Methods and materials for the growth of primate-derived primordial stem cells in feeder-free culture

• US 6,800,480 B1
• Filed: 08/29/2000
• Issued: 10/05/2004
• Est. Priority Date: 10/23/1997
• Status: Expired due to Term

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: West, Michael D., Gold, Joseph D., Murai, James T., Chiu, Choy-Pik, Inokuma, Margaret, Bodnar, Andrea G.
• Primary Examiner(s): Crouch, Deborah
• Assistant Examiner(s): WOITACH, JOSEPH T
• Application Number:
  US09/530,346
• Time in Patent Office: 1,498 Days
• Field of Search: 435/325, 435/1.1, 435/405, 435/440, 424/93.1, 424/93.2
• US Class Current: 435/325
• CPC Class Codes: C12N 2500/40 Nucleotides, nucleosides or bases
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

Methods and materials for culturing primate-derived primordial stem cells are described. In one embodiment, a cell culture medium for growing primate-derived primordial stem cells in a substantially undifferentiated state is provided which includes a low osmotic pressure, low endotoxin basic medium that is effective to support the growth of primate-derived primordial stem cells. The basic medium is combined with a nutrient serum effective to support the growth of primate-derived primordial stem cells and a substrate selected from the group consisting of feeder cells and an extracellular matrix component derived from feeder cells. The medium further includes non-essential amino acids, an anti-oxidant, and a first growth factor selected from the group consisting of nucleosides and a pyruvate salt.

Claims

1. A cellular composition comprising undifferentiated primate primordial stem (pPS) cells proliferating on an extracellular matrix, wherein the composition is free of feeder cells.

2. The composition of claim 1, wherein the extracellular matrix is a fibroblast matrix, prepared by culturing fibroblasts, lysing the fibroblasts in situ, and then washing what remains after lysis.

3. The composition of claim 1, wherein the extracellular matrix is prepared from a matrix component or combination of such components.

4. The composition of claim 3, wherein the extracellular matrix is prepared from an isolated matrix component or a combination of components selected from collagen, placental matrix, fibronectin, laminin, merosin, tenascin, heparin sulfate, condroitin sulfate, dermatan sulfate, aggrecan, biglycan, thrombospondin, vitronectin, and decorin.

5. The composition of claim 1, further comprising a nutrient medium.

6. The composition of claim 5, wherein the nutrient medium comprises sodium pyruvate and nucleosides, and has a low endotoxin level.

7. The composition of claim 5, wherein the nutrient medium comprises one or more exogenously added factors that promote undifferentiated growth of primordial stem cells, selected from TGF-β

, IL-11, IL-6, IL-6 receptor, IL-1, LIF, IL-17, LAP, MCP-1, bFGF, FGF-4, PDGF soluble receptor A, forskolin, and antibodies to IL-8, TGF-β

, BDNF, TNF-β

, VEGF, and EGF.

8. The composition of claim 5, wherein the nutrient medium is a conditioned medium.

9. The composition of claim 8, wherein the conditioned medium is obtained by collecting medium from a culture of growing feeder cells.

10. The composition of claim 1, wherein the pPS cells have been genetically altered;

or are the undifferentiated progeny of genetically altered cells.

11. The composition of claim 10, wherein the pPS cells have been genetically altered by introducing a gene encoding either the RNA component of telomerase, or telomerase reverse transcriptase (hTRT);

or are the undifferentiated progeny of such cells.

12. The composition of claim 1, wherein the undifferentiated pPS cells have been obtained from a blastocyst.

13. A composition comprising:

a) a substrate comprising an extracellular matrix;

b) a conditioned medium; and

c) proliferating undifferentiated primate primordial stem (pPS) cells;

wherein the composition is essentially free of feeder cells.

14. A cellular composition comprising undifferentiated pPS cells proliterating on an extracellular matrix, obtained by passaging pPS cells cultured on feeder cells to a new growth environment having an extracellular matrix but free of feeder cells;

and then culturing the pPS cells in the new growth environment such that they proliferate without differentiating.

15. The composition of claim 14, wherein the extracellular matrix is a fibroblast matrix, prepared by culturing fibroblasts, lysing the fibroblasts in situ, and then washing what remains after lysis.

16. The composition of claim 14, wherein the extracellular matrix is prepared from a matrix component or combination of such components.

17. The composition of claim 14, wherein the growth environment comprises one or more exogenously added factors that promote undifferentiated growth of primordial stem cells, selected from TGF-β

, IL-11, IL6, IL-6 receptor, IL-1, LIF, IL-17, LAP, MCP-1, bFGF, FGF4, PDGF soluble receptor A, forskolin, and antibodies to IL-8, TGF-β

, BDNF, TNF-β

, VEGF, and EGF.

18. The composition of claim 14, wherein the growth environment comprises a medium that has been conditioned by culturing with feeder cells.

19. The composition of claim 14, wherein the wherein the growth environment comprises added fibroblast growth factor.

20. The composition of claim 14, wherein the undifferentiated pPS cells have been obtained from a blastocyst.

21. A cell population consisting essentially of primate embryonic stem (ES) cells proliferating in culture on an extracellular matrix in a manner such that at least 50% of the proliferating ES cells are undifferentiated.

22. The cell population of claim 21, wherein the extracellular matrix is a fibroblast matrix, prepared by culturing fibroblasts, lysing the fibroblasts in situ, and then washing what remains after lysis.

23. The cell population of claim 21, wherein the extracellular matrix is prepared from a matrix component or combination of such components.

24. The cell population of claim 21, in a nutrient medium that comprises one or more exogenously added factors that promote undifferentiated growth of primordial stem cells, selected from TGF-β

, IL-11, IL-6, IL-6 receptor, IL-1, LIF, IL-17, LAP, MCP-1, bFGF, FGF-4, PDGF soluble receptor A, forskolin, and antibodies to IL-8, TGF-β

, BDNF, TNF-β

, VEGF, and EGF.

25. The cell population of claim 21, in a medium that has been conditioned by culturing with feeder cells.

26. The cell population of claim 21, wherein the undifferentiated ES cells are positive for SSEA-4, TRA-1-60, or TRA-1-81.

27. The cell population of claim 21, wherein the undifferentiated ES cells have alkaline phosphatase activity.

28. The cell population of claim 21, wherein the undifferentiated ES cells have telomerase enzymatic activity.

29. The cell population of claim 21, wherein the ES cells are human embryonic stem cells.

30. A cellular composition comprising undifferentiated human embryonic stem cells proliferating on an extracellular matrix, wherein the composition is free of feeder cells.

31. The composition of claim 30, wherein the extracellular matrix is a fibroblast matrix, prepared by culturing fibroblasts, lysing the fibroblasts in situ, and then washing what remains after lysis.

32. The composition of claim 30, wherein the extracellular matrix is prepared from a matrix component or combination of such components.

33. The composition of claim 32, wherein the extracellular matrix is prepared from an isolated matrix component or a combination of components selected from collagen, placental matrix, fibronectin, laminin, merosin, tenascin, heparin sulfate, condroitin sulfate, dermatan sulfate, aggrecan, biglycan, thrombospondin, vitronectin, and decorin.

34. The composition of claim 30, further comprising a nutrient medium.

35. The composition of claim 34, wherein the nutrient medium comprises sodium pyruvate and nucleosides, and has a low endotoxin level.

36. The composition of claim 34, wherein the nutrient medium comprises one or more exogenously added factors that promote undifferentiated growth of primordial stem cells, selected from TGF-β

, IL-11, IL-6, IL-6 receptor, IL-1, LIF, IL-17, LAP, MCP-1, bFGF, FGF-4, PDGF soluble receptor A, forskolin, and antibodies to IL-8, TGF-β

, BDNF, TNF-β

, VEGF, and EGF.

37. The composition of claim 34, wherein the nutrient medium is a conditioned medium.

38. The composition of claim 37, wherein the conditioned medium is obtained by collecting medium from a culture of growing feeder cells.

39. A cellular composition comprising undifferentiated human embryonic stem (ES) cells proliferating on an extracellular matrix, obtained by passaging human ES cells cultured on feeder cells to a new growth environment having an extracellular matrix but free of feeder cells;

and then culturing the ES cells in the new growth environment such that they proliferate without differentiating.

40. The composition of claim 39, wherein the extracellular matrix is a fibroblast matrix, prepared by culturing fibroblasts, lysing the fibroblasts in situ, and then washing what remains after lysis.

41. The composition of claim 39, wherein the extracellular matrix is prepared from a matrix component or combination of such components.

42. The composition of claim 39, wherein the growth environment comprises one or more exogenously added factors that promote undifferentiated growth of primordial stern cells, selected from TGF-β

, IL-11, IL-6, IL-6 receptor, IL-1, LIF, IL-17, LAP, MCP-1, bFGF, FGF-4, PDGF soluble receptor A, forskolin, and antibodies to IL-8, TGF-β

, BDNF, TNF-β

, VEGF, and EGF.

43. The composition of claim 39, wherein the growth environment comprises a medium that has been conditioned by culturing with feeder cells.

44. The composition of claim 39, wherein the wherein the growth environment comprises added fibroblast growth factor.