Mesoderm and definitive endoderm cell populations

• US 20060003446A1
• Filed: 06/24/2005
• Published: 01/05/2006
• Est. Priority Date: 05/17/2002
• Status: Abandoned Application

• Current Assignee: Mount Sinai School of Medicine of New York University
• Original Assignee: Mount Sinai School of Medicine of New York University
• Inventors: Gadue, Paul, Keller, Gordon
• Application Number:
  US11/165,930
• Publication Number:
  US 20060003446A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/148 Transforming growth factor alpha [TGF-a]
• 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 2501/40 Regulators of development
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem 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 5/0607 : Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/067 : Hepatocytes

Abstract

The present invention provides cell populations that are enriched for mesendoderm and mesoderm, and cell populations that are enriched for endoderm. The cell populations of the invention are useful for generating cells for cell replacement therapy.

Claims

1. A method of making a cell population enriched for endoderm cells comprising culturing embryonic stem cells in the absence of serum and the presence of activin and Wnt for about one to about six days, isolating brach⁺/HNF3β

⁺ cells, a culturing said isolated cells in the absence of serum and the presence of activin and an inhibitor of Wnt signalling for at least about one day.

2. The method of claim 1 wherein the embryonic stem cells are human embryonic stem cells.

3. The method of claim 1 wherein Wnt is Wnt 3d.

4. The method of claim 1 wherein the inhibitor of Wnt signalling is DKK-1.

5. The method of claim 1 wherein activin is present at a concentration of at least about 30 ng/ml.

6. A method of making a cell population enriched for endoderm cells comprising culturing human embryonic stem cells in the presence of serum for about two to about ten days, isolating brach⁺/HNF3β

⁺ cells, and culturing said brach⁺/HNF3β

⁺ cells in the absence of serum and the presence of activin and an inhibitor of Wnt signalling for at least about one day.

7. The method of claim 6 wherein the concentration of activin is at least about 30 ng/ml.

8. The method of claim 6 wherein the concentration of activin is about 100 ng/ml.

9. The method of claim 6 wherein the inhibitor of Wnt signalling is DKK-1.

10. The method of claim 9 wherein the concentration of DKK-1 is at least about 30 ng/ml.

11. An embryonic stem cell in which a nucleic acid encoding a selectable marker is present in the HNF3β

locus.

12. The stem cell of claim 11 in which the selectable marker is human CD4.

13. The stem cell of claim 11 in which a second nucleic acid encoding a second selectable marker is present in the brachury locus.

14. The stem cell of claim 13 in which the second selectable marker is green fluorescent protein.