Differentiation of stem cells to endoderm and pancreatic lineage

• US 20050260749A1
• Filed: 03/31/2005
• Published: 11/24/2005
• Est. Priority Date: 04/01/2004
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Odorico, Jon, Treff, Nathan, Kahan, Brenda
• Granted Patent: US 7,585,672 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61P 5/48 of the pancreatic hormones
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

Methods are described to increase the proportion of endoderm committed cells and pancreatic lineage cells in a culture of human embryonic stem cells which are undergoing differentiation. The method also results in a stem cell derived cell culture which does not have tumorigenic capability.

Claims

1. A method to enrich a culture derived from stem cells for cells of endoderm and pancreatic lineages, the method comprising the steps of

culturing stem cells into the formation of embryoid bodies;

and

selecting among embryoid bodies for the expression of the species appropriate cell surface stage-specific embryonic and culturing only the embryoid bodies which do not express cell surface stage-specific antigen for differentiation into endoderm and pancreatic cells.

2. A method as claimed in claim 1 wherein the stem cells are human embryonic stem cells and the species appropriate surface antigen is stage specific embryonic antigen-1.

3. A method as claimed in claim 2 further comprising selecting against the expression of SSEA-1 and for the expression of epithelial cell surface antigen (EpCAM).

4. A method as claimed in claim 3 wherein the selecting is performed by magnetic activated cell sorting.

5. A method to enrich a culture derived from human embryonic stem cells into cells of endoderm and pancreatic lineages, the method comprising the steps of

culturing human embryonic stem cells under conditions which permit the stem cells to begin differentiation;

and

supplementing the embryonic stem cell culture with fibroblast growth factor 10 (FGF10).

6. A method as claimed in claim 5 further comprising the steps of selecting against the expression of SSEA-1, selecting against the expression of SSEA-3, and selecting for the expression of epithelial cell surface antigen (EpCAM).

7. A method as claimed in claim 6 wherein the selecting is performed by magnetic activated cell sorting.

8. A method to enrich a culture derived from stem cells into cells of endoderm and pancreatic lineages, the method comprising the steps of

culturing stem cells under conditions which permit the stem cells to begin differentiation;

and

selecting the stem cells for lack of expression of stage specific embryonic antigen-1 (SSEA-1), for lack for expression of stage specific embryonic antigen-3 (SSEA-3), and for expression of epithelial cell surface antigen (EpCAM).

9. A method as claimed in claim 8 wherein the selection is performed by magnetic activated cell sorting.

10. A method as claimed in claim 8 wherein the stem cells are human embryonic stem cells.

11. A method as claimed in claim 8 wherein the step of culturing the stem cells which permits the stem cells to begin differentiation includes culturing the stem cells to encourage the formation of embryoid bodies.

12. A method for the creation of a stem cell derived cell population which does not have tumorigenic capability comprising the steps of

culturing stem cells under conditions which permit the stem cells to begin differentiation;

and

selecting the stem cells for lack of expression of cell surface markers associated with an undifferentiated state, and for expression of a cell surface marker indicative of a commitment to a particular differentiated lineage, the resulting cell culture not forming teratomas when injected in immunocompromised mice.

13. A method as claimed in claim 12 wherein the stem cells are human embryonic stem cells.

14. A method as claimed in claim 13 wherein the cell surface markers associated with an undifferentiated state are SSEA-1 and SSEA-3.

15. A method as claimed in claim 13 wherein the cell surface marker indicative of a commitment to a particular differentiated lineage is epithelial cell surface antigen (EpCAM).

16. A method as claimed in claim 13 wherein the selecting is performed by magnetic activated cell sorting.