Differentiation of Human Embryonic Stem Cells

• US 20120052575A1
• Filed: 08/17/2011
• Published: 03/01/2012
• Est. Priority Date: 08/31/2010
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Rezania, Alireza
• Granted Patent: US 9,528,090 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• 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 2501/19 Growth and differentiation factors [GDF]
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0608 Germ cells production of embryos, nuclear transfer A01K67/027
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells

Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells into insulin producing cells. In particular, the present invention provides a method to produce a population of cells, wherein greater than 85% of the cells in the population express markers characteristic of the definitive endoderm lineage.

Claims

1. A population of cells, wherein greater than 85% of the cells in the population express markers characteristic of the definitive endoderm lineage.

2. A method for generating a population of cells wherein greater than 85% of the cells in the population express markers characteristic of the definitive endoderm lineage, comprising the steps of:

a. Culturing a population of pluripotent stem cells,

b. Differentiating the population of pluripotent stem cells to a population of cells wherein greater than 85% of the cells in the population express markers characteristic of the definitive endoderm lineage in medium lacking serum and supplemented with BSA and a factor selected from the group consisting of insulin and IGF-1.

3. The method of claim 2, wherein the population of pluripotent stem cells are differentiated to a population of cells wherein greater than 85% of the cells in the population express markers characteristic of the definitive endoderm lineage using activin A and a Wnt ligand.

4. The method of claim 2, wherein the population of pluripotent stem cells are differentiated to a population of cells wherein greater than 85% of the cells in the population express markers characteristic of the definitive endoderm lineage using GDF-8 and at least one other factor is selected from the group consisting of:

an aniline-pyridinotriazine, a cyclic aniline-pyridinotriazine, N-{[1-(Phenylmethyl)azepan-4-yl]methyl}-2-pyridin-3-ylacetamide, 4-{[4-(4-{[2-(Pyridin-2-ylamino)ethyl]amino}-1,3,5-triazin-2-yl)pyridin-2-yl]oxy}butan-1-ol, 3-({3-[4-({2-[Methyl(pyridin-2-yl)amino]ethyl}amino)-1,3,5-triazin-2-yl]pyridin-2-yl}amino)propan-1-ol, N˜

4˜

-[2-(3-Fluorophenyl)ethyl]-N˜

2˜

-[3-(4-methylpiperazin-1-yl)propyl]pyrido[2,3-d]pyrimidine-2,4-diamine, 1-Methyl-N-[(4-pyridin-3-yl-2-{[3-(trifluoromethyl)phenyl]amino}-1,3-thiazol-5-yl)methyl]piperidine-4-carboxamide, 1,1-Dimethylethyl{2-[4-({5-[3-(3-hydroxypropyl)phenyl]-4H-1,2,4-triazol-3-yl}amino)phenyl]ethyl}carbamate, 1,1-Dimethylethyl{[3-({5-[5-(3-hydroxypropyl)-2-(methyloxy)phenyl]-1,3-oxazol-2-yl}amino)phenyl]methyl}carbamate, 1-({5-[6-({4-[(4-Methylpiperazin-1-yl)sulfonyl]phenyl}amino)pyrazin-2-yl]thiophen-2-yl}methyl)piperidin-4-ol, 1-({4-[6-({4-[(4-Methylpiperazin-1-yl)sulfonyl]phenyl}amino)pyrazin-2-yl]thiophen-2-yl}methyl)piperidine-4-carboxamide, and 2-{[4-(1-Methylethyl)phenyl]amino}-N-(2-thiophen-2-ylethyl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxamide.

5. The method of claim 4, wherein the at least one other factor is 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

0.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one.

6. The method of claim 2, wherein the population of pluripotent stem cells is differentiated in the medium lacking serum and supplemented with BSA and a factor selected from the group consisting of insulin and IGF-1 for a period of at least 6 days.

7. The method of claim 2, wherein the population of pluripotent stem cells is differentiated in the medium lacking serum and supplemented with BSA and a factor selected from the group consisting of insulin and IGF-1 for a period of at least 7 days.