Definitive endoderm

• US 10,421,942 B2
• Filed: 02/13/2017
• Issued: 09/24/2019
• Est. Priority Date: 12/23/2003
• 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., Baetge, Emmanuel E.
• Primary Examiner(s): Singh, Anoop K
• Application Number:
  US15/431,634
• Publication Number:
  US 20170159011A1
• Time in Patent Office: 953 Days
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• 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 2501/415 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 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 5/068 Stem cells; Progenitors

Abstract

Disclosed herein are cell cultures comprising definitive endoderm cells and methods of producing the same. Also disclosed herein are cell populations comprising substantially purified definitive endoderm cells as well as methods for enriching, isolating and purifying definitive endoderm cells from other cell types.

Claims

1. An in vitro method of producing human definitive endoderm cells that express SRY-box 17 (SOX17) and hepatocyte nuclear factor-3 beta (HNF3β

), the method comprising;

(a) culturing a population of human pluripotent stem cells; and

(b) differentiating the population of human pluripotent stem cells by culturing said population in a culture medium comprising equal to or less than 2%V/V serum and at least 30 ng/ml of activin A for a time sufficient to cause differentiation of said human pluripotent stem cells into human definitive endoderm cells expressing SOX17 and HNF3β

, thereby generating the human definitive endoderm cells expressing SOX17 and HNF3β

in said population.

2. The method of claim 1 wherein the culture medium further comprises:

(a) hone morphogenetic protein-4 (BMP4) in an amount sufficient to promote differentiation of the population of human pluripotent stem cells to definitive endoderm cells;

or

(b) activin B in an amount sufficient to promote differentiation of the human pluripotent stem cells to definitive endoderm cells.

3. The method of claim 1, wherein activin A in the culture medium is at a concentration of about 30 ng/ml-500 ng/ml.

4. The method of claim 2, wherein activin B in the culture medium is at a concentration of about 10 ng/ml-500 ng/ml.

5. The method of claim 2, wherein BMP4 in the culture medium is at a concentration of about 10 ng/ml-500 ng/ml of BMP4.

6. The method of claim 1, wherein the culture medium further comprises a WNT protein in an amount sufficient to promote differentiation of the population of human pluripotent stern cells to definitive endoderm cells.

7. The method of claim 6, wherein the WNT protein in Wnt3A.

8. The method of claim 1, wherein the population comprising definitive endoderm cells is substantially free of cells derived from the ectoderm lineage.