Pancreatic and Liver Endoderm Cells and Tissue by Differentiation of Definitive Endoderm Cells Obtained from Human Embryonic Stems

• US 20090298169A1
• Filed: 06/04/2007
• Published: 12/03/2009
• Est. Priority Date: 06/02/2006
• Status: Abandoned Application

• Current Assignee: The University of Georgia Research Foundation, Inc.
• Original Assignee: The University of Georgia Research Foundation, Inc.
• Inventors: Reynolds, David, Kulik, Michael, Dalton, Stephen
• Application Number:
  US12/227,913
• Publication Number:
  US 20090298169A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The invention relates to methods that allow for the efficient differentiation to form pancreatic endoderm cells from pluripotent stem cells such as human embryonic stem cells and definitive endoderm cells. The invention is directly applicable to the ultimate generation of pancreatic beta cells that could be used as part of a therapy to treat or even cure diabetes. Additionally, the present invention may be used to generate liver endoderm cells from human embryonic stem cells and definite endoderm cells as well. This invention relates to a method for generating definitive endoderm and pancreatic endoderm cells from stem cells, preferably human embryonic stem cells using defined media in the absence of feeder cells. A simply two step procedure to provide pancreatic endoderm cells from embryonic stem cells represents further embodiments of the present invention.

Claims

1. A method of producing human pancreatic endoderm cells from definitive endoderm cells comprising:

a. exposing human definitive endoderm cells to an effective amount of retinoic acid in a cell differential medium for a period of at least one day; and

b. stabilizing the differentiated cells obtained from step a by exposing said cells to a stabilizing medium in the absence of retinoic acid.

2. The method according to claim 1 wherein said definitive endoderm cells are obtained from human embryonic stem cells exposed to a P13K inhibitor.

3. The method according to claim 1 wherein said cell differential medium comprises retinoic acid at a concentration ranging from about 0.05 μ

g/ml to about 25 μ

g/ml.

4. The method according to claim 1 wherein said cell differential medium comprises retinoic acid at a concentration ranging from about 0.1 μ

g/ml to about 2 μ

g/ml.

5. The method according to claim 1 wherein said cell differentiation medium further includes fibroblast growth factor.

6. The method according to claim 1 wherein said cell differentiation medium further includes fibroblast growth factor 10 at a concentration ranging from about 2 μ

g/ml to about 100 μ

g/ml.

7. The method according to claim 1 wherein said cell differentiation medium further includes fibroblast growth factor 10 at a concentration ranging from about 2 μ

g/ml to about 100 μ

g/ml.

8. The method according to claim 1 wherein said cell differentiation medium is a basal cell medium comprising about 2% to about 20% fetal calf serum.

9. The method according to claim 1 wherein said cell differentiation medium is a basal cell medium comprising about 10% fetal calf serum.

10. The method according to claim 1 wherein said stabilizing medium is a basal cell medium comprising about 2% to about 20% fetal calf serum.

11. The method according to claim 1 wherein said stabilizing medium is a basal cell medium comprising about 10% fetal calf serum.

12. The method according to claim 1 wherein said exposing step occurs over a period of at least about 2 days.

13. The method according to claim 1 wherein said exposing step occurs over a period of at least about 4 days.

14. The method according to claim 1 wherein said exposing step occurs over a period of about 4 days.

15. The method according to claim 1 wherein said stabilizing step occurs over a period of at least about 1 day.

16. The method according to claim 1 wherein said stabilizing step occurs over a period of at least about 2 days.

17. The method according to claim 1 wherein said basal cell medium is a mixture of DMEM and F12.

18. The method according to claim 16 wherein said basal cell medium is a 50:

50 mixtures of DMEM and F12.

19. The method according to claim 1 wherein said exposing step or said stabilizing step occurs wherein definitive endoderm cells or said differentiated cells are grown on a support comprising a differentiation protein.

20. The method according to claim 1 wherein said exposing step or said stabilizing step occurs wherein definitive endoderm cells or said differentiated cells are grown on a support comprising matrigel.

21. The method according to claim 1 wherein said exposing step and said stabilizing step occurs wherein said definitive endoderm cells or said differentiated cells are grown on a support comprising a differentiation protein.

23. The method according to claim 21 wherein said fibroblast growth factor is fibroblast growth factor 10.

22. A method of producing liver endoderm cells from definitive endoderm cells comprising:

a. exposing definitive endoderm cells to an effective amount of fibroblast growth factor in a cell differential medium for a period of at least one day; and

b. stabilizing the differentiated cells obtained from step a by exposing said cells to a stabilizing medium.

24. A method of producing pancreatic endoderm cells comprising:

a. producing definitive endoderm cells from human embryonic stem cells by exposing human embryonic stem cells to a basal cell medium comprising an effective amount of a P13K inhibitor as a differentiation agent;

b. stabilizing said definitive endoderm cells from step a;

c. exposing definitive endoderm cells after step b to an effective amount of retinoic acid in a cell differential medium for a period of at least one day; and

d. stabilizing the differentiated cells obtained from step a by exposing said cells to a stabilizing medium in the absence of retinoic acid.

26. The method according to claim 24 wherein defined media includes an inhibitor of PI3K signaling.

27. The method according to claim 24 wherein said growth matrix is matrigel.

29. The method of claim 24 wherein said definitive endoderm cells are produced at a level of at least about 90% from said embryonic stem cells.

30. The method according to claim 24 wherein said definitive endoderm cells are further differentiated into pancreatic endoderm cells.

31. The method according to claim 24 wherein said cells are human cells.

25. A method of generating definitive endoderm cells from embryonic stem cells, under feeder cell-free conditions, comprising exposing plated embryonic stem cells to a defined media or MEF conditioned media using a growth matrix, and thereafter, exposing the stem cells to a differentiation media which is a defined media comprising effective amounts of Activin A, nodal, TGFβ

or other TGF component and optionally, an inhibitor of PI3kinase signaling.

28. A method of generating definitive endoderm cells from embryonic stem cells, comprising exposing said exposed stem cells in the absence of feed cells to a differentiation media comprising elevated levels of Activin A, nodal or TNFβ

and optionally, an inhibitor of PI3kinase signaling, wherein said differentiation media is a defined media free from fetal calf serum or KSR-type serum components.

32. A method of generating pancreatic endoderm cells from human embryonic stem cells said method comprising generating definitive endoderm cells from embryonic stem cells, under feeder cell-free conditions, comprising exposing embryonic stem cells to a defined media or MEF conditioned media using a growth matrix, and thereafter, exposing the stem cells to a differentiation media which is a defined media comprising effective amounts of a SMAD pathway activator and optionally, an inhibitor of PI3kinase signaling for a period of about 3-6 days to produce definitive endoderm cells and thereafter exposing said definitive endoderm cells to a defined media comprising an effective amount of retinoic acid and optionally, an effective amount of FGF10 for a further period of about 5-12 days, preferably 8-10 days to produce pancreatic endoderm cells.

33. The method according to claim 32 wherein said SMAD pathway activator is selected from the group consisting of Activin A, nodal, TGFβ

, TGF component or mixtures thereof.

34. The method according to claim 32 wherein said media further includes an effective amount of wnt3a.

35. The method according to claim 34 wherein said growth matrix is madrigel.