CELL COMPOSITIONS DERIVED FROM DEDIFFERENTIATED REPROGRAMMED CELLS

• US 20100272695A1
• Filed: 04/22/2010
• Published: 10/28/2010
• Est. Priority Date: 04/22/2009
• Status: Abandoned Application

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: D'Amour, Kevin Allen, Agulnick, Alan
• Application Number:
  US12/765,714
• Publication Number:
  US 20100272695A1
• Time in Patent Office: Days
• US Class Current: 424/93.700
• 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 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• 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 2501/70 Enzymes
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• 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

Methods and cell culture compositions, in particular, pancreatic cell culture methods and compositions, derived from dedifferentiated human reprogrammed pluripotent stem cells, such as, induced pluripotent stem (iPS) cells.

Claims

1. An in vitro cell culture comprising differentiated cells derived from dedifferentiated genetically reprogrammed cells and a Rho-kinase inhibitor.

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

(a) contacting human reprogrammed pluripotent stem cells in vitro with a first medium comprising a first agent that activates a TGFβ

receptor family member and a second agent that promotes cell-cell adhesion of the cells;

(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 PDX-1 positive pancreatic endoderm cells;

(c) transplanting the PDX-1 positive pancreatic endoderm cells of step (b) into a mammalian subject; and

(d) maturing the PDX-1 positive pancreatic endoderm cells of step (c) in vivo, thereby obtaining insulin secreting cells, wherein the insulin secreting cells secrete insulin in response to glucose stimulation.

3. The method of claim 2, wherein the first agent comprises a TGFβ

growth factor super family.

4. The method of claim 2, wherein the second agent comprises a Rho-kinase inhibitor.

5. The method of claim 4, 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 and expression vectors thereof.

6. The method of claim 5, wherein the Rho-kinase inhibitor is selected from the group consisting of Y-27632, Fasudil, H-1152P, Wf-536 and Y-30141 and derivatives thereof.

7. The method of claim 6, wherein the Rho-kinase inhibitor is selected from the group consisting of Y-27632, Fasudil and H-1152P, and derivatives thereof.

8. An in vitro cell culture comprising human cells wherein at least about 15% of the human cells are definitive endoderm cells, wherein the definitive cells are derived from dedifferentiated genetically reprogrammed cells, wherein the definitive endoderm cells are multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom.

9. An in vitro cell culture comprising human cells wherein at least about 15% of the human cells are pancreatic-duodenal homeobox factor-1 (PDX1) positive foregut endoderm cells, wherein the PDX1 positive foregut endoderm cells are derived from dedifferentiated genetically reprogrammed cells, wherein the PDX1 positive foregut endoderm cells are PDX1, SOX9, PROX1 and HNF6 co-positive.

10. An in vitro cell culture comprising human cells wherein at least about 15% of the human cells are pancreatic-duodenal homeobox factor-1 (PDX1) positive pancreatic progenitor cells, and the PDX1 positive pancreatic progenitor cells are derived from dedifferentiated genetically reprogrammed cells, wherein the PDX1 positive pancreatic progenitor cells are PDX1 and NKX6.1 co-positive.

11. An in vitro cell culture comprising human cells wherein at least about 15% of the human cells are Neurogenin 3 (NGN3) positive endocrine precursor cells, wherein the NGN3 endocrine precursor cells are derived from dedifferentiated genetically reprogrammed cells, and wherein the cells being NGN3, PAX4 and NKX2.2 co-positive.

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

(a) contacting human reprogrammed pluripotent stem 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 PDX-1 positive pancreatic endoderm cells;

(c) transplanting the PDX-1 positive pancreatic endoderm cells of step (b) into a mammalian subject; and

(d) maturing the PDX-1 positive pancreatic endoderm cells of step (c) in vivo, thereby obtaining insulin secreting cells, wherein the insulin secreting cells secrete insulin in response to glucose stimulation.

14. The method of claim 12, wherein the agent that activates a TGFβ

receptor family member comprises a TGFβ

growth factor super family.

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

(a) contacting human reprogrammed pluripotent stem cells in vitro with a first medium comprising an agent that activates a TGFβ

receptor family member and an agent that promotes proliferation of the cells;

(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 PDX-1 positive pancreatic endoderm cells;

(c) transplanting the PDX-1 positive pancreatic endoderm cells of step (b) into a mammalian subject; and

(d) maturing the PDX-1 positive pancreatic endoderm cells of step (c) in vivo, thereby obtaining insulin secreting cells, wherein the insulin secreting cells secrete insulin in response to glucose stimulation.