PLURIPOTENT STEM CELL DIFFERENTIATION

• US 20100028307A1
• Filed: 07/31/2008
• Published: 02/04/2010
• Est. Priority Date: 07/31/2008
• Status: Abandoned Application

• Current Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Original Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Inventors: O'Neil, John J.
• Application Number:
  US12/183,557
• Publication Number:
  US 20100028307A1
• Time in Patent Office: Days
• US Class Current: 424/93.700
• 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 3/10 for hyperglycaemia, e.g. antidiabetics
• 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 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2502/22 pancreatic cells
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention relates to the field of pluripotent stem cell differentiation. The present invention provides methods for the differentiation of pluripotent stem cells on a human feeder cell layer. In particular, the present invention provides an improved method for the differentiation of pluripotent stem cells into pancreatic endocrine cells using a human feeder cell layer.

Claims

1. A method for differentiating pluripotent stem cells, comprising the steps of:

a. Culturing the pluripotent stem cells,

b. Plating the pluripotent stem cells onto a human feeder cell layer, and

c. Treating the pluripotent stem cells with at least one factor that promotes the differentiation of the pluripotent stem cells.

2. The method of claim 1, wherein the human feeder cell layer comprises dermal fibroblast cells.

3. The method of claim 1, wherein the human feeder cell layer comprises foreskin fibroblast cells.

4. The method of claim 1, wherein the human feeder cell layer comprises pancreatic-derived stromal cells.

5. The method of claim 1, wherein the pluripotent stem cells are embryonic stem cells.

6. The method of claim 5, wherein the embryonic stem cells are human embryonic stem cells.

7. The method of claim 4, wherein the pancreatic-derived stromal cells are substantially negative in the expression of at least one protein marker selected from the group consisting of NCAM, ABCG2, cytokeratin 7, cytokeratin 8, cytokeratin 18, and cytokeratin 19.

8. The method of claim 4, wherein the pancreatic-derived stromal cells are substantially positive in the expression of at least one protein marker selected from the group consisting of CD44, CD73, CD90 and CD105.

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

10. The method of claim 1, wherein the pluripotent stem cells are differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage.

11. The method of claim 1, wherein the pluripotent stem cells are differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage.

12. The method of claim 1, wherein the step of culturing the pluripotent stem cells is accomplished on an extracellular matrix.

13. The method of claim 1, wherein the step of culturing the pluripotent stem cells is accomplished on a human feeder cell layer.

14. A method to treat diabetes, comprising the steps of:

a. Culturing the pluripotent stem cells,

b. Plating the pluripotent stem cells onto a human feeder cell layer,

c. Treating the pluripotent stem cells with at least one factor that promotes the differentiation of the pluripotent stem cells, and

d. Transplanting pluripotent stem cells that have been treated with at least one factor that promotes the differentiation of pluripotent stem cells into a human patient having diabetes.

15. The method of claim 14, wherein the step of treating the pluripotent stem cells with at least one factor that promotes the differentiation of the pluripotent stem cells causes the pluripotent stem cells to differentiate into cells of the pancreatic endocrine lineage.

16. The method of claim 15, wherein the differentiation of the pluripotent stem cells is accomplished in two or more steps, each step requiring the treatment of cells by at least one factor that promotes the differentiation of cells into one or more of definitive endoderm, pancreatic endoderm, and pancreatic endocrine lineage.