Methods to accelerate the isolation of novel cell strains from pluripotent stem cells and cells obtained thereby

• US 20080070303A1
• Filed: 11/21/2006
• Published: 03/20/2008
• Est. Priority Date: 11/21/2005
• Status: Abandoned Application

• Current Assignee: Advanced Cell Technology Incorporated (Astellas Pharma Incorporated)
• Original Assignee: Advanced Cell Technology Incorporated (Astellas Pharma Incorporated)
• Inventors: Murai, James, West, Michael, Sargent, Geoffrey, Kessler, Steven, Chapman, Karen
• Application Number:
  US11/604,047
• Publication Number:
  US 20080070303A1
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: A61K 35/30 Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• CPC Class Codes: A61K 35/34 Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes vascular smooth muscle A61K35/44
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61K 35/407 Liver; Hepatocytes
• CPC Class Codes: C12N 2506/02 from embryonic 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 5/0619 Neurons
• CPC Class Codes: C12N 5/0652 Cells of skeletal and connective tissues; Mesenchyme
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells
• CPC Class Codes: C12N 5/0662 Stem cells
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

This invention generally relates to methods to differentiate pluripotent stem cells, such as embryonic stem, embryonic germ, or embryo-derived cells, to obtain subpopulations of cells from heterogeneous mixtures of cells wherein the subpopulation of cells possess reduced differentiation potential compared to the original pluripotent stem cells and where the subpopulation is capable of being propagated.

Claims

1. A method for deriving cells from pluripotent stem cells wherein said derived cells possess reduced differentiation potential than said pluripotent stem cells, comprising the steps of:

(a) selecting all or a subset of differentiation conditions from a plurality of differentiation conditions that may result in the differentiation of said pluripotent stem cells;

(b) exposing said pluripotent stem cells to said selected all or a subset of differentiation conditions from step (a) for various time periods resulting in a heterogeneous population of cells comprising cells with reduced differentiation potential than said pluripotent stem cells;

(c) plating said heterogeneous population of cells to isolate a number of individual cultures of cells or a number of individual cultures of cells that are oligoclonal, wherein one or more of said cultures comprise cells with reduced differentiation potential than said pluripotent stem cells and wherein each of said individual cultures having only one cell may be propagated into a pure clonal culture of cells and wherein each of said individual cultures of cells having cells that are oligoclonal may be propagated into a larger number of cells; and

(d) propagating one or more (or all) of said individual cultures of cells in conditions selected to promote the propagation of said one or more (or all) of said individual cultures of cells.

5. The method according to any one of claims 1-4, further comprising a step of disaggregating said heterogeneous population of cells prior to plating.

6. The method according to claim 5, wherein said disaggregating step is performed by trypsinzing the heterogenous population of cells.

7. The method according to any one of claims 1-4, wherein, in said plating step, said heterogeneous population of cells is plated at limiting dilution.

8. The method according to claim 5, wherein, in said plating step, said heterogeneous population of cells, after disaggegation, is plated at limiting dilution.

9. The method according to claim 7 or claim 8, wherein limiting dilution is performed in multiwell dishes.

10. The method according to any one of claims 1-4, wherein, in said plating step, said heterogeneous population of cells is plated at low density.

11. The method according to claim 10, wherein said heterogeneous population of cells plated at low density is plated on semisolid media.

12. The method according to any one of claims 1-4, wherein said heterogeneous population of cells in step (b) are plated in juxtaposition with feeder or inducer cells.

13. The method according to any one of claims 1-4, wherein said heterogeneous population of cells form embryoid bodies prior to plating.

14. The method according to any one of claims 1-4, wherein said pluripotent stem cells are differentiated in vitro, in vivo, or in ovo.

15. The method according to any one of claims 1-4, wherein said heterogeneous population of cells are plated as single isolated cells at low density in a semisolid media.

16. The method according any one of claims 1-4, wherein, in said plating step, said heterogenous population of cells are plated as single isolated cells at low density in a hanging drop culture.

17. The method according to claim 16, further comprising the step of culturing said single isolated cells as an aggregate.

18. The method according to any one of claims 1-4, wherein said heterogeneous population of cells is cultured at low cellular density such that colonies of proliferating cells derived from a single cell can be easily identified and isolated.

19. The method according to any one of claims 1-4, wherein the cells in said individual cultures, or progeny thereof, are documented by genotype or phenotype.

20. The method according to any one of claims 1-4, wherein the cells in said individual cultures, or progeny thereof, are documented by photography.

21. The method according to any one of claims 1-4, wherein the cells in said individual cultures, or progeny thereof, are documented by immunocytochemistry.

22. The method according to any one of claims 1-4, wherein the cells in said individual cultures, or progeny thereof, are documented by hybridization of probes with RNA or cDNA transcript.

23. The method according any one of claims 1-4, wherein said pluripotent stem cells are selected from the group consisting of ES cells, EG cells, EC cells and ED cells.

24. The method according to claim 23, wherein said ED cells are selected from the group consisting of morula cells and inner cell mass cells.

25. The method according any one of claims 1, 2, 3, 4, 23 or 24, wherein said pluripotent stem cells are human cells.

26. The method according to any one of claims 1-4, wherein said pluripotent stem cells are genetically modified such that the MHC genes are deleted.

27. The method according to any one of claims 1-4, wherein said pluripotent stem cells are genetically modified such that the MHC genes are first deleted and then alleles of the MHC gene family are restored such that these stem cells are hemizygous or homozygous for one allele of the MHC gene family.

28. The method according to any one of claims 1-4, wherein said pluripotent stem cells are derived from the direct differentiation of embryonic cells without the derivation of embryonic stem cell line.

29. The method according to any one of claims 1-4, wherein said pluripotent stem cells are derived from the reprogramming of somatic cell through the exposure of said somatic cell to the cytoplasm of an undifferentiated cell.

30. The method according to any one of claims 1-4, wherein one of more cells in said individual cultures of cells are selected from the group consisting of endodermal cells, ectodermal cells and mesodermal cells.

31. The method according to any one of claims 1-4, wherein one of more cells in said individual cultures of cells are neuroglial precursor cells.

32. The method according to any one of claims 1-4, wherein one of more cells in said individual cultures of cells are hepatic cells or hepatic precursor cells.

33. The method according to any one of claims 1-4, wherein one of more cells in said individual cultures of cells are chondrocyte or chondrocyte precursor cells.

34. The method according to any one of claims 1-4, wherein one of more cells in said individual cultures of cells are myocardial or myocardial precursor cells.

35. The method according to any one of claims 1-4, wherein one of more cells in said individual cultures of cells are smooth muscle or skeletal muscle precursor cells.

36. The method according to claim 35, wherein said smooth muscle or skeletal muscle precursor cells are selected from the group consisting of somatic muscle precursor cells, muscle satellite stem cells and myoblast cells.

37. The method according to any one of claims 1-4, wherein one of more cells in said individual cultures of cells are gingival fibroblast or gingival fibroblast precursor cells.

38. The method according to any one of claims 1-4, wherein one of more cells in said individual cultures of cells are pancreatic beta cells or pancreatic beta precursor cells.

39. The method according to any one of claims 1-4, wherein one of more cells in said individual cultures of cells are dermal fibroblasts with prenatal patterns of gene expression.

40. The method according to any one of claims 1-4, wherein one of more cells in said individual cultures of cells are retinal precursor cells.

41. The method according to any one of claims 1-4, wherein one of more cells in said individual cultures of cells are hemangioblasts.

42. The method according to any one of claims 1-4, wherein said pluripotent stem cells are human pluripotent stem cells.

43. The method according to any one of claims 1-4, wherein said pluripotent stem cells are derived from a library of human embryonic stem cells, wherein said library of human embryonic stem cells comprises stem cells, each of which is hemizygous or homozygous for at least one MHC allele present in a human population, wherein each member of said library of stem cells is hemizygous or homozygous for a different set of MHC alleles relative to the remaining members of the library.

44. The method according to claim 45, wherein said library of human embryonic stem cells comprises stem cells that are hemizygous or homozygous for all MHC alleles present in a human population.

45. A method of treating a patient suffering a condition or disease such that said patient is in need of cell-based therapy, comprising the steps of:

(a) obtaining said patient;

(b) identifying MHC proteins expressed on the surface of said patient'"'"'s cells;

(c) providing a library of human cells that have reduced differentiation potential than said human embryonic stem cells made according to the method of claim 43 or 44;

(d) selecting the human cells in said library that match said patient'"'"'s MHC proteins on said patient'"'"'s cells and that are appropriate for treating said patient'"'"'s condition or disease that renders said patient in need of cell-based therapy and optionally further differentiating said human cell;

(e) administering said human cells from step (d) to said patient.

46. The method according to claim 45, wherein said method is performed in a regional center.

47. The method according to claim 46, wherein said regional center is a hospital.

48. The method according to any one of claims 1-4, wherein in the exposing step said pluripotent stem cells are exposed to said differentiation conditions for 1-100 days.

49. The method according to any one of claims 1-4, further comprising the step of determining the lineage of the derived cells.

50. A method of treating a patient suffering a condition or disease such that said patient is in need of cell-based therapy, comprising the step of administering a cell derived from a method according to any one of claims 1-4 or progeny thereof that are further differentiated.

51. The method according to any one of claims 1-4, wherein one or more of said derived cells secrete growth factors.

52. The method according to any one of claims 1-4, wherein the culture medium of one or more of said derived cells is used as a differentiation condition in any one of claims 1-4.

53. The method according to any one of claims 1-4, wherein one or more of said derived cells secrete growth factors.

54. The method according to any one of claims 1-4, wherein the culture medium of one or more of said derived cells is used as a differentiation condition in any one of claims 1-4.

55. The method according to any one of claims 1-4, wherein said pluripotent stem cells or embryoid bodies derived therefrom are exposed to a variety of differentiating conditions.

56. The method according to any one of claims 1, 2, 3, 4 or 54, wherein plating step is performed at various time intervals after exposing to the differentiating conditions.

2. A method for deriving cells from embryoid bodies derived from pluripotent stem cells wherein said derived cells possess reduced differentiation potential than said embryoid bodies derived from pluripotent stem cells, comprising the steps of:

(a) selecting all or a subset of differentiation conditions from a plurality of differentiation conditions that may result in the differentiation of said embryoid bodies derived from pluripotent stem cells;

(b) exposing said embryoid bodies derived from pluripotent stem cells to said selected all or a subset of differentiation conditions from step (a) for various time periods resulting in a heterogeneous population of cells comprising cells with reduced differentiation potential than said pluripotent stem cells;

(c) plating said heterogeneous population of cells to isolate a number of individual cultures of cells, each culture having only one cell or cells that are oligoclonal, wherein one or more of said cultures comprise cells with reduced differentiation potential than said pluripotent stem cells and wherein each of said individual cultures having only one cell may be propagated into a pure clonal culture of cells and wherein each of said individual cultures of cells that are oligoclonal may be propagated into a larger number of cells; and

(d) propagating one or more (or all) of said individual cultures of cells in conditions selected to promote the propagation of said one or more (or all) of said individual cultures of cells.

3. A method for deriving cells from pluripotent stem cells, wherein said derived cells possess reduced differentiation potential than said pluripotent stem cells, comprising the steps of:

(a) exposing said pluripotent stem cells in various differentiation conditions for various time periods resulting in a heterogeneous population of cells comprising cells with reduced differentiation potential than said pluripotent stem cells;

(b) plating said heterogeneous population of cells to isolate a number of individual cultures of cells or a number of individual cultures of cells that are oligoclonal, wherein one or more of said cultures comprise cells with reduced differentiation potential than said pluripotent stem cells and wherein each of said individual cultures having only one cell may be propagated into a pure clonal culture of cells and wherein each of said individual cultures of cells having cells that oligoclonal may be propagated into a larger number of cells; and

(c) propagating one or more (or all) of said individual cultures of cells in conditions selected to promote the propagation of said one or more (or all) of said individual cultures of cells.

4. A method for deriving cells from embryoid bodies derived from pluripotent stem cells, wherein said derived cells possess reduced differentiation potential than said pluripotent stem cells, comprising the steps of:

(a) exposing said embryoid bodies derived from pluripotent stem cells in various differentiation conditions for various time periods resulting in a heterogeneous population of cells comprising cells with reduced differentiation potential than said pluripotent stem cells;

(b) plating said heterogeneous population of cells to isolate a number of individual cultures of cells, each culture having only one cell or an oligoclonal number of cells, wherein one or more of said cultures comprise cells with reduced differentiation potential than said pluripotent stem cells and wherein each of said individual cultures having only one cell may be propagated into a pure clonal culture of cells and wherein each of said individual cultures of cells that are oligoclonal may be propagated into a larger number of cells; and

(c) propagating one or more (or all) of said individual cultures of cells in conditions selected to promote the propagation of said one or more (or all) of said individual cultures of cells.