Generation of a double insulin and somatostatin reporter line, SCSe001-A-3, for the advancement of stem cell-derived pancreatic islets.

Remarkable strides have been made over the past decade on the development of pancreatic ß-cells from human stem cells through directed differentiation, allowing for modeling of ß-cell development, function and disease. However, in vitro models and future therapeutic applications will require the use of stem cell-derived islets with multiple monohormonal endocrine cells types, including α, ß, and δ cells. Using the previously reported Mel1 InsGFP/w human embryonic stem cell (hESC) line, we have knocked-in Red Fluorescence Protein (RFP) under the control of the endogenous somatostatin promoter using CRISPR/Cas9, generating a dual insulin and somatostatin reporter hESC 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.

Retinoic acid signaling within pancreatic endocrine progenitors regulates mouse and human ß cell specification.

Retinoic acid (RA) signaling is essential for multiple developmental processes, including appropriate pancreas formation from the foregut endoderm. RA is also required to generate pancreatic progenitors from human pluripotent stem cells. However, the role of RA signaling during endocrine specification has not been fully explored. In this study, we demonstrate that the disruption of RA signaling within the NEUROG3-expressing endocrine progenitor population impairs mouse ß cell differentiation and induces ectopic expression of crucial δ cell genes, including somatostatin. In addition, the inhibition of the RA pathway in hESC-derived pancreatic progenitors downstream of NEUROG3 induction impairs insulin expression. We further determine that RA-mediated regulation of endocrine cell differentiation occurs through Wnt pathway components. Together, these data demonstrate the importance of RA signaling in endocrine specification and identify conserved mechanisms by which RA signaling directs pancreatic endocrine cell fate.

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.

RNA Splicing Factor Mutations That Cause Retinitis Pigmentosa Result in Circadian Dysregulation.

Circadian clocks regulate multiple physiological processes in the eye, but their requirement for retinal health remains unclear. We previously showed that Drosophila homologs of spliceosome proteins implicated in human retinitis pigmentosa (RP), the most common genetically inherited cause of blindness, have a role in the brain circadian clock. In this study, we report circadian phenotypes in murine models of RP. We found that mice carrying a homozygous H2309P mutation in Pre-mRNA splicing factor 8 (Prpf8) display a lengthened period of the circadian wheel-running activity rhythm. We show also that the daily cycling of circadian gene expression is dampened in the retina of Prpf8-H2309P mice. Surprisingly, molecular rhythms are intact in the eye cup, which includes the retinal pigment epithelium (RPE), even though the RPE is thought to be the primary tissue affected in this form of RP. Downregulation of Prp31, another RNA splicing factor implicated in RP, leads to period lengthening in a human cell culture model. The period of circadian bioluminescence in primary fibroblasts of human RP patients is not significantly altered. Together, these studies link a prominent retinal disorder to circadian deficits, which could contribute to disease pathology.

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.

GATA6 Plays an Important Role in the Induction of Human Definitive Endoderm, Development of the Pancreas, and Functionality of Pancreatic ß Cells.

Induced pluripotent stem cells were created from a pancreas agenesis patient with a mutation in GATA6. Using genome-editing technology, additional stem cell lines with mutations in both GATA6 alleles were generated and demonstrated a severe block in definitive endoderm induction, which could be rescued by re-expression of several different GATA family members. Using the endodermal progenitor stem cell culture system to bypass the developmental block at the endoderm stage, cell lines with mutations in one or both GATA6 alleles could be differentiated into ß-like cells but with reduced efficiency. Use of suboptimal doses of retinoic acid during pancreas specification revealed a more severe phenotype, more closely mimicking the patient's disease. GATA6 mutant ß-like cells fail to secrete insulin upon glucose stimulation and demonstrate defective insulin processing. These data show that GATA6 plays a critical role in endoderm and pancreas specification and ß-like cell functionality in humans.

High-level transgene expression in induced pluripotent stem cell-derived megakaryocytes: correction of Glanzmann thrombasthenia.

Megakaryocyte-specific transgene expression in patient-derived induced pluripotent stem cells (iPSCs) offers a new approach to study and potentially treat disorders affecting megakaryocytes and platelets. By using a Gp1ba promoter, we developed a strategy for achieving a high level of protein expression in human megakaryocytes. The feasibility of this approach was demonstrated in iPSCs derived from two patients with Glanzmann thrombasthenia (GT), an inherited platelet disorder caused by mutations in integrin αIIbß3. Hemizygous insertion of Gp1ba promoter-driven human αIIb complementary DNA into the AAVS1 locus of iPSCs led to high αIIb messenger RNA and protein expression and correction of surface αIIbß3 in megakaryocytes. Agonist stimulation of these cells displayed recovery of integrin αIIbß3 activation. Our findings demonstrate a novel approach to studying human megakaryocyte biology as well as functional correction of the GT defect, offering a potential therapeutic strategy for patients with diseases that affect platelet function.