Differentiation of human induced pluripotent stem cells as a tool to study the effects of type 2 diabetes loci.

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

A 35-day protocol to reprogram human induced pluripotent stem cells (hiPSCs) into glucose-responsive pancreatic beta-like cells has been developed. Differentiating cells will be used to study the effects of the type 2 diabetes (T2D)-associated variants on beta cell development and insulin secretory function by using CRISPR/Cas9 technology to correct the mutation and observe whether a reversal in the phenotype is seen, in the subjects own genetic background. Following CRISPR/Cas9 editing, single cell cloning, expansion and verification of edit by Sanger sequencing will be conducted. Clonal cell lines will be expanded and characterized for pluripotency and ability to generate pancreatic progenitors using flow cytometry and a hPSC scorecard assay to verify the expression of appropriate markers. Cell lines will also analyzed for potential karyotypic abnormalities and off-target CRISPR effects. AT 7 time points reflecting different developmental stages during the differentiation procedure, cells will be collected for RNA analysis. In a prior genome wide association study (GWAS) for T2D in American Indians the top signal mapped to intron 15 of KCNQ1 which resides in a gene-rich region on chromosome 11. This region is highly imprinted (genes expressed solely from maternal or paternal allele) and several genes show tissue and developmental stage-specific imprinting. A previous study found that this T2D signal has a parent-of-origin effect, with increased T2D risk and, among normal glucose tolerant individuals a lower insulin secretion, when the risk allele is inherited maternally. The genes in this region include KCNQ1, CDKN1C, TRPM5, IGF2, TH, H19, INS and others known to be important for islet development and function and may contribute to diabetes. To identify the effector gene and causal SNPs at this locus, we utilized our induced pluripotent stem cells (iPSC) to analyze imprinting for KCNQ1, CDKN1C, TRPM5 and TH during different stages of differentiation. KCNQ1 had monoallelic expression during the iPSC stage (day 0), pancreatic progenitor stage (PP, day 10) and endocrine progenitor stage (day 13-16) while there was a gradual loss of imprinting thereafter. CDKN1C had monoallelic expression during all stages of differentiation. In contrast, TRPM5 started losing imprinting during the PP stage and we observed bi-allelic expression during later stages while TH had biallelic expression during all stages. RNA sequencing data from different stages of pancreatic islet development identified 20 genes in a 1.2MB region around the diabetes signal that are expressed during various stages of islet development. We have used CRISPR/CAS9 to generate isogenic iPSCs with hemizygous deletion in a region predicted to have regulatory function and has 4 SNPs, including the GWAS index SNP, by in-vitro assays and bio-informatic analysis. Differentiating this iPSC identified increased expression of CDKN1C during islet development compared to the parental cell line where the biggest difference (2 fold) was seen on Day 13 while no difference was seen for the other genes suggestive of a potential regulatory effect.