Granted Patent
B2
US 12,441,983 · App. 18/421,847 · Granted Oct 14, 2025
Suspension culture of human embryonic stem cells
Inventors:
Ramkumar Mandalam (Union City, CA); Yan Li (Tallahassee, FL); Isabelle Nadeau-Demers (San Francisco, CA)
Assignee:
Asterias Biotherapeutics, Inc.
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Abstract
This disclosure provides an improved system for culturing human embryonic stem cells. The cells are cultured in suspension so as to maximize the production capacity of the culture environment. The new culture system of this invention allows for bulk proliferation of hES cells in a more cost-effective manner, which facilitates commercial production of important products for use in human therapy.
Claims (27)
1. A method of maintaining human pluripotent stem (hPS) cells in an undifferentiated state comprising:
a) suspending human pluripotent stem (hPS) cells in a nutrient medium to create a suspension culture, wherein the nutrient medium comprises 20 ng/mL to 500 ng/mL of fibroblast growth factor (FGF) and transforming growth factor beta (TGFβ), wherein the nutrient medium does not contain serum;
b) culturing the hPS cells in the suspension culture for a period of time; and
c) adding fresh nutrient medium to the suspension culture, wherein the fresh nutrient medium does not contain serum; wherein the hPS cells are not grown on a solid substrate.
2. The method of claim 1 , wherein the FGF comprises FGF2.
3. The method of claim 2 , wherein the concentration of FGF2 present in the nutrient medium is between 40 ng/mL and 200 ng/mL.
4. The method of claim 1 , wherein the FGF comprises FGF4.
5. The method of claim 1 , wherein the hPS cells are cultured in suspension in nutrient medium comprising 40 ng/mL to 200 ng/mL of fibroblast growth factor (FGF) and transforming growth factor beta (TGFβ) for at least two months, while maintaining the undifferentiated state.
6. The method of claim 1 , wherein the nutrient medium further comprises one or more soluble or suspended extracellular matrix components.
7. The method of claim 6 , wherein the one or more soluble or suspended extracellular matrix components are selected from the group consisting of laminin, fibronectin, proteoglycan, vitronectin and entractin.
8. The method of claim 6 , wherein the concentration of soluble or suspended extracellular matrix components present in the nutrient medium is between 10 to 33 μg/mL.
9. The method of claim 1 , wherein the nutrient medium does not comprise soluble or suspended extracellular matrix components.
10. The method of claim 1 , wherein the nutrient medium further comprises one or more additives selected from the group consisting of: stem cell factor (SCF), Flt3 ligand (Flt3L), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), thrombopoietin (TPO), epidermal growth factor (EGF), macrophage colony-stimulating factor, c-kit, forskolin, leukemia inhibitory factor (LIF), oncostatin-M, and ciliary neurotrophic factor (CNTF).
11. The method of claim 1 , wherein the addition of fresh nutrient medium comprises media exchange.
12. The method of claim 1 , wherein the addition of fresh nutrient medium comprises infusion of fresh nutrient medium into the culture.
13. The method of claim 1 , wherein the nutrient medium comprises about 0.5 ng/mL TGFβ.
14. A method of maintaining human pluripotent stem (hPS) cells in an undifferentiated state comprising:
a) suspending human pluripotent stem (hPS) cells in a nutrient medium to create a suspension culture, wherein the nutrient medium comprises 40 ng/mL to 200 ng/mL of fibroblast growth factor (FGF) and transforming growth factor beta (TGFβ), wherein the nutrient medium does not contain serum;
b) culturing the hPS cells in the suspension culture for a period of time; and
c) adding fresh nutrient medium to the suspension culture, wherein the fresh nutrient medium does not contain serum; wherein the hPS cells are not grown on a solid substrate.
15. The method of claim 14 , wherein the addition of fresh nutrient medium comprises media exchange.
16. The method of claim 14 , wherein the addition of fresh nutrient medium comprises infusion of fresh nutrient medium into the culture.
17. The method of claim 14 , wherein the nutrient medium does not comprise soluble or suspended extracellular matrix components.
18. The method of claim 14 , wherein the hPS cells are cultured in suspension in nutrient medium for at least two months, while maintaining the undifferentiated state.
19. The method of claim 14 , wherein the nutrient medium further comprises soluble or suspended extracellular matrix components.
20. The method of claim 19 , wherein the one or more soluble or suspended extracellular matrix components are selected from the group consisting of laminin, fibronectin, proteoglycan, vitronectin and entractin.
21. The method of claim 14 , wherein the nutrient medium comprises about 0.5 ng/mL TGFβ.
Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Aug 7, 2024
From: MANDALAM, RAMKUMAR; LI, YAN; NADEAU-DEMERS, ISABELLE
To: GERON CORPORATION
Reel/Frame 068213/0482 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Aug 7, 2024
From: GERON CORPORATION
To: ASTERIAS BIOTHERAPEUTICS, INC.
Reel/Frame 068213/0565 →
Continuity (6)
Continuation
11917993
Provisional Application
60693266
· Jun 22, 2005
References Cited (207)
US 5166065A
· Williams et al.
· 1992
[cited by applicant]
US 5332672A
· Conover et al.
· 1994
[cited by applicant]
US 5405772A
· Ponting
· 1995
[cited by applicant]
US 5453357A
· Hogan
· 1995
[cited by applicant]
US 5583016A
· Villeponteau et al.
· 1996
[cited by applicant]
US 5843780A
· Thomson
· 1998
[cited by applicant]
US 5914268A
· Keller et al.
· 1999
[cited by applicant]
US 5922567A
· Au-young et al.
· 1999
[cited by applicant]
US 5922597A
· Verfaillie et al.
· 1999
[cited by applicant]
US 5968829A
· Carpenter
· 1999
[cited by applicant]
US 6090622A
· Gearhart et al.
· 2000
[cited by applicant]
US 6200806B1
· Thomson
· 2001
[cited by applicant]
US 6280718B1
· Kaufman et al.
· 2001
[cited by applicant]
US 6458589B1
· Rambhatla et al.
· 2002
[cited by applicant]
US 6576464B2
· Gold et al.
· 2003
[cited by applicant]
US 6642048B2
· Xu et al.
· 2003
[cited by applicant]
US 6800480B1
· Bodnar et al.
· 2004
[cited by applicant]
US 6833269B2
· Carpenter
· 2004
[cited by applicant]
US 6875607B1
· Reubinoff et al.
· 2005
[cited by applicant]
US 7005252B1
· Thomson
· 2006
[cited by applicant]
US 7041438B2
· Carpenter et al.
· 2006
[cited by applicant]
US 7297539B2
· Mandalam et al.
· 2007
[cited by applicant]
US 7410789B2
· Schloesser et al.
· 2008
[cited by applicant]
US 7410798B2
· Mandalam et al.
· 2008
[cited by applicant]
US 7413904B2
· Gold et al.
· 2008
[cited by applicant]
US 7455983B2
· Xu et al.
· 2008
[cited by applicant]
US 7473555B2
· Mandalam et al.
· 2009
[cited by applicant]
US 7790456B2
· Terstegge et al.
· 2010
[cited by applicant]
US 9074181B2
· Mandalam et al.
· 2015
[cited by applicant]
US 20020081724A1
· Carpenter et al.
· 2002
[cited by applicant]
US 20030113910A1
· Levanduski
· 2003
[cited by applicant]
US 20030153082A1
· Bhatia
· 2003
[cited by applicant]
US 20030224411A1
· Stanton et al.
· 2003
[cited by applicant]
US 20040071672A1
· Hogan
· 2004
[cited by applicant]
US 20050164377A1
· Miyabayashi et al.
· 2005
[cited by applicant]
US 20050233446A1
· Parsons et al.
· 2005
[cited by applicant]
AU 729377B2
· 2001
[cited by applicant]
AU 2005281852B2
· 2010
[cited by applicant]
CA 2877233A1
· 2000
[cited by applicant]
CN 1424394A
· 2003
[cited by applicant]
CN 1483817A
· 2004
[cited by applicant]
CN 1545549A
· 2004
[cited by applicant]
CN 1626651A
· 2005
[cited by applicant]
EP 0695351B1
· 1999
[cited by applicant]
JP 2001017163A
· 2001
[cited by applicant]
JP 2005501554A
· 2005
[cited by applicant]
WO 9407997A1
· 1994
[cited by applicant]
WO 9721802A1
· 1997
[cited by applicant]
WO 9747734A1
· 1997
[cited by applicant]
WO 9830678A1
· 1998
[cited by applicant]
WO 9830679A1
· 1998
[cited by applicant]
WO 9843679A1
· 1998
[cited by applicant]
WO 9920741A1
· 1999
[cited by applicant]
WO 9942122A1
· 1999
[cited by applicant]
WO 9943785A1
· 1999
[cited by applicant]
WO 0017323A1
· 2000
[cited by applicant]
WO 0151610A1
· 2001
[cited by applicant]
WO 0151616A2
· 2001
[cited by applicant]
WO 0166697A2
· 2001
[cited by applicant]
WO 0181549A2
· 2001
[cited by applicant]
WO WO0231123A1
· 2002
[cited by examiner]
WO 03000868A1
· 2003
[cited by applicant]
WO 03004605A2
· 2003
[cited by applicant]
WO 03006950A2
· 2003
[cited by applicant]
WO 03020920A1
· 2003
[cited by applicant]
WO 03038070A1
· 2003
[cited by applicant]
WO 03050249A2
· 2003
[cited by applicant]
WO 03050250A2
· 2003
[cited by applicant]
WO 03050251A2
· 2003
[cited by applicant]
WO 2004007696A2
· 2004
[cited by applicant]
WO 2004055155A2
· 2004
[cited by applicant]
WO WO2005007799A2
· 2005
[cited by examiner]
WO 2005033298A1
· 2005
[cited by applicant]
WO WO2005053601A2
· 2005
[cited by examiner]
WO 2005065354A2
· 2005
[cited by applicant]
WO 2005090558A1
· 2005
[cited by applicant]
WO 2006027229A1
· 2006
[cited by applicant]
WO 2006070370A2
· 2006
[cited by applicant]
WO 2008004990A2
· 2008
[cited by applicant]
WO 2008015682A2
· 2008
[cited by applicant]
Fok, E.Y. 2004. Development of a scalable bioprocess for the culture of undifferentiated murine embryonic stem cells. Thesis submitted for Master of Applied Science degree. University of Toronto, pp. 1-103; w front matt…
[cited by examiner]
Zwaka et al. (2007) “Genetic Modification of Human Embryonic Stem Cells”, Embryonic Stem Cells, Eds. Springer, 41-52.
[cited by applicant]
Vassilieva et al. (Aug. 1, 2000) “Establishment of SSEA-1- and Oct-4-Expressing Rat Embryonic Stem-like Cell Lines and Effects of Cytokines of the IL-6 Family on Clonal Growth”, Experimental Cell Research, 258(2):361-37…
[cited by applicant]
Vogel, Gretchen (Sep. 1, 2000) “Researchers Get Green Light for Work on Stem Cells”, Science, 28(5484):1442-1443.
[cited by applicant]
Vogel, Gretchen (Feb. 11, 2000) “Stem cells. Wisconsin to distribute Embryonic Cell Lines”, Science, 287(5455):948-949.
[cited by applicant]
Wang et al. (Jun. 13, 2005) “Derivation and Growing Human Embryonic Stem Cells on Feeders Derived from Themselves”, Experimental Protocols for Embryonic Stem Cell Research, 23(9):1221-1227.
[cited by applicant]
Wang et al. (May 13, 2005) “Noggin and Bfgf Cooperate to Maintain the Pluripotency of Human Embryonic Stem Cells in the Absence of Feeder Layers”, Biochemical and Biophysical Research Communications, 330(3):934-942.
[cited by applicant]
Wartenberg et al. (2001) “Tumor-induced Angiogenesis Studied in Confrontation Cultures of Multicellular Tumor Spheroids and Embryoid Bodies Grown From Pluripotent Embryonic Stem Cells”, FASEB Journal, 15(6):995-1005.
[cited by applicant]
Williams et al. (Dec. 15, 1988) “Myeloid Leukaemia Inhibitory Factor Maintains the Developmental Potential of Embryonic Stem Cells”, Nature, 336(6200):684-687.
[cited by applicant]
Xiao et al. (Feb. 2, 2006) “Activin A Maintains Self-Renewal and Regulates Fibroblast Growth Factor, Wnt, and Bone Morphogenic Protein Pathways in Human Embryonic Stem Cells”, Stem Cells, Embryonic Stem Cells: Character…
[cited by applicant]
Xu et al. (Mar. 2005) “Basic FGF and Suppression of BMP Signaling Sustain Undifferentiated Proliferation of Human ES Cells”, Nature Methods, 2(3):185-190.
[cited by applicant]
Xu et al. (Mar. 2005) “Basic Fibroblast Growth Factor Supports Undifferentiated Human Embryonic Stem Cell Growth Without Conditioned Medium”, Stem Cells, 23(3):315-323.
[cited by applicant]
Xu et al. (Sep. 20, 2002) “Characterization and Enrichment of Cardiomyocytes Derived from Human Embryonic Stem Cells”, Circulation Research, 91(6):501-508.
[cited by applicant]
Xu et al. (Oct. 1, 2001) “Feeder-Free Growth of Undifferentiated Human Embryonic Stem Cells”, Nature Biotechnology, 19(10):971-4.
[cited by applicant]
Xu et al. (2004) “Immortalized Fibroblast-Like Cells Derived from Human Embryonic Stem Cells Support Undifferentiated Cell Growth”, Stem Cells, 22(6):972-980.
[cited by applicant]
Yan et al. (Sep. 2013) “Single-Cell RNA-Seq Profiling of Human Preimplantation Embryos and Embryonic Stem Cells”, Nature Structural & Molecular Biology, 20(9):1131-1139.
[cited by applicant]
(2011) Biographical Information for Dr. Andrea Bodnar, Bermuda Institute of Ocean Sciences Webpage, 1 Page.
[cited by applicant]
(2011) Biographical information for Dr. James Thomson, University of Wisconsin Madison Webpage, 1 Page.
[cited by applicant]
(2011) Biographical information for Dr. Joseph Itskovitz-Eldor, Rambam Medical Centre Webpage, 1 Page.
[cited by applicant]
(2011) Biographical information for Dr. Peter Grey, University of Queensland webpage, 1 Page.
[cited by applicant]
(2014) Cole Palmer Catalog, Available at: https://www.coleparmer.com/ProducUCorning-roller-bottle-850-cm2-easy-grip-cap/UX-0.
[cited by applicant]
(2023) Corning Matrigel Matrix, corning.com/catalog/cls/documents/faqs/CLS-DL-CC-026.pdf, 8 pages.
[cited by applicant]
(May 6, 2014) Denville Scientific, Available at: http://www.denvillescientific.com/node/4662, 1-2.
[cited by applicant]
Extended European Search Report Issued in EP App. No. 06785185.7, mailed on Jul. 31, 2009, 7 Pages.
[cited by applicant]
Extended European Search Report Issued in European Application No. EP19180013.5, mailed on Oct. 15, 2019, 10 Pages.
[cited by applicant]
International Preliminary Report on Patentability Issued in International Application No. PCT/US2006/023976, mailed on Dec. 24, 2007, 5 Pages.
[cited by applicant]
International Search Report and Written Opinion Issued in International Application No. PCT/US2006/023976, mailed on Aug. 13, 2007, 6 Pages.
[cited by applicant]
Lipofectamine 2000 Transfection Reagent, Thermofisher Scientific, Sep. 17, 2010, 6 Pages.
[cited by applicant]
Al-Obeidi et al. (2000) “Development of Inhibitors for Protein Tyrosine Kinases”, Oncogene, 19(49):5690-5701.
[cited by applicant]
Amit et al. (Nov. 2000) “Clonally Derived Human Embryonic Stem Cell Lines Maintain Pluripotency and Proliferative Potential for Prolonged Periods of Culture”, Developmental Biology, 227(2):271-278.
[cited by applicant]
Amit et al. (Nov. 19, 2003) “Feeder Layer- and Serum-Free Culture of Human Embryonic Stem Cells”, Biology of Reproduction, 70(3):837-845.
[cited by applicant]
Amit et al. (Jun. 1, 2003) “Human Feeder Layers for Human Embryonic Stem Cells”, Biology of Reproduction, 68(6):2150-2156.
[cited by applicant]
Anzai et al. (Feb. 1999) “Self-Renewal and Differentiation of a Basic Fibroblast Growth Factor-Dependent Multipotent Hematopoietic Cell Line Derived from Embryonic Stem Cells”, Development Growth & Differentiation, 41(1…
[cited by applicant]
Armstrong, Lyle (Apr. 5, 2007) “Government Proposals for the Regulation of Hybrid and Chimera Embryos”, House of Commons Science and Technology Committee, Fifth Report of Session Jul. 2006, vol. II, Ev76-Ev80.
[cited by applicant]
Baribault et al. (Dec. 1989) “Embryonic Stem Cell Culture and Gene Targeting in Transgenic Mice”, Molecular Biology & Medicine, 6(6):481-492.
[cited by applicant]
Beattie et al. (2005) “Activin A Maintains Pluripotency of Human Embryonic Stem Cells in the Absence of Feeder Layers”, Stem Cells, 23(4):489-495.
[cited by applicant]
Berger et al. (1997) “Self Renewal of Embryonic Stem Cells in the Absence of Feeder Cells and Exogenous Leukaemia Inhibitory Factor”, Growth Factors, 1997, 14(2-3)145-159.
[cited by applicant]
Brook et al. (May 1997) “The Origin and Efficient Derivation of Embryonic Stem Cells in the Mouse”, Proceedings of the National Academy of Sciences of the United States of America, 94(11):5709-5712.
[cited by applicant]
Carpenter et al. (Dec. 2001) “Enrichment of Neurons and Neural Precursors from Human Embryonic Stem Cells”, Experimental Neurology, 172(2):383-397.
[cited by applicant]
Carpenter et al. (2004) “Properties of Four Human Embryonic Stem Cell Lines Maintained in a Feeder-Free Culture System”, Developmental Dynamics, 229(2):243-258.
[cited by applicant]
Chambers et al. (May 30, 2003) “Functional Expression Cloning of Nanog, a Pluripotency Sustaining Factor in Embryonic Stem Cells”, Cell, , 113(5):643-655.
[cited by applicant]
Cheng et al. (2003) “Human Adult Marrow Cells Support Prolonged Expansion of Human Embryonic Stem Cells in Culture”, Stem Cells, 2003, 21(2):131-142.
[cited by applicant]
Chung et al. (2008) “Human Embryonic Stem Cell Lines Generated without Embryo Destruction”, Cell Stem Cell, Feb. 07, 2(2):113-117.
[cited by applicant]
Coutu et al. (Oct. 9, 2011) “Roles of FGF Signaling In Stem Cell Self-Renewal, Senescence and Aging”, Aging, 3(10):920-933.
[cited by applicant]
Dalton et al. (2005) “Stem Cell Chemistry”, https://cen.acs.org/articles/83/i7/STEM-CELL-CHEMISTRY.html, 6 pages.
[cited by applicant]
Dang et al. (2004) “Controlled, Scalable Embryonic Stem Cell Differentiation Culture”, Stem Cells, 22(3):275-282.
[cited by applicant]
Darden et al. (Mar. 1996) “Selected New Developments in Computational Chemistry”, Environmental Health Perspectives, 104(Suppl 1):69-74.
[cited by applicant]
Denning et al. (Feb. 2006) “Common Culture Conditions for Maintenance and Cardiomyocyte Differentiation of the Human Embryonic Stem Cell Lines, BG01 and HUES-7”, The International Journal of Developmental Biology, 50(1)…
[cited by applicant]
Dominiguez-Bendala et al. (2013) “Islet Cell Therapy and Pancreatic Stem Cells”, Handbook of Stem Cells, Chapter 70, 835-853.
[cited by applicant]
Dravid et al. (2005) “Defining the Role of Wnt/r1-Catenin Signaling in the Survival, Proliferation, and Self-Renewal of Human Embryonic Stem Cells”, Stem Cells, 23(10):1489-1501.
[cited by applicant]
Drukker et al. (2003) “Genetic manipulation of human embryonic stem cells”, Human Embryonic Stem Cells, 265-284.
[cited by applicant]
Dvorak et al. (2005) “Expression and Potential Role of Fibroblast Growth Factor 2 and Its Receptors in Human Embryonic Stem Cells”, Stem Cells, 23(8):1200-1211.
[cited by applicant]
Eiges et al. (Apr. 3, 2001) “Establishment of Human Embryonic Stem Cell-Transfected Clones Carrying a Marker for Undifferentiated Cells”, Current Biology, 11(7):514-518.
[cited by applicant]
Evans et al. (Jul. 1981) “Establishment in Culture of Pluripotential Cells from Mouse Embryos”, Nature, 292(5819):154-156.
[cited by applicant]
Fok et al. (Aug. 4, 2005) “Shear-Controlled Single-Step Mouse Embryonic Stem Cell Expansion and Embryoid Body-Based Differentiation”, Stem Cells, 23(9):1333-1342.
[cited by applicant]
Genbacev et al. (May 2005) “Serum-Free Derivation of Human Embryonic Stem Cell Lines on Human Placental Fibroblast Feeders”, Fertility and Sterility, 83(5):1517-1529.
[cited by applicant]
Gendall et al. (May 1997) “Isolation and Characterization of a Leukemia Inhibitory Factor-Independent Embryonic Stem Cell Line”, The International Journal of Biochemistry & Cell Biology, 29(5):829-840.
[cited by applicant]
Gerecht-Nir et al. (Jun. 5, 2004) “Bioreactor Cultivation Enhances the Efficiency of a Human Embryoid Body (Heb) Formation and Differentiation”, Biotechnology and Bioengineering, 86(5):493-502.
[cited by applicant]
Guo et al. (Apr. 12, 2016) “Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass”, Stem Cell Reports, 6(4):437-446.
[cited by applicant]
Hovatta et al. (2003) “A Culture System Using Human Foreskin Fibroblasts as Feeder Cells Allows Production of Human Embryonic Stem Cells”, Human Reproduction, 18(7):1404-1409.
[cited by applicant]
Itskovitz-Eldor et al. (Feb. 2000) “Differentiation of Human Embryonic Stem Cells into Embryoid Bodies Compromising the Three Embryonic Germ Layers”, Molecular Medicine, , 6(2):88-95.
[cited by applicant]
James et al. (2005) “Tgfβ/Activin/Nodal Signaling is Necessary for the Maintenance of Pluripotency in Human Embryonic Stem Cells”, Development, 132(6):1273-1282.
[cited by applicant]
Kehat et al. (Aug. 2001) “Human Embryonic Stem Cells can Differentiate into Myocytes with Structural and Functional Properties of Cardiomyocytes”, Journal of Clinical Investigation, 108(3):407-414.
[cited by applicant]
Kehoe et al. (Oct. 17, 2009) “Scalable Stirred-Suspension Bioreactor Culture of Human Pluripotent Stem Cells”, Tissue Engineering: Part A, , 16(2):405-421.
[cited by applicant]
Keirstead et al. (May 11, 2005) “Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cell Transplants Remyelinate and Restore Locomotion After Spinal Cord Injury”, The Journal of Neuroscience, 25(19):4694-4705.
[cited by applicant]
Keller, Gordonm (Dec. 1995) “In Vitro Differentiation of Embryonic Stem Cells”, CurrentBiology, 7(6):862-869.
[cited by applicant]
Zandstra et al. (2000) “Leukemia Inhibitory Factor (LIF) Concentration Modulates Embryonic Stem Cell Self-Renewal and Differentiation Independently of Proliferation”, Biotechnology and Bioengineering, 69(6):607-617.
[cited by applicant]
Klimanskaya et al. (Nov. 23, 2006) “Human Embryonic Stem Cell Lines Derived from Single Blastomeres”, Nature, 444(7118):481-485.
[cited by applicant]
Klimanskaya et al. (Mar. 8, 2005) “Human Embryonic Stem Cells Derived without Feeder Cells”, Lancet, 365(9471):1636-1641.
[cited by applicant]
Koshimizu et al. (1996) “Functional Requirement of Gp130-Mediated Signaling for Growth and Survival of Mouse Primordial Germ Cells in Vitro and Derivation of Embryonic Germ (EG) Cells”, Development, 122(4):1235-1242.
[cited by applicant]
Koshimizu et al. (1995) “Retinoic Acid Is a Potent Growth Activator of Mouse Primordial Germ Cells in Vitro”, Developmental Biology, 168(2):683-685.
[cited by applicant]
Kurosawa et al. (May 2007) “Methods for Inducing Embryoid Body Formation: In Vitro Differentiation System of Embryonic Stem Cells”, Journal of Bioscience and Bioengineering, 103(5):389-398.
[cited by applicant]
Lai et al. (Mar. 6, 2012) “SRY (Sex Determining Region Y)-Box2 (Sox2)/Poly ADP-Ribose Polymerase 1 (Parp1) Complexes Regulate Pluripotency”, Proceedings of the National Academy of Sciences of the United States of Americ…
[cited by applicant]
Lebkowski et al. (2001) “Human Embryonic Stem Cells: Culture, Differentiation, and Genetic Modification for Regenerative Medicine Applications”, The Cancer Journal, 7(Suppl 2):S83-S93.
[cited by applicant]
Levenstein et al. (Mar. 2006) “Basic FGF Support of Human Embryonic Stem Cell Self-Renewal”, Stem Cells, 24(3):16 Pages.
[cited by applicant]
Li et al. (Jun. 21, 2005) “Expansion of Human Embryonic Stem Cells in Defined Serum-Free Medium Devoid of Animal-Derived Products”, Biotechnology and Bioengineering, 91(6):688-698.
[cited by applicant]
Lim et al. (Sep. 2002) “Proteome Analysis of Conditioned Medium From Mouse Embryonic Fibroblast Feeder Layers Which Support the Growth of Human Embryonic Stem Cells”, Proteomics, 2(9):1187-1203.
[cited by applicant]
Ludwig et al. (Jan. 1, 2006) “Derivation of Human Embryonic Stem Cells in Defined Conditions”, Nature Biotechnology, 24(2):185-187.
[cited by applicant]
Matsuda et al. (1999) “STAT3 Activation is Sufficient to Maintain an Undifferentiated State of Mouse Embryonic Stem Cells”, The EMBO Journal, 18(15):4261-4269.
[cited by applicant]
Matsui et al. (Sep. 4, 1992) “Derivation of Pluripotential Embryonic Stem Cells from Murine Primordial Germ Cells in Culture”, Cell, 70(5):841-847.
[cited by applicant]
Mered et al. (Oct. 1980) “Cell Growth Optimization in Microcarrier Culture”, In Vitro, 16(10):859-865.
[cited by applicant]
Mitsui et al. (May 30, 2003) “The Homeoprotein Nanog is Required for Maintenance of Pluripotency in Mouse Epiblast and ES Cells”, Cell, ,13(5):631-642.
[cited by applicant]
Miyamoto et al. (2004) “Human Placenta Feeder Layers Support Undifferentiated Growth of Primate Embryonic Stem Cells”, Stem Cells, 22(4):433-440.
[cited by applicant]
Nichogiannopoulou, Aliki (Oct. 10, 2012) “Patentability of Human Embryonic Cells at the EPO in Light of CJEU Decision C-34/10”, 23rd Forum on Biotech Patenting, 33 Pages.
[cited by applicant]
Nichols et al. (Nov. 1994) “Derivation of Germline Competent Embryonic Stem Cells with a Combination of Interleukin-6 and Soluble Interleukin-6 Receptor”, Experimental Cell Research, 215(1):237-239.
[cited by applicant]
Nichols et al. (1990) “Establishment of Germ-Line-Competent Embryonic Stem (ES) Cells Using Differentiation Inhibiting Activity”, Development, 110(4):1341-1348.
[cited by applicant]
Oh et al. (2006) “Human Embryonic Stem Cells: Technological Challenges towards Therapy”, Clinical and Experimental Pharmacology and Physiology, 33:489-495.
[cited by applicant]
Ornitz et al. (Mar. 9, 2001) “Fibroblast Growth Factors”, Genome Biology, 2(3):1-12.
[cited by applicant]
Pease et al. (Oct. 1990) “Isolation of Embryonic Stem (ES) Cells in Media Supplemented With Recombinant Leukemia Inhibitory Factor (LIF)”, Developmental Biology, 141(2):344-352.
[cited by applicant]
Pebay et al. (2005) “Essential Roles of Sphingosine-1-Phosphate and Platelet-Derived Growth Factor in the Maintenance of Human Embryonic Stem Cells”, Stem Cells, 23(10):1541-1548.
[cited by applicant]
Pera et al. (Jul. 2009) “Gene Expression Profiles of Human Inner Cell Mass Cells and Embryonic Stem Cells”, Differentiation, 78(1):18-23.
[cited by applicant]
Prowse et al. (Mar. 2005) “A Proteome Analysis of Conditioned Media from Human Neonatal Fibroblasts Used in the Maintenance of Human Embryonic Stem Cells”, Proteomics, 5(4):978-989.
[cited by applicant]
Pyle et al. (Jan. 29, 2006) “Neurotrophins Mediate Human Embryonic Stem Cell Survival”, Nature Biotechnology, 24(3):344-350.
[cited by applicant]
(Apr. 2000) Reubinoff et al., “Embryonic Stem Cell Lines From Human Blastocysts: Somatic Differentiation in Vitro”, Nature Biotechnology, 18(4):399-404.
[cited by applicant]
Richards et al. (2003) “Comparative Evaluation of Various Human Feeders for Prolonged Undifferentiated Growth of Human Embryonic Stem Cells”, Stem Cells, 21(5):546-556.
[cited by applicant]
Richards et al. (Sep. 2002) “Human Feeders Support Prolonged Undifferentiated Growth of Human Inner Cell Masses and Embryonic Stem Cells”, Nature Biotechnology, 20(9):933-936.
[cited by applicant]
Robertson, Elizabeth J. (1997) “Derivation and Maintenance of Embryonic Stem Cell Cultures”, Methods in Molecular Biology, 75:173-184.
[cited by applicant]
Rose et al. (Jan. 1994) “Oncostatin M (OSM) Inhibits the Differentiation of Pluripotent Embryonic Stem Cells In Vitro”, Cvtokine, 6(1):48-54.
[cited by applicant]
Rosler et al. (Feb. 2004) “Long-Term Culture of Human Embryonic Stem Cells in Feeder-Free Conditions”, Developmental Dynamics, 229(2):259-274.
[cited by applicant]
Sato et al. (Jan. 2004) “Maintenance of Pluripotency In Human and Mouse Embryonic Stem Cells Through Activation of Wnt Signaling by a Pharmacological GSK-3-Specific Inhibitor”, Nature Medicine, 10(1):55-63.
[cited by applicant]
Schechter et al. (2002) “Growth of Purified Lacrimal Acinar Cells in Matrigel Raft Cultures”, Experimental Eye Research, 74(3):349-60.
[cited by applicant]
Schulz et al. (2004) “Differentiation of Human Embryonic Stem Cells to Dopaminergic Neurons in Serum-Free Suspension Culture”, Stem Cells, 22(7):1218-1238.
[cited by applicant]
Shamblott et al. (Nov. 1998) “Derivation of Pluripotent Stem Cells from Cultured Human Primordial Germ Cells”, Proceedings of the National Academy of Sciences of the United States of America, 95(23):13726-13731.
[cited by applicant]
Shamblott et al. (Jan. 2, 2001) “Human Embryonic Germ Cell Derivatives Express a Broad Range of Developmentally Distinct Markers and Proliferate Extensively In Vitro”, Proceedings of the National Academy of Sciences of …
[cited by applicant]
Smith et al. (Dec. 15, 1988) “Inhibition of Pluripotential Embryonic Stem Cell Differentiation by Purified Polypeptides”, Nature, 336(6200):688-690.
[cited by applicant]
Sottile et al. (2003) “In Vitro Osteogenic Differentiation of Human ES Cells”, Cloning & Stem Cells, 5(2):149-155.
[cited by applicant]
Stojkovic et al. (Mar. 2005) “An Autogeneic Feeder Cell System That Efficiently Supports Growth of Undifferentiated Human Embryonic Stem Cells”, Stem Cells, 23(3):306-314.
[cited by applicant]
Stojkovic et al. (2004) “Derivation, Growth and Applications of Human Embryonic Stem Cells”, Reproduction, 128(3):259-267.
[cited by applicant]
Strelchenko et al. (2006) “Embryonic Stem Cells from Morula”, Methods in Enzymology, 418:93-108.
[cited by applicant]
Thomson et al. (Nov. 6, 1998) “Embryonic Stem Cell Lines Derived from Human Blastocysts”, Science, 282(5391):1145-1147.
[cited by applicant]
Thomson et al. (Aug. 1995) “Isolation of a Primate Embryonic Stem Cell Line”, Proceedings of the National Academy of Sciences of the United States of America, 92(17):7844-7848.
[cited by applicant]
Thomson et al. (1998) “Primate Embryonic Stem Cells”, Current Topics in Developmental Biology, 38:133-165.
[cited by applicant]
Trosko et al. (2000) “Gap Junctions and the Regulation of Cellular Functions of Stem Cells During Development and Differentiation”, Methods, 20(2):245-64.
[cited by applicant]
Tsukada et al. (2006) “Acidic Fibroblast Growth Factor Promotes Hepatic Differentiation of Monkey Embryonic Stem Cells”, In Vitro Cellular & Developmental Biology—Animal, 41:83-88.
[cited by applicant]
Vallier et al. (2005) “Activin/Nodal and FGF Pathways Cooperate to Maintain Pluripotency of Human Embryonic Stem Cells”, Journal of Cell Science, 118(Pt 19):4495-4509.
[cited by applicant]
Vallier et al. (Sep. 22, 2004) “Nodal Inhibits Differentiation of Human Embryonic Stem Cells Along the Neuroectodermal Default Pathway”, Developmental Biology, 275(2):403-421.
[cited by applicant]
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