Stem cells for modeling type 2 diabetes

Patent metadata

Field	Value
Publication number	US-2016177273-A1
Application number	US-201514974785-A
Country	US
Kind code	A1
Filing date	Dec 18, 2015
Priority date	Dec 19, 2014
Publication date	Jun 23, 2016
Grant date	—

Abstract

The invention provides stem cell derived beta-pancreatic cells and animal models of T2D in which cells have been grafted. The stem cells bear a mutated form of SLC30A8 conferring protection or susceptibility to T2D. The cells and animal models can be used for drug screening as well as to provide insights into the mechanism of T2D and potentially new therapeutic and diagnostic targets.

First claim

What is claimed is:

1 . A human induced pluripotent stem cell (iPSC) comprising in its genome a first and a second allele of the SLC30A8 gene the same or different from each other.

2 . The iPSC of claim 1 , wherein the the first and the second allele are the same.

3 . The iPSC of claim 1 , wherein the the first and the second allele are different.

4 . The iPSC of claim 2 , wherein the first and the second allele encode arginine (R) at amino acid position 325 (R325).

5 . The iPSC of claim 2 , wherein the first and the second allele encode tryptophan (W) at amino acid position 325 (W325).

6 . The iPSC of claim 2 , wherein the first and the second allele encode a stop codon at amino acid position 138 (R138* stop ).

7 . The iPSC of claim 3 , wherein the first allele encodes arginine (R) at amino acid position 325 (R325) and the second allele encodes tryptophan (W) at amino acid position 325 (W325).

8 . The iPSC of claim 3 , wherein the first allele encodes arginine (R) at amino acid position 325 (R325) and the second allele encodes a stop codon at amino acid position 138 (R138* stop ).

9 . The iPSC of claim 1 as a clonal cell line.

10 . The iPSC of claim 1 generated by homologous recombination with a gene targeting construct bearing the mutation flanked by homology arms, wherein the recombination is enhanced by CRISPR/Cas9-mediated cleavage between segments of the genome corresponding to the homology arms.

11 . The iPSC of claim 10 , wherein the gene targeting construct is a single-stranded donor oligonucleotide.

12 . A cell culture comprising a population of cells according to claim 1 in culture medium maintaining the pluripotent state.

13 . The cell culture of claim 12 , wherein the culture medium comprises mTeSR1™ medium and the cells are cultured on a surface coating matrix.

14 . A method of modeling a beta-pancreatic cell susceptible for or protective against type II diabetes, comprising culturing a cell according to claim 1 under conditions promoting differentiation of the cell to a stem cell derived beta-pancreatic cell.

15 . The method of claim 14 , wherein the cell is cultured under successive conditions as follows:
RPMI medium supplemented by Activin A, CHIR99021, and B27; RPMI medium supplemented with activin A and B27; RPMI medium supplemented with FGF10, KAAD-cyclopamine, and B27; DMEM medium supplement with Noggin, KAAD-cyclopamine, retinoic acid, SB431542, and B27; CMRL medium supplemented with ALK5i, ILV, Noggin, and B27; and CMRL medium supplemented with excendin-4, nicotinamide, IBMX, FRKL, and B27.

16 . The method of claim 14 , wherein the differentiation goes through the following stages: definitive endoderm, primitive gut tube, posterior foregut, pancreatic endoderm, and endocrine.

17 . A cell produced by the method of claim 14 , characterized by one or more of the following properties:
expression of nuclear protein NKX6-1, PDX-1, zinc transporter 8, and/or urocortin-3; insulin packaged into secretory granules; glucose-responsive insulin secretion; glucose-sensitive calcium flux; and/or glucose-sensitive C-peptide secretion.

18 . The cell of claim 17 , wherein the cell secretes insulin responsive to successive glucose challenges.

19 . A cell culture comprising a population of cells of claim 17 and culture medium.

20 . The cell culture of claim 19 , wherein the culture medium comprises CMRL medium.

21 . A method of screening a compound for activity for treating type 2 diabetes comprising:
(a) contacting the stem cell derived beta-pancreatic cell of claim 17 with the compound; (b) determining glucose-induced insulin secretion relative to a control cell without the compound; and (c) selecting a compound wherein glucose-induced insulin secretion is changed relative to a control cell without the compound.

22 . A method of producing a mouse model of type 2 diabetes, comprising grafting a cell according to claim 17 into an immunodeficient mouse, wherein the grafted cell propagates, thereby forming a pancreatic tissue.

23 . The method of claim 22 , wherein the grafting comprises dissociating the cell from other cells in culture, treating the cell with a matrix promoting substance, and transplanting the treated cell into the mouse under the kidney capsule, wherein the cell propagates, thereby forming the pancreatic tissue.

24 . The method of claim 22 , wherein the mouse is a severe combined immunodeficient (SCID) mouse.

25 . The method of claim 22 , wherein the mouse is a non-obese diabetic (NOD) mouse.

26 . A method of screening a compound, comprising:
(a) contacting a mouse produced by the method of claim 22 with the compound; (b) determining a level of insulin or glucose relative to a control mouse not treated with the compound; and (c) selecting a compound that changes the level of insulin or glucose relative to a control mouse not treated with the compound.

Assignees

• Regeneron Pharma

Inventors

• Gong Guochun
• Lai Ka-Man Venus
• Valenzuela David M

Classifications

• A61K35/39

  Pancreas; Islets of Langerhans (Langerhans cells of epidermis A61K35/36) · CPC title

• A61K35/545

  Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells · CPC title

• A01K67/0271

  Chimeric vertebrates, e.g. comprising exogenous cells · CPC title

• A01K2267/0362

  Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes · CPC title

• A01K2207/12

  Animals modified by administration of exogenous cells · CPC title