PDX1-expressing dorsal and ventral foregut endoderm

• US 20070122905A1
• Filed: 10/27/2006
• Published: 05/31/2007
• Est. Priority Date: 10/27/2005
• Status: Abandoned Application

• Current Assignee: CyThera, Inc. (Vertex Pharmaceuticals Incorporated)
• Original Assignee: CyThera, Inc. (Vertex Pharmaceuticals Incorporated)
• Inventors: Agulnick, Alan, D'Amour, Kevin, Eliazer, Susan, Baetge, E.
• Application Number:
  US11/588,693
• Publication Number:
  US 20070122905A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• 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 2501/405 Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• 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 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• 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 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: C12N 5/068 Stem cells; Progenitors

Abstract

Disclosed herein are cell cultures comprising dorsal and/or ventral PDX1-positive foregut endoderm cells and methods of producing the same. Also disclosed herein are cell populations comprising substantially purified dorsal and/or ventral PDX1-positive foregut endoderm cells as well as methods for enriching, isolating and purifying dorsal and/or ventral PDX1-positive foregut endoderm cells from other cell types. Methods of identifying differentiation factors capable of promoting the differentiation of dorsal and/or ventral PDX1-positive foregut endoderm cells, are also disclosed.

Claims

1. A cell culture comprising human cells wherein at least about 26% of said human cells are pancreatic-duodenal homoebox factor-1 (PDX1) positive, dorsally-biased, foregut endoderm cells that express at least one marker selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2, SLC27A2, SERPINF2, DUSP9, CDH6 and SOX9, said PDX1-positive, dorsally-biased, foregut endoderm cells being multipotent cells that can differentiate into cells of the dorsal pancreatic bud.

2. The cell culture of claim 1, wherein said marker is selected from the group consisting of SERPINF2, DUSP9, CDH6 and SOX9.

3. The cell culture of claim 1, wherein said marker is a cell surface marker selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.

4. The cell culture of claim 1, wherein said PDX1-positive, dorsally-biased, foregut endoderm cells express at least one marker selected from the group consisting of ADORA2A, CD47, EPB41L1, MAG, SFRP5, SLC16AI0, SLC16A2, SLCIA3, SLC3OA4, SLICK, SLITRK4, XPR1, HOXA1, PDEI IA, FAM49A and WNT5A.

5. The cell culture of claim 4, wherein said marker is selected from the group consisting of HOXAI, PDEI IA, FAM49A and WNT5A.

6. The cell culture of claim 4, wherein said marker is a cell surface marker selected from the group consisting of ADORA2A, CD47, EPB41L1, MAG, SFRP5, SLC16A10, SLC16A2, SLCIA3, SLC3OA4, SLICK, SLITRK4 and XPRI.

7. The cell culture of claim 1, wherein at least about 30% of said human cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

8. The cell culture of claim 1, wherein at least about 40% of said human cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

9. The cell culture of claim 1, wherein at least about 50% of said human cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

10. The cell culture of claim 1, wherein at least about 60% of said human cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

11. The cell culture of claim 1, wherein at least about 75% of said human cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

13. The cell culture of claim 1, wherein the expression of PDX1 is greater than the expression of a marker selected from the group consisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in said PDX1-positive, dorsally biased, foregut endoderm cells.

14. The cell culture of claim 1, wherein said cell culture is substantially free of cells selected from the group consisting of visceral endodermal cells, parietal endodermal cells and neural cells.

12. The cell culture of claim I, wherein human feeder cells are present in said culture, and wherein at least about 2% of human cells other than said human feeder cells are PDX1-positive, dorsally biased, foregut endoderm cells.

15. The cell culture of claim I further comprising a retinoid.

16. The cell culture of claim 15, wherein said retinoid is retinoic acid (RA).

17. The cell culture of claim 16 further comprising B27.

18. A cell culture comprising human cells wherein at least about 2% of said human cells are pancreatic-duodenal homoebox factor-β

(PDX1) positive, ventrally-biased, foregut endoderm cells that express at least one marker is selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2, SLC27A2, SERPINF2, DUSP9, CDH6 and SOX9, said PDX1-positive, ventrally-biased, foregut endoderm cells being multipotent cells that can differentiate into cells of the ventral pancreatic bud.

19. The cell culture of claim 18, wherein said marker is selected from the group consisting of SERPINF2, DUSP9, CDH6 and SOX9.

20. The cell culture of claim 18, wherein said marker is a cell surface marker selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPFNF2 and SLC27A2.

21. The cell culture of claim 18, wherein said PDX1-positive, ventrally-biased, foregut endoderm cells do not substantially express one or more markers selected from the group consisting of ADORA2A, CD47, EPB41L1, MAG, SFRP5, SLC16AI0, SLC16A2, SLCIA3, SLC3OA4, SLICK, SLITRK4, XPRI, HOXAI, PDEI IA, FAM49A and WNT5A.

22. The cell culture of claim 18, wherein at least about 5% of said human cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

23. The cell culture of claim 18, wherein at least about 10% of said human cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

24. The cell culture of claim 18, wherein at least about 25% of said human cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

25. The cell culture of claim 18, wherein at least about 50% of said human cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

26. The cell culture of claim 18, wherein at least about 75% of said human cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

27. The cell culture of claim 18, wherein human feeder cells are present in said culture, and wherein at least about 2% of human cells other than said human feeder cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

28. The cell culture of claim 18, wherein the expression of PDX1 is greater than the expression of a marker selected from the group consisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in said PDX1-positive, ventrally-biased, foregut endoderm cells.

29. The cell culture of claim 18, wherein said cell culture is substantially free of cells selected from the group consisting of visceral endodermal cells, parietal endodermal cells and neural cells.

30. The cell culture of claim 18 further comprising a retinoid.

31. The cell culture of claim 30, wherein said retinoid is retinoic acid (RA).

32. The cell culture of claim 31 further comprising B27.

33. A cell population comprising cells wherein at least about 90% of said cells are human PDX1-positive, dorsally-biased, foregut endoderm cells that express at least one marker selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2, SLC27A2, SERPINF2, DUSP9, CDH6 and SOX9, said PDX1-positive, dorsally-biased, foregut endoderm cells being multipotent cells that can differentiate into cells of the dorsal pancreatic bud.

34. The cell population of claim 33, wherein said marker is selected from the group consisting of SERPINF2, DUSP9, CDH6 and SOX9.

35. The cell population of claim 33, wherein said marker is a cell surface marker selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.

36. The cell population of claim 33, wherein said PDX1-positive, dorsally-biased, foregut endoderm cells express at least one marker selected from the group consisting of ADORA2A, CD47, EPB41LI, MAG, SFRP5, SLC16A10, SLC16A2, SLCIA3, SLC3OA4, SLICK, SLITRK4, XPRI, HOXAI, PDEI IA, FAM49A and WNT5A.

37. The cell population of claim 36, wherein said marker is selected from the group consisting of HOXA I, PDE I IA, FAM49A and WNT5A.

38. The cell population of claim 36, wherein said marker is a cell surface marker selected from the group consisting of ADORA2A, CD47, EPB41L1, MAG, SFRP5, SLC16A10, SLCI6A2, SLCIA3, SLC3OA4, SLICK, SLITRK4 and XPRI.

39. The cell population of claim 33, wherein at least about 95% of said cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

40. The cell population of claim 33, wherein at least about 98% of said cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

41. The cell population of claim 33, wherein the expression of PDX1 is greater than the expression of a marker selected from the group consisting of AFP, SOX7, SOX1, ZIC1 and NFM in said PDX1-positive, dorsally-biased, foregut endoderm cells.

42. A cell population comprising cells wherein at least about 90%.of said cells are human PDX1-positive, ventrally-biased, foregut endoderm cells that express at least one marker selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2, SLC27A2, SERPINF2, DUSP9, CDH6 and SOX9, said PDX1-positive, ventrally-biased, foregut endoderm cells being multipotent cells that can differentiate into cells of the ventral pancreatic bud.

43. The cell population of claim 42, wherein said marker is selected from the group consisting of SERPINF2, DUSP9, CDH6 and SOX9.

44. The cell population of claim 42, wherein said marker is a cell surface marker selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.

45. The cell population of claim 42, wherein said PDX1-positive, ventrally-biased, foregut endoderm cells do not substantially express one or more markers selected from the group consisting of ADORA2A, CD47, EPB41LI, MAG, SFRP5, SLC16AI0, SLC16A2, SLCIA3, SLC3OA4, SLICK, SLITRK4, XPRI, HOXA1, PDEI IA, FAM49A and WNT5A.

46. The cell population of claim 42, wherein at least about 95% of said cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

47. The cell population of claim 42, wherein at least about 98% of said cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

48. The cell population of claim 42, wherein the expression of PDX1 is greater than the expression of a marker selected from the group consisting of AFP, SOX7, SOX I, ZIC1 and NFM in said PDX1-positive, ventrally-biased, foregut endoderm cells.