Generation of a human Tropomyosin 1 knockout iPSC line.

The CHOPWT17_TPM1KOc28 iPSC line was generated to interrogate the functions of Tropomyosin 1 (TPM1) in primary human cell development. This line was reprogrammed from a previously published wild type control iPSC line.

Generation of a human Tropomyosin 1 knockout iPSC line.

The CHOPWT17_TPM1KOc28 iPSC line was generated to interrogate the functions of Tropomyosin 1 ( TPM1 ) in primary human cell development. This line was reprogrammed from a previously published wild type control iPSC line.

A Non-Coding Disease Modifier of Pancreatic Agenesis Identified by Genetic Correction in a Patient-Derived iPSC Line.

GATA6 is a critical regulator of pancreatic development, with heterozygous mutations in this transcription factor being the most common cause of pancreatic agenesis. To study the variability in disease phenotype among individuals harboring these mutations, a patient-induced pluripotent stem cell model was used. Interestingly, GATA6 protein expression remained depressed in pancreatic progenitor cells even after correction of the coding mutation. Screening the regulatory regions of the GATA6 gene in these patient cells and 32 additional agenesis patients revealed a higher minor allele frequency of a SNP 3' of the GATA6 coding sequence. Introduction of this minor allele SNP by genome editing confirmed its functionality in depressing GATA6 expression and the efficiency of pancreas differentiation. This work highlights a possible genetic modifier contributing to pancreatic agenesis and demonstrates the usefulness of using patient-induced pluripotent stem cells for targeted discovery and validation of non-coding gene variants affecting gene expression and disease penetrance.

A Dual Reporter EndoC-ßH1 Human ß-Cell Line for Efficient Quantification of Calcium Flux and Insulin Secretion.

Human in vitro model systems of diabetes are critical to both study disease pathophysiology and offer a platform for drug testing. We have generated a set of tools in the human ß-cell line EndoC-ßH1 that allows the efficient and inexpensive characterization of ß-cell physiology and phenotypes driven by disruption of candidate genes. First, we generated a dual reporter line that expresses a preproinsulin-luciferase fusion protein along with GCaMP6s. This reporter line allows the quantification of insulin secretion by measuring luciferase activity and calcium flux, a critical signaling step required for insulin secretion, via fluorescence microscopy. Using these tools, we demonstrate that the generation of the reporter human ß-cell line was highly efficient and validated that luciferase activity could accurately reflect insulin secretion. Second, we used a lentiviral vector carrying the CRISPR-Cas9 system to generate candidate gene disruptions in the reporter line. We also show that we can achieve gene disruption in ~90% of cells using a CRISPR-Cas9 lentiviral system. As a proof of principle, we disrupt the ß-cell master regulator, PDX1, and show that mutant EndoC-ßH1 cells display impaired calcium responses and fail to secrete insulin when stimulated with high glucose. Furthermore, we show that PDX1 mutant EndoC-ßH1 cells exhibit decreased expression of the ß-cell-specific genes MAFA and NKX6.1 and increased GCG expression. The system presented here provides a platform to quickly and easily test ß-cell functionality in wildtype and cells lacking a gene of interest.

Modeling Monogenic Diabetes using Human ESCs Reveals Developmental and Metabolic Deficiencies Caused by Mutations in HNF1A.

Human monogenic diabetes, caused by mutations in genes involved in beta cell development and function, has been a challenge to study because multiple mouse models have not fully recapitulated the human disease. Here, we use genome edited human embryonic stem cells to understand the most common form of monogenic diabetes, MODY3, caused by mutations in the transcription factor HNF1A. We found that HNF1A is necessary to repress an alpha cell gene expression signature, maintain endocrine cell function, and regulate cellular metabolism. In addition, we identified the human-specific long non-coding RNA, LINKA, as an HNF1A target necessary for normal mitochondrial respiration. These findings provide a possible explanation for the species difference in disease phenotypes observed with HNF1A mutations and offer mechanistic insights into how the HNF1A gene may also influence type 2 diabetes.