Stem cells for modeling type 2 diabetes

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.

1 .- 13 . (canceled) 
     
     
         14 . A method of modeling a beta-pancreatic cell, comprising culturing a population of human induced pluripotent stem cells (hiPSCs) under conditions promoting differentiation of the cell to a stem cell derived beta-pancreatic cell wherein each cell comprises in its genome a heterozygous or homozygous R325W allele of the human SLC30A8 gene or each cell comprises in its genome a heterozygous or homozygous R138 *   stop  allele of the human SLC30A8 gene. 
     
     
         15 . The method of  claim 14 , wherein the cells are 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  that 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 diabetes type 2 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 diabetes type 2, comprising grafting a cell according to  claim 17  into an immunodeficient mouse, wherein cell propagates forming beta-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 forming 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 wherein the level of insulin or glucose is changed relative to a control mouse not treated with the compound.   
     
     
         27 . The method of  claim 14 , wherein each cell comprises in its genome a heterozygous or homozygous R325W allele of the human SLC30A8 gene. 
     
     
         28 . The method of  claim 27 , wherein each cell comprises in its genome a homozygous R325W allele of the human SLC30A8 gene. 
     
     
         29 . The method of  claim 27 , wherein each cell comprises in its genome a heterozygous R325W allele of the human SLC30A8 gene. 
     
     
         30 . The method of  claim 27 , wherein the hiPSCs are a clonal cell line. 
     
     
         31 . The method of  claim 27 , wherein the hiPSCs are generated by homologous recombination with a gene targeting construct comprising a sequence encoding R325W, wherein the sequence is flanked by homology arms, and wherein the recombination is enhanced by CRISPR/Cas9-mediated cleavage between segments of the endogenous hiPSC genome homologous to the homology arms. 
     
     
         32 . The method of  claim 31 , wherein the gene targeting construct is a single-stranded donor oligonucleotide. 
     
     
         33 . The method of  claim 14 , wherein each cell comprises in its genome a heterozygous or homozygous R138* stop  allele of the human SLC30A8 gene. 
     
     
         34 . The method of  claim 33 , wherein each cell comprises in its genome a homozygous R138* stop  allele of the human SLC30A8 gene. 
     
     
         35 . The method of  claim 33 , wherein each cell comprises in its genome a heterozygous R138* stop  allele of the human SLC30A8 gene. 
     
     
         36 . The method of  claim 33 , wherein the hiPSCs are a clonal cell line. 
     
     
         37 . The method of  claim 33 , wherein the hiPSCs are generated by homologous recombination with a gene targeting construct comprising a sequence encoding R138* stop , wherein the sequence is flanked by homology arms, and wherein the recombination is enhanced by CRISPR/Cas9-mediated cleavage between segments of the endogenous hiPSC genome homologous to the homology arms. 
     
     
         38 . The method of  claim 37 , wherein the gene targeting construct is a single-stranded donor oligonucleotide.