Mesoderm and definitive endoderm cell populations

• US 9,745,553 B2
• Filed: 03/11/2016
• Issued: 08/29/2017
• Est. Priority Date: 06/24/2005
• Status: Active Grant

• Current Assignee: Icahn School of Medicine at Mount Sinai
• Original Assignee: Icahn School of Medicine at Mount Sinai
• Inventors: Keller, Gordon, Gadue, Paul
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US15/067,728
• Publication Number:
  US 20160186134A1
• Time in Patent Office: 536 Days
• 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 the steps of (a) culturing pluripotent stem cells in the presence of activin to induce differentiation into endoderm lineages, (b) further culturing the cells of step (a) in the presence of BMP-4 and bFGF and in the absence of serum to induce differentiation into a hepatic lineage, and (c) culturing the cells from step (b) with one or more cytokines that promote hepatocyte growth to produce a population enriched for hepatocytes.

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

3. The method of claim 1 where activin is present at a concentration of about 30 ng/ml to about 100 ng/ml.

4. The method of claim 1 wherein activin is present at a concentration of about 50 ng/ml.

5. The method of claim 1 wherein BMP-4 is present at a concentration of about 10 ng/ml to about 30 ng/ml.

6. The method of claim 1 wherein BMP-4 is present at a concentration of about 30 ng/ml.

7. The method of claim 1 wherein bFGF is present at a concentration of about 1.0 ng/ml to about 10 ng/ml.

8. The method of claim 1 wherein bFGF is present at a concentration of about 10 ng/ml.

9. The method of claim 1 wherein the cytokines are selected from the group consisting of VEGF, human recombinant HGF, EGF, bFGF, and TGFalpha.

10. The method of claim 9 wherein VEGF is present at a concentration of about 10 ng/ml.

11. The method of claim 9 wherein human recombinant HGF is present at a concentration of about 20 ng/ml.

12. The method of claim 9 wherein EGF is present at a concentration of about 10 ng/ml.

13. The method of claim 9 wherein bFGF is present at a concentration of about 10 ng/ml.

14. The method of claim 9 wherein TGFalpha is present at a concentration of about 20 ng/ml.