGT 72 factor TGGAGG GTTGCC AGATGA ACTGTT TGCTGC GGGTGC

Neurotrophin- NT-4/5 TCCTCC 73 GCACAT 74 4/5 TTTTCC AGGACT TTCTCC GTTTAG TCCTCC CCTTGC

Tyrosine TrkA GTGGCT 7s TGTTGG 76 receptor GTCAAG AGAGCT kinase-A GCACTG GGTACC AAGG AGG

Bone BMP2 CTGTCT 77 TTCTGA 78 morphogenetic TCTAGT GTCACT protein-2 GTTGCT AACCTG GCTTCC GTGTCC

Bone BMP4 ACACTG 79 TTCCAG 8 O morphogenetic TGAGGA CCCACG protein- 4 GTTTCC TCACTG ATCACG AAGTCC

Transforming TGFC. GTATCC 81 AGACCA 82 growth factor- TGGTAG CTGTCT C. CTGTGT CAGAGT GTCAGG GGCAGC

Transforming TGFB CCACCT 83 GGACTG 84 growth factor- GCAAGA ATCCCA B CCATCG TTGATT ACATGG TCCACG

Wascular WEGF TTTCTG 85 TGGCTT 86 endothelial CTCTCT GTCACA growth factor TGGGTG TCTGCA CACTGG AGTACG

Placental PGF CACTTG 87 TGCCTT 88 growth factor CTTC TT TGTCGT GCAGGT CTCCAG CCTAGC AATAGG US 8,980,630 B2 27 28 TABLE 1 - continued Primer Sequences

Alolore- Forward Forward Rewerse Reverse viated Primer Primer primer Primer Full Name Name s" ->3' SEO ID NO 5'-->3' SEO ID NO

Fibroblast FGF2 AAGCGG 89 TCCGAG 9 O growth factor- CTCTAC TTTATA 2 TGCAAG CTGCCC AACGGC AGTTCG

Fibroblast FGF4 GACTAC 91 GGAAGT 92 growth factor- CTGCTG GGGTTA 4. GGCCTC CCTTCA AAAAG TGGTCG G

Platelet PDGF AGCGAC 93 TCCAGG 94 derived growth TGGCTC TGCTCC factor GAAGTC TCTAAC AGATCC CT CACC

Epidermal EGF CTGTAC 9s CATAGT 96 growth factor TCAGTG AAGCAA TCACAG ATCGTG CACAGC CCGTGC

Glyceraldehyde GAPDH TGTGAA 97 ATGCCA 98 3- CGGATT GTGAGC phosphatedehy TGGCCG TTCCCG drogenase TATCGG TTCAGC

Immunocytochemistry 30 three days. AMM consisted of 10% FBS/DMEM and 10 Cells are fixed in 4% PFA and Stored in PBS at 4°C. until ug/mL insulin in a base of DMEM. ADSCs are maintained at stained. Fixed cultures are blocked with 5% donor goat 37°C. for 3 weeks with media changed everythird day. Cells serum/0.3% Triton PBS for 45 minutes, followed by incuba are fixed with 4% PFA and stored at 4° C. until stained. tion in the following primary antibodies for 24 hours at 4°C.: Control cultures are grown in 10% FBS/DMEM. Oil Red Ois fibronectin (rabbit), 1:1000, Sigma, nestin (rabbit), 1:200, 35 utilized to visualize fat droplets. Santa Cruz, Biotechnologies, Santa Cruz, Calif., vimentin Osteogenic Induction (mouse, rabbit), 1:250-1:500, Santa Cruz, Biotechnologies, Osteogenic differentiation is performed as described pre neurofilament-M (mouse, rabbit), 1:250-1:500, Chemicon, viously (Woodbury et al., 2002). ADSCs are maintained in Temecula, Calif., and tau (mouse), 1:250, Santa Cruz. osteogenic induction media up to 6 weeks with fresh media After extensive washing, cultures are incubated at 37°C. for 40 added every third day. Cells are fixed with 4% PFA and stored 60 minutes with the species-specific Alexa Fluor 594 or 488 at 4° C. until stained. Control cultures are grown in 10% 1:750, Molecular Probes, Eugene, Oreg. secondary anti FBS/DMEM. Alizarin red is utilized to visualize mineralized bodies. Primary antibodies are omitted in all negative con matrix. RNA is obtained at 4 and 6 weeks post-induction and trols. 4,6-Diamidino-2-phenyindole (DAPI) 1 ug/mL, subjected to RT-PCR, examining expression of bone-specific Sigma is used as a nuclear counterstain. All samples are 45 genes. cover slipped with Fluoromount G. Electron Microscopy Hepatogenic Induction Systems. Fort Washington, Pa. and visualized with an ADSCs are seeded onto fibronectin-coated dishes at aden inverted fluorescent microscope Zeiss Axiovert, Thomwood, sity of ~3,000 cell/cm and grown to 70% confluency in N.Y.). serum containing media (SCM). Cultures are Subsequently Neural Induction 50 transferred to hepatocyte induction media (HIM) consisting Neuronal differentiation is performed as previously of 20 ng/ml hepatocyte growth factor (HGF), 10 ng/ml oncos described (Kramer et al., 2006) with modification. The media tatin M(OSM), 10 ng/ml FGF-4, 10 Mdexamethasone, 1% is supplemented with 10 ng/mlbFGF at 24 hours post-induc FBS in low glucose (LG) DMEM and maintained in this tion and every 48 hours there after. Cells in neural induction media until harvested. Control cultures are maintained in 1% media (NIM) are maintained under a humidified atmosphere 55 FBS/LG-DMEM without additional factors. Media is of 5% CO, in air at 30° C. Neural differentiation is deter changed every 3-4 days. Hepatic differentiation of the ADSCs mined by morphological changes and immunocytochemical is determined by Dil-Ac-LDL (low-density lipoprotein) analysis. In addition, RT-PCR is performed on mRNA iso uptake Biomedical Technologies, Stoughton, Mass. and lated from ADSCs cultured in NIM for 7 days, examining Periodic Schiff Acid (PAS) staining for glycogen. RNA is expression of neural specific genes. 60 obtained at 2 and 3 weeks posted induction and subjected to Adipogenic Induction RT-PCR, examining expression of liver-specific genes. Prior to adipogenic differentiation, ADSCs are rinsed Reporter Vector Construction and Transfection into extensively with PBS. ADSCs are exposed to an adipogenic ADSCS induction media (AIM) consisting of 10% FBS/DMEM, 500 The pEF1/eGFP vector used for transfection of ADSCs is uMIBMX, 1 uM dexamethasone, 10 ug/mL insulin and 100 65 generated from two commercially available vectors. The GFP uMindomethacin. Following three days in the AIM, ADSCs expression vector is constructed from elements derived from are cultured in adipogenic maintenance media (AMM) for two commercially available vectors. pEfl/myc/his (Invitro US 8,980,630 B2 29 30 gen) containing the elongation factor 1 promoter (EF1) Example 2 upstream of a multiple cloning site (MCS) serves as the vector backbone. The eGFP gene, isolated from pVivo2GFPLacz Amnion-Derived Stem Cell Isolation and (InvivoGen) is inserted into the MCS, allowing regulated Characterization expression by the EF1 promoter. The EF1GFP chimeric vec tor is introduced into proliferating ADSCs using Lipo Amniotic membranes are isolated from embryonic day fectamine (Invitrogen) as recommended by the manufacturer. 18.5 (E18.5) rats. To avoid contamination with previously Stable transfectants are selected using G418. Clonal lines identified fetal stem cell populations residing in the rat pla expressing high levels of GFP are identified via microscopic centa or umbilical cord, amnion tissue is isolated from the 10 dorsal part of the amniotic sac. The amnion is mechanically examination. For transplantation studies GFPADSC clone 2 peeled from the chorion and Subsequently cut into Small is utilized, which demonstrates the stem cells characteristics pieces and placed into tissue culture. Within 24 hours, cells of self-renewal and multi-differentiation in vitro. This clone begin to emerge from the tissue explants (FIG. 1A). By 72 uniformly expresses GFP and stable reporter gene expression hours numerous ameboid shaped cells migrate from the tissue is maintained at least 25 passages, the longest time examined. 15 pieces. Cell doublets are clearly visible, indicating cell pro Transuterine Intraventricular Injection liferation (FIG. 1B). Immunocytochemical analysis of pri Timed pregnant rats, 15.5 days postcoitum (E15.5) serve as mary cultures reveal that by 24 hours fibronectin-positive (+) hosts. Animals are sedated by an i.p. injection of ketamine (50 cells are visible at the edge of the tissue explants. In contrast, mg/kg) Xylazine (2.6 mg/kg) acepromazine (0.65 mg/kg). A 3 cytokeratin-19 (CK-19)+ epithelial cell remain within the cm Ventral midline incision exposes the abdominal cavity, tissue pieces and do not migrate from the explants. At 72 revealing the uterine horns and enclosed embryos. Guided by hours close to 100% of the migrating cells are vimentin (FIG. fiber optic transillumination, 23 Jul of ADSCs cell suspension 2, A-C) and fibronectin positive (FIG. 2, D-F). (100,000-150,000 cells in total) are pressure-injected into the Explants are removed after one week and the remaining lateral ventricles offetal brains using a glass capillary pipette. cells are trypsinized and replated in 100 mm tissue culture Successful injections are evidenced by rapid diffusion of the 25 dishes. By passage 5, the cell cultures predominately consist fast green dye throughout the Ventricular system. Embryos of rapidly dividing ameboid shaped cells, which are termed that do not receive donor cells are sacrificed in utero by amnion-derived stem cells (ADSCs). intraventricular infusion of 9% saline. Initial characterization of the ADSCs is performed by flow Tissue Processing cytometry. ADSCs express a number of mesenchymal cell For prenatal time points, dams are euthanized, embryos are 30 surface markers, including CD29 and CD90, but are negative retrieved and brains are microdissected. Brain tissue is for the lymphohematopoietic markers CD45 and CD11b immersion-fixed in 4% paraformaldehyde (PFA) for 24 hours (FIG. 7). at 4°C., rinsed 3x with PBS, then fixed for an additional 24 To further characterize ADSC gene expression, RNA is hours in 30%. Sucrose/4% PFA. Tissue is then Stored in 30% isolated from cultures at passage 3 and 12 and analyzed by 35 RT-PCR. ADSCs express a number of transcription factors, sucrose/PBS until processing. Postnatal animals are eutha including nanog and Sox2, involved in maintaining the pluri nized by an injection of pentobarbital (0.5 ml, 50 mg/ml), potency and self-renewal of ES cells (FIG. 3A). In addition, perfused with saline, followed by 4% PFA. Brains are ADSCs express telomerase, a protein associated with the removed and postfixed in 4% PFA for 24 hours, and subse immortalization of ES cells (FIG. 3A). The expression of quently stored in 30% sucrose/PBS until they are sectioned. 40 telomerase is consistent with the proliferative capacity of All samples are Sagitally, coronally or horizontally cryosec these cells; ADSCs are expanded greater than 50 passages in tioned at 16 um, and processed immunohistochemically. culture. Immunohistochemistry and Microscopic Analysis. PCR analysis reveals that cultured ADSCs express genes of Slides containing 16 um sections are rinsed extensively all three germinal layers. Specifically, ADSCs express a num with PBS. Slides are placed in citric acid buffer solution (pH 45 ber of neuroectodermal genes of varying function (FIG. 3B). 6.0), microwaved until boiling and allowed to cool slowly to Transcription factors involved in neural differentiation, ambient temperature. The microwave step is repeated three such as Nurr1 and NeuroD are express, and mRNA for the times for optimal antigen retrieval. Tissues are blocked with neuronal structural proteins MAP2 and NF-M are also 5% donor goat serum for 45 minutes. Sections are subse detected. ADSCs also contain transcripts for GFAP and quently incubated for 24 hours with primary antibodies: GFP 50 Olig1, markers of astrocytes and oligodendrocytes, respec (mouse, rabbit), 1:2501:500, Chemicon, vimentin (mouse, tively. ADSCs do not express a number of other neural and rabbit), 1:2501:500, Santa Cruz, Biotechnologies. Nestin ectodermal markers including, tyrosine hydroxylase, tau and (mouse), 1:250, DSHB), B-III tubulin (mouse), 1:500, pancytokeratin (data not shown). Chemicon, glial fibrillary acidic protein (GFAP) (rabbit), Consistent with the flow cytometry data, ADSCs express a 1:1000, Sigma), NeuN (mouse), 1:250, Chemicon, 55 number of mesenchymal genes. For example, vimentin, an adenomatous polyposis coli (APC) (mouse), 1:200, Calbio intermediate filament found in mesenchymal cells (FIG. 3C), chem and von Willebrand factor (vWF) (rabbit), 1:500, as well as other genes normally express bone (osterix and Chemicon. After numerous washes, the sections are incu alkaline phosphatase), fat (adipsin) and Smooth muscle bated with secondary antibodies: species specific Alexa Fluor (SM22C.) are detected. The cells also contained mRNA for 594 or 488 is used (1:500, Molecular Probes). 4.6 Diamidino 60 numerous endodermal genes (FIG. 3D) including, C.-1-antit 2phenyindole (DAPI)1 g/mL or propidium iodide (PI) 20 rypsin, an enzyme found in both the liver and lung. Further ug/ml dH20 are used as a nuclear counterstains. Negative more, ADSCs express a number of neurotrophin genes (FIG. controls performed by omitting the primary antibody. All 3E), including brain-derived neurotrophic factor (BDNF), a samples are cover slipped with Fluoromount G. Electron molecule involved in neuron Survival and synaptic plasticity, Microscopy Systems and visualized with an inverted fluo 65 as well as a number of growth factors (FIG.3F) that may play rescent microscope Zeiss AXioVert. Z sectioning is utilized roles in embryonic development. Expression of osteopontin, to colocalize all cellular markers. TrkB, TrkC and GDNF in cultured ADSCs is not detected. US 8,980,630 B2 31 32 To confirm PCR results and examine the co-expression of droplets, as indicated by positive Oil Red O staining. With markers of different embryonic layers within single cells, increasing time in culture, the lipid droplets coalesce, even immunocytochemistry is performed. Subpopulations of tually filling most of the cytoplasm (FIGS. 6, E and F). In ADSCs are observed to be multidifferentiated, simulta contrast, control cultures display no signs of lipid accumula neously expressing products of two distinct embryonic germ 5 tion (FIG. 6G). layers. For example, single ADSCs co-express the mesoder mal protein, vimentin and the neuroectodermal protein, Example 5 NF-M (FIG. 4, A-D). Other cells co-express the mesenchy mal protein, fibronectin and the neural progenitor marker, Endodermal Differentiation of ADSCs nestin (FIG. 4, E-H). The multidifferentiated state of sub 10 populations of ADSCs raises the possibility that ADSCs are To further gauge plasticity, ADSC capacity for endodermal also multipotent, capable of differentiating to diverse cell differentiation is examined. After exposure to hepatocyte types in vitro. To test this hypothesis, the ADSCs are sub induction media (HIM) ADSCs assume a cobblestone jected to a number of defined induction protocols designed to appearance (FIG. 6I), similar to mature hepatocytes. Differ foster differentiation into distinct somatic cell types. 15 entiated cells demonstrate the ability to take up LDL from the media (FIGS. 6, H and I), an established hepatocyte trait. In Example 3 contrast, control cells retain their ameboid morphology and do not take up LDL (FIG. 6J). In addition, PAS staining Neural Differentiation of ADSCs demonstrates that induced ADSC accumulate glycogen, another indication of hepatic differentiation. Moreover, liver To examine neuroectodermal potential, ADSCs are specific genes, including albumin and C-1-antitrypsin are exposed to a defined neural induction medium (NIM). up-regulated (FIG. 6K). ADSCs responds slowly to the induction media assuming elongated fibroblastic morphologies within 24 hours (FIGS. Example 6 5, A and B). Over the next several days, greater than 75% of 25 the cells convert from flat, ameboid figures to cells displaying Clonal Analysis of ADSCs compact, light refractile cell bodies (FIG.5C). ADSCs elabo rate long processes (FIG.5C) some extending more than 500 The observed plasticity of ADSCs may reflect the pluripo um from the cell body. In many cases, the cellular processes tency of single cells or the presence of multiple progenitor form networks (FIG. 5D) with neighboring cells similar to 30 cell types in the population. To distinguish between these those found in primary neural cultures. possibilities, several clonal lines derived from single cells are Morphological changes of differentiated ADSCs are established. Similar to parental populations of ADSCs, clonal accompanied by consistent changes in gene expression. lines consist of Small ameboid shaped cells. Clonal lines are ADSCs maintain the expression of some neuronal genes Such further analyzed by flow cytometry, RT-PCR and in vitro as NF-M, and up-regulate other neural genes (FIG. 5, E-H). 35 differentiation as described above. For instance, tau, a microtubule associated protein found in Flow cytometry reveals that clonal populations (C1, C2) of mature neurons, is expressed only in NIM-treated cultures ADSC have similar cell surface marker profiles when com (FIG. 5, E-H). Differentiated ADSCs up-regulate additional pare to parental ADSC populations (P5). All cultures are neural specific genes including GAP43, while down-regulat predominately positive for CD90 and CD29 (FIGS. 7, A and ing mesenchymal genes such as SM22C. (FIG.5I). Moreover, 40 B). Some variability exists with regard to CD44 and CD31 Sox2 and NeuroD, two transcription factors found in undif (FIGS. 7, Cand D), but all populations are uniformly negative ferentiated neural stem cells, are appropriately down-regu for the lymphohematopoietic markers CD45 and CD11b lated in differentiating ADSCs. (FIGS. 7, E and F). PCR analysis of clonal ADSCs recapitulates the expression Example 4 45 profile found in the parental population (FIG. 8A). Clonal lines express genes representing all three germinal layers. Mesodermal Differentiation of ADSCs Immunocytochemical analysis reveals that close to 100% of the cells express the mesenchymal proteins vimentin and The potential of ADSC to differentiate into prototypical fibronectin. Moreover, consistent with the multidifferentiated mesenchymal cell types: osteoblasts and adipocytes is also 50 state of the parental cultures, immunocytochemistry demon examined. ADSCs exposed to osteogenic induction media strates that Subpopulations of clonal ADSCs co-express mes (OIM) for four weeks attain cuboidal morphologies and begin enchymal and neuroectodermal proteins. to form nodules, consistent with osteoblastic differentiation. To assess plasticity, two clonal ADSC cell lines (C1, C2) At this time they are not depositing mineralized matrix. How are exposed to defined induction protocols to determine their ever, by six weeks treated cultures exhibit extensive mineral 55 ability to multidifferentiate in vitro. In neural induction media ized matrix deposition, as denoted by alizarin red staining clonal lines attain a neural phenotype. Morphology of NIM (FIGS. 6, A and B), consistent with a mature osteoblast phe treated clonal ADSCs is similar to that obtained with parental notype. As expected, control cultures of ADSCs do not population; treated cells display phase bright, refractile cell assume cuboidal morphologies or lay down mineralized bodies (FIG. 8B) and long network forming processes. Simi matrix (FIG. 6C). PCR analysis reveals appropriate changes 60 lar to parental populations, upregulation of neural-specific in gene expression with increased expression of osteopontin, genes is observed (see FIG. 5). Clonal lines exposed to adi and down-regulation of neural NF-M and endodermal C-1- pogenic and osteogenic induction media accumulate fat drop antitrypsin (FIG. 6D). Consistent with differentiation, down lets (FIG. 8B), and lay down mineralized matrix respectively regulation of the pluripotency gene nanog is also observed. (FIG. 8B). Moreover, under conditions conducive for hepatic ADSCs incubated in adipogenic induction media (AIM) 65 differentiation, the clonal lines differentiate into presumptive acquired characteristics specific to mature adipocytes. After hepatoctyes and exhibit LDL uptake (FIG. 8B). Collectively, two weeks, cells begin to accumulate Small cytoplasmic lipid this data Suggests that parental ADSC cultures contain a US 8,980,630 B2 33 34 population of true stem cells. Clonal lines derived from these that migrates around blood vessels attained crescent mor cells possess the ability to undergo long-term self-renewal phologies and express VWF (FIG. 12IL, arrow), a blood gly and multidifferentiation. coprotein involved in coagulation. These morphologic and phenotypic observations are consistent with an endothelial Example 7 cell differentiation of donor ADSCs. Some ADSCs that engraft in the brain attain neuronal mor phologies: Small cell body with long process extensions (FIG. In Vitro Characterization of ADSCs 5AC, arrow). Despite these morphological changes, no donor To determine whether ADSCs assume neuronal functions cells are found to upregulate any neural or glial markers, in vivo and assess potential use for cellular therapy, donor 10 including B-III Tubulin (FIG.13DG, arrows)and GFAP (FIG. cells are characterized after transplantation into the embry 13HK, arrows). Transplanted ADSCs are also negative for the onic day 15.5 (E15.5) rat brain. For transplantation studies a mature neural marker NeuN and the oligodendrocyte marker. clonal population of ADSCs which is utilized uniformly Example 10 express GFP (FIG. 9AC). This clonal population demon strates the stem cell characteristics of self-renewal and multi 15 potency. Close to 100% of the cells express vimentin (FIG. Long-Term Survival of Donor Cells 9DG), while a subset express the neural progenitor marker Even in the absence of neuronal differentiation, donor nestin. (FIG.9HK, arrowheads). Overexpression of the GFP ADSCs are able to survive in cortical (FIG. 14AC, arrows) gene does not affect the cells ability to respond morphologi and vascular regions (FIG. 14D, arrow) up to two and half cally to the in vitro neural induction protocol (FIG.9L). months postnatal, the longest time examined. No evidence of a host inflammatory response or immunological rejection is Example 8 observed, at early (P7) or later postnatal time points, as indi Transplantation of GFP-Expressing ADSCs into the cated by the absence of ED1+reactive microglia cells (FIG. 25 14EH). Embryonic Brain We claim: One hundred to one hundred and fifty thousand donor 1. A method for obtaining a multipotent amnion-derived GFP-expressing cells suspended in 2 to 3 ul of DMEM are stem cell (ADSC) comprising: injected into the telencephalic ventricles of each E15.5 recipi a. Separating an amniotic membrane tissue sample from ent embryo. Invasion, migration, localization, phenotypic 30 chorion of a mammalian embryo, expression and long-term Survival are assessed at various b. culturing the amniotic membrane tissue sample in cul times post-transplantation. ture media without any enzymes or reagents to digest the Initially, short-term Survival and engraftment 24 hours amniotic membrane tissue sample: post-transplantation is characterized. At E16.5 donor cells are c. preparing a single-cell culture of adherent ADSCs iso diffused throughout the ventricular system (FIG.10A). Some 35 lated from the amniotic membrane tissue sample; cells can also be identified within the parenchymal of the d. culturing the ADSCs; and brain at this early time point (FIG. 10A, inset). By E17.5 e. obtaining or isolating the ADSCs. discrete spherical clusters consisting of GFP+ cells are 2. The method of claim 1, wherein the amniotic membrane observed within the ventricles (FIG. 10B, arrow). A few clus tissue sample is washed and fragmented after step a. and 40 before step b. ters appear to have fused with the walls of the ventricles (FIG. 3. The method of claim 1, wherein the amniotic membrane 10C) and individual donor cells can be seen in the brain tissue sample is cultured in Dulbecco's modified Eagle's parenchyma (FIG. 11C, arrows). Widespread distribution of medium (DMEM) supplemented with 20% fetal bovine transplanted ADSCs is detected at later time points. serum (FBS). At E20.5 donor cells are consistently observed in multiple 4. A method of cryopreserving an amniotic membrane brain areas, including the cortex (FIG. 11 AC, arrow) and 45 tissue sample comprising: midbrain (FIG. 11EG, arrow). Distribution of GFP+ cells at a. Washing an amniotic membrane tissue sample; later postnatal time points seems to be random. In some b. Suspending the amniotic membrane tissue sample in a animals, cells can be localized to the hippocampus and stria cryopreservation medium consisting essentially of 60% tum while in others they are observed in the. Donor cells are Dulbecco's Modified Eagles Media (DMEM)/30% often associated with blood vessels (FIG. 11D, arrow). These 50 Fetal Bovine Serum (FBS) or 30% Liforcel serum sub cells have crescent morphologies, similar to endothelial cells. stitute/10% Dimethyl Sulfoxide (DMSO); and Example 9 c. storing the tissue sample at a temperature below about -80° C. 5. A method of cryopreserving ADSCs comprising: Phenotypic Characterization of Transplanted ADSCs 55 ... washing a population of ADSCs: The majority of donor cells that migrate into the paren t . Suspending the population of ADSCs in a cryopreserva chyma by P7, or 1 week postnatal, do not integrate within the tion medium comprising 60% Dulbecco's Modified normal cytoarchitecture of the brain. In most cases the Eagles Media (DMEM)/30%. Fetal Bovine Serum (FBS) ADSCs assume either elongated (FIG. 12A, arrows) or ame 60 or 30% Liforcel serum substitute/10% Dimethyl Sulfox boid (FIG. 4E, arrows) morphologies. Mirroring the expres ide (DMSO); and sion pattern in culture, the majority of ADSC express vimen c. storing the tissue sample at a temperature below about tin (FIG. 12AD, arrows), while a subset also express Nestin -80° C. (FIG. 12EH, arrows). Interestingly, a subpopulation of cells