CELL COMPOSITIONS DERIVED FROM DEDIFFERENTIATED REPROGRAMMED CELLS

• US 20130164787A1
• Filed: 02/06/2013
• Published: 06/27/2013
• Est. Priority Date: 04/22/2009
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: Agulnick, Alan D., Kelly, Olivia, Robins, Allan, Ohi, Yuki, Schultz, Thomas
• Granted Patent: US 9,109,245 B2
• Time in Patent Office: Days
• US Class Current: 435/71.1
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• 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 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/195 Heregulin, neu differentiation factor
• 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 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• 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

Abstract

Disclosed herein are cell culture compositions, for example, pancreatic cell culture compositions, derived from dedifferentiated human reprogrammed pluripotent stem cells, such as induced pluripotent stem (iPS) cells, and methods for producing and using such cell culture compositions.

Claims

1. An in vitro human pancreatic endoderm cell population comprising differentiated cells derived from dedifferentiated genetically reprogrammed cells and an ERBB receptor tyrosine kinase activating agent.

2. The pancreatic endoderm cell population of claim 1, wherein the ERRB receptor tyrosine kinase activating agent comprises an EGF growth factor or ligand.

3. The pancreatic endoderm cell population of claim 2, wherein the EGF growth factor or ligand comprises a heregulin isoform selected from a group consisting of heregulin-1, heregulin-2, and heregulin-3 and heregulin-4.

4. The pancreatic endoderm cell population of claim 3, wherein the heregulin isoform comprises heregulin-1 and heregulin-4.

5. The pancreatic endoderm cell population of claim 3, wherein ligand the heregulin isoform comprises heregulin-4.

6. A method for producing insulin, said method comprising the steps of:

a. contacting a foregut endoderm cell culture derived from dedifferentiated genetically reprogrammed cells in vitro with an ERBB receptor tyrosine kinase activating agent, thereby producing a cell population comprising endocrine cell and non-endocrine cell subpopulations; and

b. maturing the subpopulations of step (a) in vivo, thereby obtaining insulin secreting cells, wherein the insulin secreting cells secrete insulin in response to glucose stimulation.

7. The method of claim 6 further comprising contacting the foregut endoderm cell culture with a rho-kinase inhibitor.

8. The method of claim 7, wherein the rho-kinase inhibitor is selected from the group consisting of Y-27632, Fasudil, H-1152P, Wf-536, Y-30141, antisense nucleic acids for ROCK, RNA interference inducing nucleic acids, competitive peptides, antagonist peptides, inhibitory antibodies, antibody-ScFV fragments, dominant negative variants, derivatives thereof and expression vectors thereof.

9. The method of claim 7, wherein the rho-kinase inhibitor is selected from the group consisting of Y-27632, Fasudil, H-1152P, Wf-536, Y-30141 and derivatives thereof.

10. The method of claim 9, wherein the Rho-kinase inhibitor is selected from the group consisting of Y-27632, Fasudil, H-1152P and derivatives thereof.

11. A method for producing insulin, said method comprising the steps of:

a. contacting dedifferentiated genetically reprogrammed cells in vitro with a first medium comprising an agent that activates a TGFβ

receptor family member;

b. culturing, in vitro, the cells of step (a) in a second medium lacking the agent that activates a TGFβ

receptor family member, thereby generating foregut endoderm cells;

c. contacting the foregut endoderm cells of step (b) with an ERBB receptor tyrosine kinase activating agent, thereby generating a cell population comprising endocrine cell and non-endocrine cell subpopulations; and

d. maturing the cell subpopulations of step (c) in vivo, thereby obtaining insulin secreting cells, wherein the insulin secreting cells secrete insulin in response to glucose stimulation.

12. The method of claim 11, wherein the non-endocrine cells are CHGA negative (CHGA−

) cells.

13. The method of claim 11, wherein the endocrine cells are CHGA positive (CHGA+) cells.

14. The method of claim 11, wherein the CHGA negative (CHGA−

) cells further expresses NKX6.1.

15. The method of claim 11 further comprising contacting the foregut endoderm cells with a rho-kinase inhibitor.

16. The method of claim 15, wherein the rho-kinase inhibitor is selected from the group consisting of Y-27632, Fasudil, H-1152P, Wf-536, Y-30141, antisense nucleic acids for ROCK, RNA interference inducing nucleic acid, competitive peptides, antagonist peptides, inhibitory antibodies, antibody-ScFV fragments, dominant negative variants, derivatives thereof and expression vectors thereof.