Identification of unique cell type responses in pancreatic islets to stress.

Diabetes involves the death or dysfunction of pancreatic ß-cells. Analysis of bulk sequencing from human samples and studies using in vitro and in vivo models suggest that endoplasmic reticulum and inflammatory signaling play an important role in diabetes progression. To better characterize cell type-specific stress response, we perform multiplexed single-cell RNA sequencing to define the transcriptional signature of primary human islet cells exposed to endoplasmic reticulum and inflammatory stress. Through comprehensive pair-wise analysis of stress responses across pancreatic endocrine and exocrine cell types, we define changes in gene expression for each cell type under different diabetes-associated stressors. We find that ß-, α-, and ductal cells have the greatest transcriptional response. We utilize stem cell-derived islets to study islet health through the candidate gene CIB1, which was upregulated under stress in primary human islets. Our findings provide insights into cell type-specific responses to diabetes-associated stress and establish a resource to identify targets for diabetes therapeutics.

Single-nucleus multi-omics of human stem cell-derived islets identifies deficiencies in lineage specification.

Insulin-producing ß cells created from human pluripotent stem cells have potential as a therapy for insulin-dependent diabetes, but human pluripotent stem cell-derived islets (SC-islets) still differ from their in vivo counterparts. To better understand the state of cell types within SC-islets and identify lineage specification deficiencies, we used single-nucleus multi-omic sequencing to analyse chromatin accessibility and transcriptional profiles of SC-islets and primary human islets. Here we provide an analysis that enabled the derivation of gene lists and activity for identifying each SC-islet cell type compared with primary islets. Within SC-islets, we found that the difference between ß cells and awry enterochromaffin-like cells is a gradient of cell states rather than a stark difference in identity. Furthermore, transplantation of SC-islets in vivo improved cellular identities overtime, while long-term in vitro culture did not. Collectively, our results highlight the importance of chromatin and transcriptional landscapes during islet cell specification and maturation.

Genetic architecture and temporal analysis of Caenorhabditis briggsae hybrid developmental delay.

Identifying the alleles that reduce hybrid fitness is a major goal in the study of speciation genetics. It is rare to identify systems in which hybrid incompatibilities with minor phenotypic effects are segregating in genetically diverse populations of the same biological species. Such traits do not themselves cause reproductive isolation but might initiate the process. In the nematode Caenorhabditis briggsae, a small percent of F2 generation hybrids between two natural populations suffer from developmental delay, in which adulthood is reached after approximately 33% more time than their wild-type siblings. Prior efforts to identify the genetic basis for this hybrid incompatibility assessed linkage using one or two genetic markers on chromosome III and suggested that delay is caused by a toxin-antidote element. Here, we have genotyped F2 hybrids using multiple chromosome III markers to refine the developmental delay locus. Also, to better define the developmental delay phenotype, we measured the development rate of 66 F2 hybrids and found that delay is not restricted to a particular larval developmental stage. Deviation of the developmental delay frequency from hypothetical expectations for a toxin-antidote element adds support to the assertion that the epistatic interaction is not fully penetrant. Our mapping and refinement of the delay phenotype motivates future efforts to study the genetic architecture of hybrid dysfunction between genetically distinct populations of one species by identifying the underlying loci.

Advances Toward Engineering Functionally Mature Human Pluripotent Stem Cell-Derived ß Cells.

Human stem cell-derived ß (SC-ß) cells have the potential to revolutionize diabetes treatment through disease modeling, drug screening, and cellular therapy. SC-ß cells are likely to represent an early clinical translation of differentiated human pluripotent stem cells (hPSC). In 2014, two groups generated the first in vitro-differentiated glucose-responsive SC-ß cells, but their functional maturation at the time was low. This review will discuss recent advances in the engineering of SC-ß cells to understand and improve SC-ß cell differentiation and functional maturation, particularly new differentiation strategies achieving dynamic glucose-responsive insulin secretion with rapid correction to normoglycemia when transplanted into diabetic mice.

Single-Cell Transcriptome Profiling Reveals ß Cell Maturation in Stem Cell-Derived Islets after Transplantation.

Human pluripotent stem cells differentiated to insulin-secreting ß cells (SC-ß cells) in islet organoids could provide an unlimited cell source for diabetes cell replacement therapy. However, current SC-ß cells generated in vitro are transcriptionally and functionally immature compared to native adult ß cells. Here, we use single-cell transcriptomic profiling to catalog changes that occur in transplanted SC-ß cells. We find that transplanted SC-ß cells exhibit drastic transcriptional changes and mature to more closely resemble adult ß cells. Insulin and IAPP protein secretions increase upon transplantation, along with expression of maturation genes lacking with differentiation in vitro, including INS, MAFA, CHGB, and G6PC2. Other differentiated cell types, such as SC-α and SC-enterochromaffin (SC-EC) cells, also exhibit large transcriptional changes. This study provides a comprehensive resource for understanding human islet cell maturation and provides important insights into maturation of SC-ß cells and other SC-islet cell types to enable future differentiation strategy improvements.

Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells.

Generation of pancreatic ß cells from human pluripotent stem cells (hPSCs) holds promise as a cell replacement therapy for diabetes. In this study, we establish a link between the state of the actin cytoskeleton and the expression of pancreatic transcription factors that drive pancreatic lineage specification. Bulk and single-cell RNA sequencing demonstrated that different degrees of actin polymerization biased cells toward various endodermal lineages and that conditions favoring a polymerized cytoskeleton strongly inhibited neurogenin 3-induced endocrine differentiation. Using latrunculin A to depolymerize the cytoskeleton during endocrine induction, we developed a two-dimensional differentiation protocol for generating human pluripotent stem-cell-derived ß (SC-ß) cells with improved in vitro and in vivo function. SC-ß cells differentiated from four hPSC lines exhibited first- and second-phase dynamic glucose-stimulated insulin secretion. Transplantation of islet-sized aggregates of these cells rapidly reversed severe preexisting diabetes in mice at a rate close to that of human islets and maintained normoglycemia for at least 9 months.

SIX2 Regulates Human ß Cell Differentiation from Stem Cells and Functional Maturation In Vitro.

Generation of insulin-secreting ß cells in vitro is a promising approach for diabetes cell therapy. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are differentiated to ß cells (SC-ß cells) and mature to undergo glucose-stimulated insulin secretion, but molecular regulation of this defining ß cell phenotype is unknown. Here, we show that maturation of SC-ß cells is regulated by the transcription factor SIX2. Knockdown (KD) or knockout (KO) of SIX2 in SC-ß cells drastically limits glucose-stimulated insulin secretion in both static and dynamic assays, along with the upstream processes of cytoplasmic calcium flux and mitochondrial respiration. Furthermore, SIX2 regulates the expression of genes associated with these key ß cell processes, and its expression is restricted to endocrine cells. Our results demonstrate that expression of SIX2 influences the generation of human SC-ß cells in vitro.

Gene-edited human stem cell-derived ß cells from a patient with monogenic diabetes reverse preexisting diabetes in mice.

Differentiation of insulin-producing pancreatic ß cells from induced pluripotent stem cells (iPSCs) derived from patients with diabetes promises to provide autologous cells for diabetes cell replacement therapy. However, current approaches produce patient iPSC-derived ß (SC-ß) cells with poor function in vitro and in vivo. Here, we used CRISPR-Cas9 to correct a diabetes-causing pathogenic variant in Wolfram syndrome 1 (WFS1) in iPSCs derived from a patient with Wolfram syndrome (WS). After differentiation to ß cells with our recent six-stage differentiation strategy, corrected WS SC-ß cells performed robust dynamic insulin secretion in vitro in response to glucose and reversed preexisting streptozocin-induced diabetes after transplantation into mice. Single-cell transcriptomics showed that corrected SC-ß cells displayed increased insulin and decreased expression of genes associated with endoplasmic reticulum stress. CRISPR-Cas9 correction of a diabetes-inducing gene variant thus allows for robust differentiation of autologous SC-ß cells that can reverse severe diabetes in an animal model.

A hydrogel platform for in vitro three dimensional assembly of human stem cell-derived islet cells and endothelial cells.

Differentiation of stem cells into functional replacement cells and tissues is a major goal of the regenerative medicine field. However, one limitation has been organization of differentiated cells into multi-cellular, three-dimensional assemblies. The islets of Langerhans contain many endocrine and non-endocrine cell types, such as insulin-producing ß cells and endothelial cells. Despite the potential importance of endothelial cells to islet function, facilitating interactions between endothelial cells and islet endocrine cell types already differentiated from human embryonic stem cells has been difficult in vitro. We have developed a strategy of assembling human embryonic stem cell-derived islet cells with endothelial cells into three-dimensional aggregates on a hydrogel. The resulting islet organoids express ß cell and other endocrine markers and are functional, capable of undergoing glucose-stimulated insulin secretion. This assembly was not observed on traditional tissue culture plastic and in aggregates generated in suspension culture, highlighting how physical culture conditions greatly influence the interactions among these cell types. These results provide a platform for evaluating the effects of the islet tissue microenvironment on human embryonic stem cell-derived ß cells and other islet endocrine cells to develop tissue engineered islets. STATEMENT OF SIGNIFICANCE: Differentiation of insulin-producing cells and tissues from human pluripotent stem cells is being investigated for diabetes cell replacement therapies. Despite successes generating ß cells, the cell type responsible for glucose-stimulated insulin secretion within the islets of Langerhans found in the pancreas, successful assembly with other non-endocrine cell types, particularly endothelial cells, has been technically challenging. The present study provides a platform for the assembly of endothelial cells with SC-ß and other endocrine cells, producing islet organoids that are functional and express ß cell markers, that can be used to study the islet microenvironment and islet tissue engineering.

Acquisition of Dynamic Function in Human Stem Cell-Derived ß Cells.

Recent advances in human pluripotent stem cell (hPSC) differentiation protocols have generated insulin-producing cells resembling pancreatic ß cells. While these stem cell-derived ß (SC-ß) cells are capable of undergoing glucose-stimulated insulin secretion (GSIS), insulin secretion per cell remains low compared with islets and cells lack dynamic insulin release. Herein, we report a differentiation strategy focused on modulating transforming growth factor ß (TGF-ß) signaling, controlling cellular cluster size, and using an enriched serum-free media to generate SC-ß cells that express ß cell markers and undergo GSIS with first- and second-phase dynamic insulin secretion. Transplantation of these cells into mice greatly improves glucose tolerance. These results reveal that specific time frames for inhibiting and permitting TGF-ß signaling are required during SC-ß cell differentiation to achieve dynamic function. The capacity of these cells to undergo GSIS with dynamic insulin release makes them a promising cell source for diabetes cellular therapy.