In vitro differentiation of pluripotent stem cells to pancreatic endoderm cells (PEC) and endocrine cells

• US 8,859,286 B2
• Filed: 12/13/2013
• Issued: 10/14/2014
• Est. Priority Date: 03/14/2013
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: Agulnick, Alan D.
• Primary Examiner(s): FOX, ALLISON M
• Application Number:
  US14/106,330
• Publication Number:
  US 20140271566A1
• Time in Patent Office: 305 Days
• US Class Current: 435/377
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• 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 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• 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 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• 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 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0012 : Cell encapsulation
• CPC Class Codes: C12N 5/0676 : Pancreatic cells
• CPC Class Codes: C12N 5/0678 : Stem cells; Progenitor cells; Precursor cells

Abstract

A human immature endocrine cell population and methods for making an immature endocrine cell population are provided. Specifically, immature beta cells and methods for production of immature beta cells are described. Immature beta cells co-express INS and NKX6.1 and are uni-potent and thereby develop into mature beta cells when implanted in vivo. The mature beta cells in vivo are capable of producing insulin in response to glucose stimulation.

Claims

1. A method of producing Chromogranin A (CHGA)-negative cells comprising, contacting human definitive endoderm lineage cells in vitro with a TGFβ

superfamily member and a Wnt family member thereby producing CHGA-negative cells.

2. The method of claim 1, wherein the TGFβ

superfamily member is Activin A.

3. The method of claim 1, wherein the Wnt family member is Wnt3a.

4. The method of claim 1, further comprising contacting the human definitive endoderm lineage cells in vitro with an ERBB receptor kinase activating agent.

5. The method of claim 4, wherein the ERBB receptor kinase activating agent is Heregulin.

6. The method of claim 1, further comprising allowing the CHGA-negative cells to form unipotent immature beta cells.

7. The method of claim 6, wherein said unipotent human immature beta cell expresses INS and NKX6.1.

8. A method for producing mature beta cells in vivo, said method comprising:

a) contacting human definitive endoderm lineage cells in vitro with a TGFβ

superfamily member and a Wnt family member, thereby producing CHGA-negative cells;

b) differentiating the CHGA-negative cells into immature beta cells;

c) transplanting the immature beta cells of step (b) into a mammalian subject;

d) allowing the immature beta cells to mature in vivo to produce a population of cells comprising insulin secreting beta cells.

9. The method of claim 8, wherein the TGFβ

superfamily member is Activin A.

10. The method of claim 8, wherein the human definitive endoderm lineage cells are contacted with at least 50 ng/mL of the TGFβ

superfamily member.

11. The method of claim 8, wherein the Wnt family member is Wnt3a.

12. The method of claim 8, wherein the human definitive endoderm lineage cells are contacted with at least 25 ng/mL of the Wnt family member.

13. The method of claim 8, further comprising contacting the human definitive endoderm lineage cells in vitro with an ERBB receptor kinase activating agent.

14. The method of claim 13, wherein the ERBB receptor kinase activating agent is Heregulin.

15. The method of claim 13, wherein the human definitive endoderm lineage cells are contacted with 5-15 fold less of the ERBB receptor kinase activating agent than the TGFβ

superfamily member and the Wnt family member.

16. The method of claim 8, wherein the immature beta cells express INS and NKX6.1.

17. The method of claim 8, wherein the immature beta cells express INS, NKX6.1 and MAFB.

18. A method of producing unipotent human immature beta cells comprising:

a) contacting human definitive endoderm lineage cells in vitro with Activin A and Wnt3a, thereby producing CHGA-negative cells, and then

b) differentiating the CHGA-negative cells into unipotent human immature beta cells.

19. The method of claim 18, wherein said unipotent human immature beta cells express INS and NKX6.1.

20. The method of claim 18, wherein step (a) further comprises contacting the human endoderm lineage cells in vitro with Heregulin.