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(12) United States Patent (10) Patent No.: US 8,980,630 B2 Woodbury Et Al

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(12) United States Patent (10) Patent No.: US 8,980,630 B2 Woodbury Et Al 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 N NS S S. Š U.S. Patent Mar. 17, 2015 Sheet 2 of 14 US 8,980,630 B2 U.S. Patent US 8,980,630 B2 O ??GjoineN ?LlunN U.S. Patent Mar. 17, 2015 Sheet 4 of 14 US 8,980,630 B2 U.S. Patent US 8,980,630 B2 U.S. Patent Mar. 17, 2015 Sheet 7 of 14 US 8,980,630 B2 al s w s &********-4………...... s R s --------------S ~~~~~~~~)*~~~~~~~~*~~~~.~~~~~~~~~~~~.~~~~;~~~~*~~~~.~~~~,~~~~~~~~.~~~~*~ cott's vs. &#2 c 3 girl 23 ti ..!!!--****** No-oooo , s: as wer, as t U.S. Patent Mar. 17, 2015 Sheet 8 of 14 US 8,980,630 B2 S. O c t Yuanme Yumeaume Name? U.S. Patent Mar. 17, 2015 Sheet 9 of 14 US 8,980,630 B2 U.S. Patent Mar. 17, 2015 Sheet 10 of 14 US 8,980,630 B2 U.S. Patent Mar. 17, 2015 Sheet 11 of 14 US 8,980,630 B2 U.S. Patent Mar. 17, 2015 Sheet 12 of 14 US 8,980,630 B2 U.S. Patent Mar. 17, 2015 Sheet 13 of 14 US 8,980,630 B2 U.S. Patent Mar. 17, 2015 Sheet 14 of 14 US 8,980,630 B2 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 cardiovascular disease. 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.
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