Compositions for the in vitro derivation and culture of embryonic stem (es) cell lines with germline transmission capability and for the culture of adult stem cells

• US 20050059145A1
• Filed: 12/20/2002
• Published: 03/17/2005
• Est. Priority Date: 12/21/2001
• Status: Abandoned Application

• Current Assignee: Thromb-X NV
• Original Assignee: Thromb-X NV
• Inventors: Schoonjans, Luc
• Application Number:
  US10/499,428
• Publication Number:
  US 20050059145A1
• Time in Patent Office: Days
• US Class Current: 435/353
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: C07K 14/5415 Leukaemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/2303 Interleukin-3 (IL-3)
• CPC Class Codes: C12N 2501/2306 Interleukin-6 (IL-6)
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/33 Insulin
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• 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
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors

Abstract

The present invention describes novel compositions for deriving, maintaining and growing pluripotent and germ-line competent mammalian embryonic stem cells. The compositions of this invention refers to compositions comprising a 1) conditioned medium of a cell line expressing limited amounts of Leukemia Inhibitory Factor (LIF), 2) conditioned medium from a cell line transfected with mammalian LIF and 3) a medium supplemented with recombinant rabbit LIF. The present invention describes novel compositions for deriving, maintaining and growing adult human stem cells and/or adult early progenitor cells, preferably under stroma free conditions and without added LIF and/or cytokines or growth factors. The media of the present invention are used or for the generation of pluripotent and germ line competent embryonic stem cells of mammals of which these cells were not obtained up to now. The media of the present invention are used or for the generation of adult human stem cells and/or adult early progenitor cells. The present invention is also directed to a novel rabbit LIF and to nucleotides encoding the Rabbit LIF and methods for the expression of recombinant rabbit LIF in the Pichia pastoris expression system.

Claims

1. The composition according to claim 1, wherein said

composition contains less than 0.05 ng/ml Leukemia Inhibitory Factor (LIF).

36. The composition according to claim 1, wherein said composition is suitable for stem cells of a non-human mammal selected from the group consisting of primates, pigs, cows, horses, goats, sheep, cats, dogs, rabbits and rodents.

37. The composition according to claim 1, wherein said composition is suitable for stem cells of Rattus sp.

38. The composition according to claim 1, wherein said composition is suitable for stem cells of Mus sp.

39. The composition according to claim 1, wherein said composition is suitable for stem cells of a Mus musculus mouse strain of any of the strains with a genetic background background selected from the group consisting of 129/SvEv, C57BL/6N, C57BL/6J-HPRT, BALB/cAnN, CBA/CaOla, 129/SvJ, DBA/2N, DBA/1Ola, C3H/HeN, C57BL/6JOla, FVB/N and Swiss Webster.

40. The composition according to claim 1, wherein said composition is suitable for stem cells of a mouse with a genetic background of 129/SvEv or a genetic background of C57BL/6N.

41. The composition according to claim 1, wherein said conditioned medium of cells is from an immortal fibroblast cell line.

42. The composition according to claim 1, wherein said conditioned medium of cells is from the rabbit Rab9 immortal fibroblast cell line (ATCC catalogue number CRL-1414).

43. The composition according to claim 1, which is suitable for the derivation, maintenance or growth of pluripotent embryonic stem cells of mammals and is supplemented with one or more compounds of the group consisting of interleukin, oncostatin, ciliary neurotrophic factor, stem cell factor, basic fibroblast growth factor, and cardiotrophin.

44. The composition according to claim 1, which is suitable for the stroma free expansion of mammalian adult stem cells and early progenitor cells and wherein said composition is not enriched with cytokines.

2-35. -35. (canceled)

45. A method of deriving, maintaining or growing pluripotent embryonic stem cells from a mammal or expanding mammalian adult stem cells or early progenitor cells comprising the step of cultivating the cells for at least one passage in a composition comprising a conditioned medium of a fibroblast cell line and containing less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF).

46. The method according to claim 45, wherein said stem cells are non-human pluripotent embryonic stem cells.

47. The method according to claim 45, wherein said stem cells are germ line competent embryonic stem cells.

48. The method according to claim 45, wherein said stem cells are germ line competent embryonic stem cells from a non-human mammal selected from the group consisting of primates, pigs, cows, horses, goats, sheep, cats, dogs, rabbits and rodents.

49. The method according to claim 45, wherein said stem cells are germ line competent embryonic stems from mice with a genetic background of C57BL/6J-HPRT, BALB/cAnN, CBA/CaO1a, 129/SvJ, DBA/2N, DBA/1Ola, C3H/HeN, C57BL/6JOla, FVB/N and Swiss Webster.

50. The method according to claim 45, wherein said adult stem cells are hematopoietic stem cells and wherein said adult early progenitor cells are hematopoietic precursor cells.

51. The method according to claim 45, which comprises the step of cultivating the adult stem cells or early progenitor cells until an expansion by at least 25 fold is obtained or until the amount of nucleated cells are being expanded by at least 10 fold.

52. A germ line competent embryonic stem cells obtainable by the method according to claim 45, wherein said cells are from a non-human mammal selected from the group consisting of primates, pigs, cows, horses, goats, sheep, cats, dogs rabbits and rats.