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

已经开发了将人诱导多能干细胞（hiPSC）重编程为葡萄糖响应性胰腺β样细胞的35天方案。分化细胞被用于研究2型糖尿病（T2 D）相关变体对β细胞发育和胰岛素分泌功能的影响，方法是使用CRISPR/Cas9技术纠正突变，并观察受试者自身的遗传背景中是否出现表型逆转。在CRISPR/Cas9编辑之后，进行单细胞克隆、扩增和通过桑格测序验证编辑，并且扩增克隆细胞系并使用流式细胞术和hPSC记分卡测定来表征多能性和产生胰腺祖细胞的能力以验证适当标志物的表达。还分析了细胞系的潜在核型异常和脱靶CRISPR效应。在反映分化过程中不同发育阶段的7个时间点，收集细胞用于RNA分析。 在美国西南部印第安人中，与2型糖尿病（T2 D）的最强关联映射到KCNQ 1的内含子15，其中前导SNP（rs 2299620）位于强连锁不平衡（LD）区域。该区域是高度印记的（基因仅从母本或父本等位基因表达），并且一些基因显示组织和发育阶段特异性印记。先前的一项研究发现，这种T2 D信号具有父母的起源效应，增加T2 D风险，并且在正常葡萄糖耐受个体中，当风险等位基因是母系遗传时，胰岛素分泌较低。为了确定该位点的效应基因和因果SNP，我们利用我们的诱导多能干细胞（iPSC）分析KCNQ 1，CDKN 1C，TRPM 5和TH在不同分化阶段的印记。KCNQ 1在iPSC阶段（第0天）、胰腺祖细胞阶段（PP，第10天）和内分泌祖细胞阶段（第13-16天）具有单等位基因表达，而此后印记逐渐丧失。CDKN 1C在所有分化阶段都有单等位基因表达。相比之下，TRPM 5在PP阶段开始失去印记，我们在后期阶段观察到双等位基因表达，而TH在所有阶段都有双等位基因表达。来自胰岛发育不同阶段的RNA测序数据在糖尿病信号周围的1.2MB区域中鉴定了20个基因，这些基因在胰岛发育的不同阶段表达。 我们目前认为我们已经将KCNQ 1的功能区定位到了一个包含rs 2299620和3个额外SNP的400 bp片段上。为了证明该片段的功能性，我们使用CRISPR-CAS 9来产生在这4个SNP处具有靶向编辑的iPSC，以及强LD中的3个另外的SNP，并使用这些等基因iPSC衍生的胰岛作为模型系统来研究变体的作用。这些胰岛的流式细胞术鉴定了使用具有风险单倍型的iPSC产生的胰岛中较低百分比的β样细胞（共表达INS和NKX 6 -1的细胞），表明这些T2 D相关SNP对β细胞质量的影响。阶段特异性基因表达分析确定了内分泌祖细胞（EP）阶段INS基因表达的倍数增加的显着差异。由于内分泌细胞类型定型发生在EP阶段，INS表达的增加与β细胞定型相关，因此在此阶段胰岛素表达的较低增加与风险单倍型可以部分解释变体对成熟胰岛组成的影响。在具有风险单倍型的EP中，我们还鉴定了H19的表达增加，H19定位在SNP的上游，并且已知其影响β细胞质量。 增强子甲基化调控基因表达。因此，我们评估了所有13个CpG位点（CpG-func）的阶段特异性甲基化，这些位点包括功能区（rs 2299620和rs74606911之间）和rs 2299620上游的3个CpG位点（CpG-cnc）。在胰岛分化的不同阶段，我们鉴定了CpG-func的动态甲基化变化，而CpG-cnc没有变化（保持高甲基化，平均甲基化>90%）。CpG-func在iPSC阶段被高甲基化，但在EP阶段变得低甲基化。有趣的是，当在风险和非风险单倍型之间进行比较时，仅在EP阶段期间在CpG-func中观察到甲基化的强烈差异。这些数据支持KCNQ 1的T2 D SNP影响胰岛组成（β细胞质量），并且这种影响在发育早期表现出来，可能通过对CpG甲基化和基因调控的影响。

A 35-day protocol to reprogram human induced pluripotent stem cells (hiPSCs) into glucose-responsive pancreatic beta-like cells has been developed. Differentiating cells are being 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 are conducted and clonal cell lines are 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 are also analyzed for potential karyotypic abnormalities and off-target CRISPR effects. AT 7 time points reflecting different developmental stages during the differentiation procedure, cells are collected for RNA analysis. The strongest association for type 2 diabetes (T2D) in Southwestern American Indians maps to intron 15 of KCNQ1 where the lead SNP (rs2299620) is in a region of strong linkage disequilibrium (LD). 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.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 currently believe we have mapped the functional region at KCNQ1 to an 400 bp fragment containing rs2299620 and 3 additional SNPs. TO demonstrate the functionality of this fragment, we used CRISPR-CAS9 to generate iPSCs with targeted edits at these 4 SNPs, and 3 additional SNPs in strong LD, and used these isogenic iPSC-derived pancreatic islets as a model system to study the effect of the variants. Flow cytometry of these islets identified a lower percentage of beta-like cells (cells co-expressing INS and NKX6-1) in islets generated using iPSCs with the risk haplotype, suggesting an effect of these T2D associated SNPs on beta cell mass. Stage specific gene expression analyses identified a significant difference in the fold increase in INS gene expression during the endocrine progenitor (EPs) stage. As endocrine cell type commitment occurs during EP stage and increase in INS expression correlates with beta cell commitment, the lower increase in insulin expression during this stage with the risk haplotype could in part explain the effect of the variants on mature islet composition. In EPs with the risk haplotype, we also identified increased expression of H19 which maps upstream of the SNPs and is known to affect beta-cell mass. Methylation at enhancers regulates gene expression. Therefore, we assessed stage specific methylation at all 13 CpG sites (CpG-func) that encompass the functional region (between rs2299620 and rs74606911) and 3 CpG sites (CpG-ctrl) immediately upstream of rs2299620. We identified dynamic methylation changes at CpG-func whereas no changes at CpG-ctrl (remained hypermethylated, average methylation >90%) during different stages of pancreatic islet differentiation. CpG-func were hypermethylated during the iPSC stage but became hypomethylated during the EP stage. Interestingly, a strong difference in methylation was seen in CpG-func only during the EP stage when compared between the risk and non-risk haplotypes. These data support that the T2D SNPs at KCNQ1 affect islet composition (beta-cell mass) and this effect is manifested early during development, likely via an effect on CpG methylation and gene regulation.