Method of Inducing Embryonic Stem Cells Into Pancreatic Cells

• US 20080286867A1
• Filed: 04/14/2006
• Published: 11/20/2008
• Est. Priority Date: 04/15/2005
• Status: Active Grant

• Current Assignee: Beijing Hua Yuan BO Chuang Technology Limited
• Original Assignee: Peking University
• Inventors: Shi, Yan, Jiang, Wei, Hou, Lingling, Ding, Mingxiao, Deng, Hongkui, Tang, Fuchou
• Granted Patent: US 7,776,597 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• 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 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention provided a simple three-step approach based on the combinational induction with activin A, all-trans retinoic acid and, optionally, other maturation factors which are able to induce embryonic stem cells to differentiate into insulin-producing cells. A kit used to induce embryonic stem cells to differentiate into insulin-producing cells was also provided.

Claims

1. A method for inducing embryonic stem cell, comprising in sequence the steps of:

culturing embryonic stem cells in a cell culture medium to form embryonic bodies;

incubating the embryonic bodies with Activin A; and

incubating the embryonic bodies with RA to develop insulin-producing precursor cells.

2. The method of claim 1, wherein the culture medium is DMEM containing about 10% fetus calf serum when embryonic stem cells are from mice and the culture medium is CDM when embryonic stem cells are from human.

3. The method of claim 1, wherein the concentration of Activin A is about 50-300 ng/ml culture medium;

and the concentration of RA is about 1×

10<sup>−

7</sup>-1×

10<sup>−

5</sup> mol/L culture medium.

4. The method of claim 3, further comprising the step of coating a culture vessel used to contain the culture medium with Metrigel before the step of culturing embryonic stem cells in the cell culture medium to form embryonic bodies.

5. The method of claim 4, wherein the mass percentage of the metrigel is about 1% of the culture medium.

6. The method of claim 5, wherein the embryonic bodies are incubated with Activin A for 20 hours-4 days, then cultured in a culture medium without Activin A for 6-8 hours, and then incubated with RA for 20 hours-4 days to develop insulin-producing precursor cells.

7. The method of claim 6, further comprising the step of incubating the resultant insulin-producing precursor cells in a culture medium containing maturation factor(s) to expand insulin-producing precursor cells.

8. The method of claim 7, wherein the maturation factor(s) include bFGF.

9. The method of claim 8, wherein the concentration of bFGF is 8-12 ng/ml.

10. The method of claim 9, further comprising the step of incubating the expanded insulin-producing precursor cells in a culture medium containing N2 supplement, B27 supplement, Laminin, and nicotinamide to develop insulin producing cells.

11. The method of claim 10, wherein the culture medium is DMEM/F12 or CDM containing N2 supplement, B27 supplement, 1 μ

g/ml of Laminin, 10 ng/ml of bFGF, Insulin-Transferrin-Selenium-A (1;

100) and 10 mmol/L of nicotinamide.

12. A kit for inducing embryonic stem cell, comprising Activin A and RA.

13. The kit of claim 12, further comprising Metrigel.

14. The kit of claim 13, further comprising a first cell culture medium.

15. The kit of claim 14, further comprising serum.