PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM

• US 20100233755A1
• Filed: 03/22/2010
• Published: 09/16/2010
• Est. Priority Date: 10/27/2005
• Status: Abandoned Application

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: BAETGE, EMMANUEL E., AGULNICK, ALAN D., ELIAZER, SUSAN, D'AMOUR, KEVIN ALLEN
• Application Number:
  US12/729,084
• Publication Number:
  US 20100233755A1
• Time in Patent Office: Days
• US Class Current: 435/29
• 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 10% of said human cells are pancreatic-duodenal homoebox factor-1 (PDX1) positive, endoderm cells that express at least one marker selected from the group consisting of SOX9, HNF6, and PROX1, and which express the PDX1 protein.

2. The cell culture of claim 1, wherein at least 40% of said human cells are PDX1-positive endoderm cells.

3. The cell culture of claim 1, wherein at least 50% of said human cells are PDX1-positive endoderm cells.

4. The cell culture of claim 1, wherein at least 60% of said human cells are PDX1-positive endoderm cells.

5. The cell culture of claim 1, wherein at least 75% of said human cells are PDX1-positive endoderm cells.

6. The cell culture of claim 1, wherein human feeder cells are present in said culture, and wherein at least 10% of human cells other than said human feeder cells are PDX1-positive endoderm cells.

7. 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 endoderm cells, wherein expression is determined by quantitative PCR.

8. 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.

9. The cell culture of claim 1 further comprising a retinoid.

10. The cell culture of claim 9, wherein said retinoid is retinoic acid (RA).

11. The cell culture of claim 10 further comprising B27.

12. The cell culture of claim 1, wherein said human cells are non-recombinant.

13. The cell culture of claim 1, wherein said endoderm cells are multipotent cells that can further differentiate to cells derived from the foregut.

14. A cell population comprising cells wherein at least 90% of said cells are human PDX1-positive, endoderm cells that express at least one marker selected from the group consisting of SOX9, HNF6, and PROX1, and which express the PDX1 protein.

15. The cell population of claim 14, wherein at least 98% of said cells are PDX1-positive endoderm cells.

16. The cell population of claim 14, 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 endoderm cells, wherein expression is determined by quantitative PCR.

17. The cell population of claim 14, wherein said human cells are non-recombinant.

18. The cell population of claim 14, wherein said endoderm cells are multipotent cells that can further differentiate to cells derived from the foregut.

19. A method of producing PDX1-positive endoderm cells, said method comprising:

(a) culturing a population of pluripotent human cells in the presence of exogenous TGFβ

superfamily growth factor;

(b) culturing the cells from step (a) in the presence of an exogenous FGF-family growth factor; and

(c) culturing the cells from step (b) in the presence of a retinoid, thereby producing multipotent PDX1-positive endoderm cells.

20. The method of claim 19, wherein said TGFβ

superfamily growth factor comprises activin A.

21. The method of claim 19, wherein the FGF-family growth factor comprises FGF-7.

22. The method of claim 19, wherein said retinoid is retinoic acid (RA).

23. The method of claim 22, wherein RA is provided in a concentration ranging from 0.01 μ

M to about 50 μ

M.

24. The method of claim 19, wherein at least 25% of said cell population expresses PDX1.

25. A method of screening growth factors capable of promoting the differentiation of PDX1-negative definitive endoderm cells to PDX1-positive definitive endoderm cells, said method comprising:

(a) culturing a population of PDX1-negative definitive endoderm cells;

(b) determining the expression of PDX1 in said population;

(c) providing one or more candidate growth factors to said population;

(d) determining the expression of PDX1 in said population after addition of the one or more candidate growth factors;

(e) comparing expression of PDX1 at step (b) with expression of PDX1 at step (d);

(f) evaluating whether expression of PDX1 at step (d) is increased as compared to expression of PDX1 at step (b) thereby determining that the candidate differentiation factor is capable of promoting the differentiation of PDX1-negative definitive endoderm cells to PDX1-positive definitive endoderm cells.