PDX1 expressing endoderm

• US 8,633,024 B2
• Filed: 02/28/2008
• Issued: 01/21/2014
• Est. Priority Date: 04/27/2004
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: D'Amour, Kevin Allen, Agulnick, Alan D., Eliazer, Susan, Baetge, Emmanuel E.
• Primary Examiner(s): Singh, Anoop
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US12/039,701
• Publication Number:
  US 20090042287A1
• Time in Patent Office: 2,154 Days
• Field of Search: 435/325, 435/366, 435/377
• US Class Current: 435/377
• 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: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• 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 2501/155 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 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2502/02 embryonic cells
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2503/00 Use of cells in diagnostics
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0018 Culture media for cell or tissue culture media for specific animal cell type C12N5/06
• 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 5/0608 Germ cells production of embryos, nuclear transfer A01K67/027
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0679 Cells of the gastro-intestinal tract
• CPC Class Codes: C12Q 1/6881 : for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/158 : Expression markers

Abstract

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

Claims

1. An in vitro method of producing human PDX1-positive foregut endoderm cells, said method comprising:

(a) culturing a population of human pluripotent stem cells,

(b) differentiating the human pluripotent stem cells in the presence of an exogenous TGFβ

superfamily growth factor to obtain definitive endoderm cells,

(c) removing the exogenous TGFβ

superfamily growth factor from the definitive endoderm cells, and

(d) differentiating the definitive endoderm cells of step (c) in the presence of a retinoid to obtain PDX1-positive foregut endoderm cells.

2. The in vitro method of claim 1, wherein step (d) further comprises providing an FGF-family growth factor.

3. The in vitro method of claim 2, wherein the FGF-family growth factor is provided at approximately the same time as said retinoid.

4. The in vitro method of claim 2, wherein said FGF-family growth factor comprises FGF-10.

7. The in vitro method of claim 1, wherein the expression of PDX1 is determined by immunocytochemistry.

8. The in vitro method of claim 1, wherein said retinoid is retinoic acid (RA).

9. The in vitro method of claim 8, wherein RA is provided in a concentration ranging from 0.01 μ

M to 50 μ

M.

10. The in vitro method of claim 8, wherein RA is provided in a concentration ranging from 0.1 μ

M to 10 μ

M.

11. The in vitro method of claim 1, wherein the exogenous TGFβ

superfamily growth factor comprises activin A.

5. An in vitro method of producing human PDX1-positive endoderm cells, said method comprising:

(a) culturing a population of pluripotent human stem cells,

(b) differentiating the human pluripotent stem cells in the presence of an exogenous TGFβ

superfamily growth factor to obtain definitive endoderm cells,

(c) removing the exogenous TGFβ

superfamily growth factor from the definitive endoderm cells, and

(d) differentiating the definitive endoderm cells of step (c) in the presence of a retinoid to obtain a population of PDX1-positive endoderm cells, wherein at least 25% of said cell population expresses PDX1.

12. The in vitro method of claim 5, wherein the expression of PDX1 is determined by immunocytochemistry.

13. The in vitro method of claim 5, wherein said retinoid is RA.

14. The in vitro method of claim 13, wherein RA is provided in a concentration ranging from 0.01 μ

M to 50 μ

M.

15. The in vitro method of claim 13, wherein RA is provided in a concentration ranging from 0.1 μ

M to 10 μ

M.

16. The in vitro method of claim 5, wherein the exogenous TGFβ

superfamily growth factor comprises activin A.

6. An in vitro method of producing human PDX1-positive endoderm cells, said method comprising:

(a) culturing a population of pluripotent human stem cells,

(b) differentiating the population of pluripotent human cells in the presence of an exogenous TGFβ

superfamily growth factor to obtain definitive endoderm cells,

(c) removing the exogenous TGFβ

superfamily growth factor from the definitive endoderm cells, and

(d) differentiating the definitive endoderm cells of step (c) in the presence of a retinoid to obtain a population of PDX1-positive endoderm cells, wherein the expression of PDX1 is greater than the expression of a marker selected from the group consisting of α

-fetoprotein, SOX7, SOX1, ZIC1 and NFM in said population of PDX1-positive endoderm cells as determined by quantitative polymerase chain reaction.

17. The in vitro method of claim 6, wherein said retinoid is RA.

18. The in vitro method of claim 17, wherein RA is provided in a concentration ranging from 0.01 μ

M to 50 μ

M.

19. The in vitro method of claim 17, wherein RA is provided in a concentration ranging from 0.1 μ

M to 10 μ

M.

20. The in vitro method of claim 6, wherein the exogenous TGFβ

superfamily growth factor comprises activin A.