MEDIUM COMPOSITION COMPRISING NEUROPEPTIDE Y FOR THE GENERATION, MAINTENANCE, PROLONGED UNDIFFERENTIATED GROWTH OF PLURIPOTENT STEM CELLS AND METHOD OF CULTURING PLURIPOTENT STEM CELL USING THE SAME

• US 20120171766A1
• Filed: 06/16/2010
• Published: 07/05/2012
• Est. Priority Date: 06/16/2009
• Status: Active Grant

• Current Assignee: Korea Research Institute of Bioscience & Biotechnology
• Original Assignee: Korea Research Institute of Bioscience & Biotechnology
• Inventors: Cho, Yee Sook, Son, Mi-Young
• Granted Patent: US 8,628,963 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/835 Neuropeptide Y [NPY]; Peptide YY [PYY]
• 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
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The present invention relates to a medium composition comprising neuropeptide Y, effective for proliferation and maintenance of undifferentiated pluripotent stem cells, and a method for culturing undifferentiated pluripotent stem cells using the same. The present invention improves the culture conditions for undifferentiated pluripotent stem cells, and ultimately, the present invention can be effectively used for the development of large-scale culture systems, thereby acquiring clinically applicable pluripotent stem cells. Further, the present invention relates to a dedifferentiation medium composition comprising neuropeptide Y (NPY), and a method for inducing dedifferentiation (or reprogramming) using the same. The present invention improves the culture conditions for dedifferentiation and contributes to develop technology of producing clinically applicable induced pluripotent stem cells, thereby being used for the development of stem cell therapy.

Claims

1-33. -33. (canceled)

34. A culture medium comprising neuropeptide Y as an undifferentiation factor.

35. The culture medium according to claim 34, which maintains pluripotent stem cells in an undifferentiated state.

36. The culture medium according to claim 34, which induces cell dedifferentiation.

37. The culture medium according to claim 34, wherein a concentration of the neuropeptide Y is in the range from 0.01 to 100 μ

M.

38. The culture medium according to claim 34, wherein the medium further comprises one or more selected from the group consisting of N2 supplement, B27 supplement, bFGF and TGFβ

without animal serum and feeder cell-derived factors.

39. The culture medium according to claim 38, wherein the N2 and B27 supplements are provided in a ratio of 1:

1, and bFGF and TGFβ

are provided at a concentration of 4-100 ng/ml and 1-10 ng/ml, respectively.

40. A method for culturing pluripotent stem cells in an undifferentiated state, comprising the step of culturing pluripotent stem cells in the culture medium according to claim 34 to maintain the pluripotent stem cells in an undifferentiated state.

41. The method according to claim 40, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

42. The method according to claim 40, wherein the pluripotent stem cells are derived from human.

43. The method according to claim 40, wherein the maintenance of pluripotent stem cells in an undifferentiated state is confirmed by the increased expression of one or more genes selected from the group consisting of ALP (alkaline phosphatase), OCT4, SOX2, hTERT (human telomerase reverse transcriptase), TDGF (teratocarcinoma-derived growth factor) and SSEA-4 as compared to expression in the pluripotent cells cultured in culture minimum medium not comprising neuropeptide Y.

44. A method for establishing pluripotent stem cell lines maintaining an undifferentiated state of the pluripotent stem cells, comprising the step of culturing pluripotent stem cells in the culture medium according to claim 34.

45. A method for establishing dedifferentiated cell lines, comprising the step of culturing differentiated cells in the medium according to claim 34.

46. The method according to claim 45, wherein the differentiated cells are somatic cells or progenitor cells, and the dedifferentiated cell lines are induced pluripotent stem cells.

47. The method according to claim 45, wherein the differentiated cells are derived from human.

48. An in vitro cell culture comprising pluripotent stem cells in a culture medium according to claim 34, wherein the medium is free of animal serum and feeder cell-derived factors and has never been exposed to animal serum and feeder cell-derived factors.

49. The in vitro cell culture according to claim 48, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

50. The in vitro cell culture according to claim 48, wherein the pluripotent stem cells are derived from human.

51. An in vitro cell culture comprising differentiated cells in a culture medium according to claim 34, wherein the differentiated cells are dedifferentiated.

52. The in vitro cell culture according to claim 51, wherein the differentiated cells are somatic cells or progenitor cells.

53. The in vitro cell culture according to claim 51, wherein the differentiated cells are derived from human.