Production of insulin producing cells

• US 9,085,757 B2
• Filed: 06/17/2011
• Issued: 07/21/2015
• Est. Priority Date: 06/17/2010
• Status: Active Grant

• Current Assignee: Regents of The University of Minnesota (University of Minnesota)
• Original Assignee: Regents of The University of Minnesota (University of Minnesota)
• Inventors: Firpo, Meri, Geng, Zhaohui
• Primary Examiner(s): Shen, Bin
• Application Number:
  US13/702,031
• Publication Number:
  US 20130243735A1
• Time in Patent Office: 1,495 Days
• Field of Search: 435/377
• US Class Current: 1/1
• 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: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• 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 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/91 Heparin
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells

Abstract

The invention provides methods for differentiating stem cells along the pancreatic lineage as well as large scale culture methods. The present invention further provides pancreatic progenitor cells derived from stem cells to provide pancreatic cells to a subject.

Claims

1. An in vitro method to differentiate stem cells to pancreatic progenitor cells comprising the steps of:

a) contacting the stem cells with at least one member of the TGFβ

family of cytokines and at least one member of the Wnt family of proteins,

b) contacting the cells obtained from step a) with at least one member of the TGFβ

family of cytokines, at least one member of the Wnt family of proteins, and an agent that inhibits sonic hedgehog activity; and

c) contacting the cells obtained from step b) with a member of the Epidermal growth factor (EGF) family of proteins;

so as to yield pancreatic progenitor cells.

2. The method of claim 1, wherein the at least one member of the TGFβ

family of cytokines is activinA or nodal.

3. The method of claim 1, wherein the at least one member of the Wnt family is Wnt 3 or Wnt 3A.

4. The method of claim 1, wherein the at least one member of the EGF family is EGF.

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

6. The method of claim 1, wherein the stem cells are induced pluripotent stem (iPS) cells.

7. The method of claim 1 further comprising contacting the cells obtained from step c) with at least one member of the TGFβ

family of cytokines, betacellulin, exendin4 or a combination thereof to yield cells expressing insulin.

8. The method of claim 7, wherein the at least one member of the TGFβ

family of cytokines is GDF-11.

9. The method of claim 7, wherein the cells expressing insulin or having increased expression of insulin secrete insulin, c-peptide or a combination thereof.

10. The method of claim 7, wherein the insulin is insulin-1.

11. The method of claim 1, wherein the agent that inhibits sonic hedgehog activity is cyclopamine or an anti-SHH antibody.

12. The method of claim 1, wherein the stem cells are mammalian cells.

13. The method of claim 12, wherein the mammalian cells are human cells.

14. The method of claim 1, wherein the differentiation occurs in a cell culture dish.

15. The method of claim 1, wherein the differentiation occurs in bioreactor.

16. An in vitro method to differentiate stem cells to pancreatic progenitor cells comprising the steps of:

a) contacting the stem cells with Activin A and Wnt3 a,

b) contacting the cells obtained from step a) with Activin-A, Wnt3 a, and an agent that inhibits sonic hedgehog activity; and

c) contacting the cells obtained from step b) with EGF;

so as to yield pancreatic progenitor cells.