Expansion of definitive endoderm cells

• US 8,658,151 B2
• Filed: 10/20/2009
• Issued: 02/25/2014
• Est. Priority Date: 12/23/2003
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: Kelly, Olivia, Baetge, Emmanuel E., Carpenter, Melissa
• Primary Examiner(s): Singh, Anoop
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US12/582,600
• Publication Number:
  US 20100041150A1
• Time in Patent Office: 1,589 Days
• Field of Search: 424/93.1, 424/93.7
• US Class Current: 424/93.1
• 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 2501/155 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 2502/13 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

Abstract

Disclosed herein are cell cultures comprising expanded definitive endoderm cells as well as methods for expanding definitive endoderm cells in culture.

Claims

1. A method of expanding human definitive endoderm cells in culture, said method comprising the steps of:

(a) providing a cell culture comprising at least 20% human definitive endoderm cells, wherein said human definitive endoderm cells are multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom, and

(b) culturing the human definitive endoderm cells provided from step (a) under conditions that permit the expansion of said human definitive endoderm cells.

2. The method of claim 1, wherein the step of culturing said human definitive endoderm cells comprises passaging said definitive endoderm cells, thereby producing a plurality of cell cultures comprising human definitive endoderm cells.

3. The method of claim 2, wherein the step of passaging said human definitive endoderm cells comprises providing at least one protease to said cell culture.

4. The method of claim 3, wherein said at least one protease comprises trypsin.

5. The method of claim 2, wherein the step of passaging said human definitive endoderm cells comprises mechanically disrupting contacts between said human definitive endoderm cells.

6. The method of claim 2, wherein the step of passaging said human definitive endoderm cells comprises incubating said human definitive endoderm cells in a cell dispersal buffer.

7. The method of claim 2, wherein said human definitive endoderm cells are attached to a substrate.

8. The method of claim 7, wherein the step of passaging said human definitive endoderm cells comprises detaching said human definitive endoderm cells from said substrate.

9. The method of claim 7, wherein said substrate is a surface of a tissue culture flask.

10. The method of claim 2, wherein the step of passaging said human definitive endoderm cells comprises providing at least one enzyme to said cell culture.

11. The method of claim 1, wherein said human definitive endoderm cells are derived from human embryonic stem cells (hESCs).