Method for inducing differentiation of embryonic stem cells into functioning cells

• US 20040072344A1
• Filed: 07/25/2003
• Published: 04/15/2004
• Est. Priority Date: 01/25/2002
• Status: Abandoned Application

• Current Assignee: Kazutomo Inoue, Yugengaisha Okuma Contactlens Kenkyujo
• Original Assignee: Kazutomo Inoue, Yugengaisha Okuma Contactlens Kenkyujo
• Inventors: Ishii, Michiyo, Gu, Yanjun, Kim, Dohoon, Inoue, Kazutomo
• Application Number:
  US10/626,772
• Publication Number:
  US 20040072344A1
• Time in Patent Office: Days
• US Class Current: 435/366
• 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 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/46 Amines, e.g. putrescine
• 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 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/33 Insulin
• CPC Class Codes: C12N 2501/392 Sexual steroids
• CPC Class Codes: C12N 2501/58 Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
• 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 5/0619 Neurons
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention provides a 4-step method for inducing differentiation of embryonic stem cells into functioning cells comprising 1) expanding ES cells; 2) inducing Embryoid Bodies in the presence of leukemia inhibitory factor and basic FGF; 3) selection expanding of the EBs and 4) differentiation. According to the present invention, ES cells can be differentiated into either insulin producing pancreatic islet like cell clusters or nerve like cells. Thus obtained functioning cells may be potential sources of donor cells in cell transplant therapy for many patients.

Claims

1. A method for inducing differentiation of mammalian embryonic stem cells into functioning cells, which comprises the steps of:

1) culturing the mammalian embryonic stem cells together with feeder cells with a medium comprising leukemia inhibitory factor;

2) culturing the cells obtained from step

     1) in absence of feeder cells with a medium comprising leukemia Inhibitory factor and basic FGF in a suspension culture condition to give embryonic bodies;

3) culturing the obtained embryonic bodies with a selection-expanding medium; and

4) culturing the obtained cell clusters with a differentiation medium to give functioning cells.

2. The method of claim 1, wherein the medium used in step 2) comprises about 100-10000 U/ml of leukemia inhibitory factor.

3. The method of claim 1, wherein the medium used in step 2) comprises about 2-100 ng/ml of bFGF.

4. The method of claim 1, wherein the medium used in step 3) comprises nicotinamide, insulin and fibronectine in a serum-free cell culture medium.

5. The method of claim 1, wherein the functioning cells are insulin producing pancreatic islet-like cell clusters.

6. The method of claim 5 wherein the medium used in step 4) comprises nicotinamide, insulin and laminin in a serum-free cell culture medium.

7. The method of claim 1, wherein the functioning cells are nerve-like cells.

8. The method of claim 7 wherein the medium used in step 4) comprises L-lysine, insulin and laminin in a serum-free cell culture medium.

9. Functioning cells induced from mammalian ES cells by the method of claim 1.

10. Insulin secreting cell clusters induced from mammalian ES cells by the method of claim 5.

11. Nerve-like cells induced from mammalian ES cells by the method of claim 7.

12. A method for treating a mammalian patient having disorders in pancreatic islet function, which comprises implanting pancreatic islet-like cell clusters induced from allogenic ES cells by the method of claim 5 to the patient.

13. The method of claim 12, wherein the patient is a type I diabetic patient.

14. A method for treating a mammalian patient having disorders in nerve function, which comprises implanting nerve-like cells induced from allogenic ES cells by the method of claim 7 to the patient.