Methods for generating stem cell-derived beta cells and methods of use thereof

• US 10,253,298 B2
• Filed: 12/18/2015
• Issued: 04/09/2019
• Est. Priority Date: 12/18/2014
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Melton, Douglas A., Gurtler, Mads
• Primary Examiner(s): Bertoglio, Valarie E
• Application Number:
  US14/975,457
• Publication Number:
  US 20160177269A1
• Time in Patent Office: 1,208 Days
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: G01N 33/507 Pancreatic cells

Abstract

Disclosed herein are methods for generating SC-β cells, and isolated populations of SC-β cells for use in various applications, such as cell therapy.

Claims

1. A method comprising contacting a cell population comprising PDX1+ pancreatic progenitor cells that do not express NKX6.1 with a culture medium comprising rho-associated protein kinase (Rock) inhibitor and Activin A, wherein the contacting results in one or more cells in the cell population differentiating to PDX1+ pancreatic progenitor cells that express NKX6.1.

2. The method of claim 1, further comprising washing the cell population after the PDX1+ pancreatic progenitor cells that do not express NKX6.1 have differentiated to PDX1+ pancreatic progenitor cells that express NKX6.1, wherein said washing is effective to remove Activin A from contact with the cell population.

3. The method of claim 1, wherein the Rock inhibitor comprises Y-27632.

4. The method of claim 1, wherein the culture medium comprises 10 μ

M of the Rock inhibitor.

5. The method of claim 1, wherein the culture medium comprises 5 ng/ml of Activin A.

6. The method of claim 1, wherein the cell population comprising PDX1+ pancreatic progenitor cells that do not express NKX6.1 is exposed to the Rock inhibitor and Activin A for a period of 6 days.

7. The method of claim 1, wherein the method further i) prevents or minimizes cell cluster disintegration, ii) promotes cell survival, iii) increases number of PDX1+ pancreatic progenitor cells that express NKX6.1 in resulting cell population, and combinations thereof.

8. A method comprising contacting a cell population comprising PDX1+, NKX6.1+ pancreatic progenitor cells with a culture medium comprising rho-associated protein kinase (Rock) inhibitor and staurosporine, wherein the contacting results in one or more cells in the cell population differentiating to chromogranin A, NKX6.1+ endocrine progenitor cells.

9. The method of claim 8, further comprising differentiating the chromogranin A, NKX6.1+ endocrine progenitor cells to SC-β

cells, wherein the SC-β

cells exhibit co-expression of NKX6.1 and C-peptide.

10. The method of claim 8, wherein the culture medium comprises 3 nM of staurosporine.

11. The method of claim 8, wherein between at least 75% and 80% of the cell population differentiates to chromogranin A, NKX6.1+ endocrine progenitor cells before differentiating to SC-β

cells.