Treatment of pluripotent cells

• US 9,845,460 B2
• Filed: 11/20/2013
• Issued: 12/19/2017
• Est. Priority Date: 04/24/2008
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Davis, Janet, Liu, Jiajian
• Primary Examiner(s): Chen, Shin Lin
• Application Number:
  US14/085,068
• Publication Number:
  US 20140080210A1
• Time in Patent Office: 1,490 Days
• Field of Search: 435366, 435377, 514 1
• CPC Class Codes: A61K 31/00 Medicinal preparations containing organic active ingredients
• 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 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• 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 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/48 Regulators of apoptosis
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2506/00 Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
• CPC Class Codes: C12N 2506/02 from embryonic cells
• 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
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention is provides a method for treating human pluripotent cells. In particular, the methods of the invention are directed to the treatment of human pluripotent cells, whereby the human pluripotent cells can be efficiently expanded in culture and differentiated by treating the pluripotent cells with an inhibitor of glycogen synthase kinase 3β (GSK-3B) enzyme activity.

Claims

1. A method to expand and differentiate human embryonic stem cells, comprising the steps of:

a. Culturing human embryonic stem cells, and

b. Treating the human embryonic stem cells with an inhibitor of glycogen synthase kinase 3β

(GSK-3B) enzyme activity, wherein the inhibitor is 3-[1-(3-hydroxy-propyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione.

2. The method of claim 1, wherein the human embryonic stem cells express at least one of the following pluripotency markers:

ATP-binding cassette, subfamily G, member 2 (ABCG2), crypto, forkhead box D3 (FoxD3), Connexin43, Connexin45, POU domain transcription factor Oct4, sex-determining region Y-box2 (SOX-2), Nanog, human telomerase reverse transcriptase (hTERT), undifferentiated embryonic cell transcription factor 1 (UTF-1), zinc finger protein 42 (ZFP42), stage-specific embryonic antigen 3 (SSEA-3), stage-specific embryonic antigen 4 (SSEA-4), tumor-related antigen 1-60 (Tra1-60), and tumor-related antigen 1-81 (Tra1-81).

3. The method of claim 1, wherein the human embryonic stem cells are differentiated into cells expressing markers characteristic of the definitive endoderm lineage.

4. The method of claim 1, wherein the human embryonic stem cells are treated with the inhibitor of GSK-3B enzyme activity for about one to about 72 hours.

5. The method of claim 1, wherein the human embryonic stem cells are treated with the inhibitor of GSK-3B enzyme activity for about 12 to about 48 hours.

6. The method of claim 1, wherein the human embryonic stem cells are treated with the inhibitor of GSK-3B enzyme activity for about 48 hours.

7. The method of claim 1, wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 100 nM to about 10004.

8. The method of claim 1, wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 1 μ

M to about 10 μ

M.

9. The method of claim 1, wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 10 μ

M.