Methods of isolation, expansion and differentiation of fetal stem cells from chorionic villus, amniotic fluid, and placenta and therapeutic uses thereof

• US 20050124003A1
• Filed: 11/15/2002
• Published: 06/09/2005
• Est. Priority Date: 11/15/2001
• Status: Active Grant

• Current Assignee: Children's Medical Center Corporation
• Original Assignee: Children's Medical Center Corporation
• Inventors: Atala, Anthony, De Coppi, Paolo
• Granted Patent: US 8,021,876 B2
• Time in Patent Office: Days
• US Class Current: 435/7.200
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: C12N 5/0605 Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696

Abstract

The present invention is directed to pluripotent fetal stem cells derived from chorionic villus, amniotic fluid, and placenta and the methods for isolating, expanding and differentiating these cells, and their therapeutic uses such as manipulating the fetal stem cells by gene transfection and other means for therapeutic applications.

Claims

1. A method for producing a population of cells enriched for pluripotent fetal stem cells, comprising selecting c-kit positive cells from a chorionic villus, amniotic fluid, or placenta sample.

7. The method of claim 1, wherein the selecting is by flow cytometry.

8. The method of claim 1, wherein the selecting is by fluorescence activated cell sorting or high gradient magnetic selection.

9. The method of claim 1, further comprising the step of further enriching for the pluripotent fetal stem cells by additionally selecting for cells expressing markers expressed by SSAE and/or SSAE4 embryonic stem cells.

10. The method of claims 1 or 9, further comprising the steps of further enriching a population of pluripotent fetal stem cells by eliminating from the population those cells that express SSAE1 marker.

11. The method of claim 1, wherein the chorionic villus, amniotic fluid or placenta sample is cryopreserved prior to the selection step.

12. The method of claim 1, further comprising cyropreserving the c-kit positive cells.

20. A pluripotent fetal stem cell prepared according to the method of claims 1 to 11.

2. The method of claim 1, wherein the selecting is performed using an antibody against c-kit.

3. The method of claim 2, wherein the antibody is a monoclonal antibody.

4. The method of claim 2, wherein the monoclonal antibody is a mouse monoclonal IgG against an antigenic epitope of human c-kit.

5. The method of claim 2, wherein the antibody is fluorochrome conjugated.

6. The method of claim 2, wherein the antibody is conjugated to magnetic particles.

13. A method of proliferating a population of cells enriched for pluripotent fetal stem cells comprising:

(a) selecting at least one c-kit positive cell from a chorionic villus, amniotic fluid or placenta sample;

(b) introducing said at least one selected cell to a culture medium; and

(c) proliferating said at least one selected cell in the culture medium.

14. A method of differentiating c-kit positive pluripotent fetal stem cells comprising providing a chorionic villus, amniotic fluid or placenta sample and inducing differentiation of c-kit positive cells within said sample by exposing the sample to one or more differentiation-inducing agents.

15. The method of claim 14, wherein the differentiation-inducing agents result in a cell having a phenotype selected from the group consisting of osteogenic phenotype, hematopoietic phenotype, adipogenic phenotype, myogenic phenotype, hepatic phenotype, neurogenic phenotype, and endothelial phenotype.

16. A method of differentiating c-kit positive pluripotent fetal stem cells comprising:

(a) providing a chorionic villus, amniotic fluid or placenta sample;

(b) obtaining cells from said sample; and

(c) inducing differentiation of c-kit positive cells from step (b) within said sample by exposing said cells to one or more differentiation-inducing agents.

17. The method of claim 16, wherein the cells are cultured prior to exposure to the differentiation-inducing agent.

18. The method of claim 16, wherein the differentiation-inducing agents result in a cell having a phenotype selected from the group consisting of osteogenic phenotype, hematopoietic phenotype, adipogenic phenotype, myogenic phenotype, hepatic phenotype, neurogenic phenotype and endothelial phenotype.

19. A method for storing pluripotent fetal stem cells comprising the steps of:

(a) obtaining a chorionic villus, amniotic fluid or placenta sample from a human subject;

(b) isolating a substantially enriched population the c-kit positive pluripotent fetal stem cells from the sample; and

(c) cryopreserving the isolated substantially enriched population of c-kit positive pluripotent fetal stem cells.

21. An isolated pluripotent fetal stem cell which is c-kit positive.

22. A method of treating disease in a human comprising administering a substantially enriched population of pluripotent c-kit positive human fetal stem cells into an individual in need thereof.

26. The method of claims 22 or 25, wherein the disease is Parkinson'"'"'s Disease.

23. A method of transplanting an enriched population of pluripotent c-kit positive human fetal stem cells into a human in need thereof.

24. A composition suitable for bonemarrow transplantation comprising an enriched population of pluripotent c-kit positive human fetal stem cells.

25. A method of treating disease in a human comprising administering to a human in need thereof a substantially enriched population of cells comprising pluripotent c-kit positive human fetal stem cells which have been differentiated to a lineage selected from osteogenic, hematopoietic, adipogenic, myogenic, hepatic, neurogenic and endothelial cell lineage.

27. The method of claim 25, wherein the disease is Parkinson'"'"'s Disease and the cell lineage is neurogenic cell lineage.

27-1. A composition suitable for bonemarrow transplantation comprising a substantially enriched population of cells comprising pluripotent c-kit positive human fetal stem cells which have been differentiated to a lineage selected from osteogenic, hematopoietic, adipogenic, myogenic, hepatic, neurogenic and endothelial phenotype.

28. A method of transplanting into a human in need thereof a substantially enriched population of cells comprising pluripotent c-kit positive human fetal stem cells which have been differentiated to a lineage selected from osteogenic, hematopoietic, adipogenic, myogenic, hepatic, neurogenic and endothelial phenotype.

44. The method of claim 28, wherein the sample is subsequently cryopreserved.

28-2. A method of obtaining a population of cells enriched for pluripotent fetal stem cells, comprising isolating a tissue specimen from the chorionic villus of a human placenta.

30. A method of obtaining a population of cells enriched for pluripotent fetal stem cells, comprising isolating a tissue specimen containing said cells from human placenta.

41. The method of claim 30, wherein negative selection is used to enrich c-kit positive cells from the chorionic villus.

31. A method of obtaining a population of cells enriched for pluripotent fetal stem cells, comprising selecting. c-kit positive cells from a chorionic villus.

42. The method of claim 31, wherein negative selection is used to enrich c-kit positive cells from the amniotic fluid.

43. The method of claim 31, wherein negative selection is used to enrich c-kit positive cells from the placenta.

32. A method of obtaining a population of cells enriched for pluripotent fetal stem cells, comprising selecting c-kit positive cells from amniotic fluid.

33. A method of obtaining a population of cells enriched for pluripotent fetal stem cells, comprising selecting c-kit positive cells from placenta.

34. A method of obtaining a population of cells enriched for pluripotent fetal stem cells, comprising the steps of:

(a) cryopreseverving a tissue specimen from the chorionic villus;

(b) thawing the cryopreserved specimen at a later date; and

(c) selecting c-kit positive cells.

35. A method of obtaining a population of cells enriched for pluripotent fetal stem cells, comprising the steps of:

(a) cryopreseverving a specimen of the amniotic fluid;

(b) thawing the cryopreserved specimen at a later date; and

(c) selecting c-kit positive cells.

36. A method of obtaining a population of cells enriched for pluripotent fetal stem cells, comprising the steps of:

(a) cryopreseverving a tissue specimen from a placenta;

(b) thawing the cryopreserved specimen at a later date; and

(c) selecting c-kit positive cells.

37. A method of producing a population of cells enriched for pluripotent fetal stem cells comprising,

(a) isolating c-kit positive cells from the chorionic villus;

and

(b) proliferating said cells in culture medium.

38. A method of producing differentiated tissue comprising,

(a) providing a tissue specimen from chorionic villus;

(b) culturing the tissue under conditions that cause c-kit positive cells to proliferate; and

(c) upon induction cause the c-kit positive cells to differentiate.

39. A method of producing differentiated tissue comprising,

(a) providing a tissue specimen from amniotic fluid;

(b) culturing the tissue under conditions that cause c-kit positive cells to proliferate; and

(c) upon induction cause the c-kit positive cells to differentiate.

40. A method of producing differentiated tissue comprising,

(a) providing a tissue specimen from placenta;

(b) culturing the tissue under conditions that cause c-kit positive cells to proliferate; and

(c) upon induction cause the c-kit positive cells to differentiate.

45. A tissue-engineered