Differentiation of human embryonic stem cells

• US 9,725,699 B2
• Filed: 03/18/2014
• Issued: 08/08/2017
• Est. Priority Date: 04/28/2006
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Inventors: Xu, Jean, Rezania, Alireza
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US14/217,711
• Publication Number:
  US 20150159139A1
• Time in Patent Office: 1,239 Days
• Field of Search: 435377
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• 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 2501/42 Notch; Delta; Jagged; Serrate
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides an improved method for the formation of pancreatic endoderm, pancreatic hormone expressing cells and pancreatic hormone secreting cells. The present invention also provides methods to promote the differentiation of pluripotent stem cells without the use of a feeder cell layer.

Claims

1. A method for differentiating human definitive endoderm into pancreatic endoderm cells:

(a) culturing human definitive endoderm cells in a medium supplemented with retinoic acid or a retinoic acid analog;

(b) removing the retinoic acid or retinoic acid analog from the culture of step (a); and

(c) subsequently culturing the cells from step (b) in a medium supplemented with at least one fibroblast growth factor to obtain pancreatic endoderm cells.

2. The method of claim 1, wherein the method comprises culturing the human definitive endoderm cells in a medium supplemented with retinoic acid.

3. The method of claim 1, wherein the method comprises culturing the human definitive endoderm cells in a medium supplemented with a retinoic acid analog.

4. The method of claim 1, wherein the fibroblast growth factor is FGF-2.

5. The method of claim 1, wherein the fibroblast growth factor is FGF-4.

6. The method of claim 1, wherein the fibroblast growth factor is FGF-10.

7. The method of claim 1, wherein the culture medium in step (a) is supplemented with about 0% to about 2% of serum.

8. The method of claim 1, wherein the culture medium in step (c) is supplemented with about 0% to about 2% of serum.