Methods and products for embryonic stem cell culture

• US 20080248567A1
• Filed: 12/19/2007
• Published: 10/09/2008
• Est. Priority Date: 12/22/2006
• Status: Abandoned Application

• Current Assignee: University of Vermont & State Agricultural College
• Original Assignee: University of Vermont & State Agricultural College
• Inventors: Singla, Dinender Kumar
• Application Number:
  US12/002,971
• Publication Number:
  US 20080248567A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2502/99 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

The invention relates to methods and media for preparing and maintaining self-renewing pluripotent embryonic stem cells. The methods include, in some embodiments, culturing embryonic stem cells in culture medium that includes culture medium conditioned by cells that express wnt3a.

Claims

1. A method of maintaining a self-renewing pluripotent embryonic stem cell culture, the method comprising culturing one or more pluripotent embryonic stem cells in culture medium comprising medium conditioned by cells that express wnt3a, under conditions suitable for growth and proliferation of the self-renewing pluripotent embryonic stem cell culture.

2. The method of claim 1, wherein the cells that express wnt3a are L3 cells.

5. The method of claim 1, wherein the culture medium is from about 0.1% to about 100% (v/v) medium conditioned by cells that express wnt3a.

9. The method of claim 1, wherein the pluripotent embryonic stem cells are cultured in the absence of feeder cells.

10. The method of claim 1, wherein the culture medium does not comprise leukemia inhibitory factor (LIF).

20. An isolated, self renewing, pluripotent embryonic stem (ES) cell culture prepared using the method of claim 1.

21. The isolated, self-renewing, pluripotent ES cell culture of claim 20, wherein the cells that express wnt3a are L3 cells.

24. The isolated, self-renewing, pluripotent ES cell culture of claim 20, wherein the culture medium is from about 0.1% to about 100% (v/v) medium conditioned by cells that express wnt3a.

28. The isolated, self-renewing, pluripotent ES cell culture of claim 20, wherein the pluripotent embryonic stem cells are cultured in the absence of feeder cells.

29. The isolated, self-renewing, pluripotent ES cell culture of claim 20, wherein the culture medium does not comprise leukemia inhibitory factor (LIF).

3-4. -4. (canceled)

6-8. -8. (canceled)

11-19. -19. (canceled)

22-23. -23. (canceled)

25-27. -27. (canceled)

30-38. -38. (canceled)

39. A method of preparing a differentiated embryonic stem cell culture, the method comprising, maintaining the self-renewing pluripotent embryonic stem cell culture using the method of claim A1 and stimulating differentiation of the self-renewing pluripotent embryonic stem cell culture to prepare a differentiated embryonic stem cell culture.

40. The method of claim 39, wherein the differentiated embryonic stem cells are cardiac cells, hepatocytes, or neurons.

41. The method of claim 39, wherein the cells that express wnt3a are L3 cells.

44. The method of claim 39, wherein the culture medium is from about 0.1% to about 100% (v/v) medium conditioned by cells that express wnt3a.

49. The method of claim 39, wherein the culture medium does not comprise leukemia inhibitory factor (LIF).

42-43. -43. (canceled)

45-48. -48. (canceled)

50-85. -85. (canceled)

86. A culture medium for maintaining a self-renewing pluripotent embryonic stem cell culture, the culture medium comprising at least 30-50% (v/v) of medium conditioned by cells expressing wnt3a.

87. The culture medium of claim 86, wherein the cells that express wnt3a are L3 cells.

88. The culture medium of claim 86, wherein the culture medium is at least about 50% (v/v) medium conditioned by cells that express wnt3a.

89. The culture medium of claim 86, wherein the culture medium comprises DMEM.

90. A conditioned medium for maintaining a self-renewing pluripotent embryonic stem cell culture, wherein the conditioned medium comprises at least about 50-200 ng/ml wnt3a.