Mesoderm and definitive endoderm cell populations

• US 8,283,168 B2
• Filed: 06/23/2006
• Issued: 10/09/2012
• Est. Priority Date: 05/17/2002
• Status: Active Grant

• Current Assignee: Icahn School of Medicine at Mount Sinai
• Original Assignee: Mount Sinai School of Medicine (Sinai Health System)
• Inventors: Keller, Gordon M., Gadue, Paul
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US11/993,432
• Publication Number:
  US 20110212061A1
• Time in Patent Office: 2,300 Days
• Field of Search: 435/377, 435/325
• US Class Current: 435/377
• 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. The present invention further provides a method of generating hepatocytes, cell populations enriched for hepatocytes, and a method of hepatocyte replacement therapy.

Claims

1. A method of making a cell population enriched for hepatocytes comprising culturing embryonic stem cells in the presence of activin or nodal to provide brach⁺/HFN3β

⁺/cKit⁺ cells, isolating the brach⁺/HFN3 β

⁺/cKit⁺ cells, culturing said brach⁺/HFN3β

⁺/cKit⁺ cells in the presence of BMP-4 and bFGF in serum-free media to produce hepatoblasts, and culturing the hepatoblasts in serum-free media supplemented with one or more cytokines that support hepatocyte growth for an amount of time sufficient to produce a population enriched for hepatocytes.

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

3. The method of claim 1 wherein the embryonic stem cells are cultured in the presence of activin or nodal for about 2 to about 10 days.

4. The method of claim 1 wherein the embryonic stem cells are cultured in the absence of serum.

5. A method of making a cell population enriched for hepatocytes comprising culturing pluripotent stem cells in the presence of activin to provide brach⁺/HFN3 β

⁺/cKit⁺ cells, culturing said brach⁺/HFN3β

⁺/cKit⁺ cells in the presence of BMP-4 and bFGF in serum-free media to produce hepatoblasts, and culturing the hepatoblasts in serum-free media supplemented with one or more cytokines that support hepatocyte growth for an amount of time sufficient to produce a population enriched for hepatocytes.

6. The method of claim 5 wherein the stem cells are human stem cells.

7. The method of claim 5 wherein the stem cells are cultured in the presence of activin for about 2 to about 10 days.

8. The method of claim 5 wherein the stem cells are cultured in the absence of serum.