Single cell regulatory architecture of human pancreatic islets suggests sex differences in ß cell function and the pathogenesis of type 2 diabetes.

Biological sex affects the pathogenesis of type 2 and type 1 diabetes (T2D, T1D) including the development of ß cell failure observed more often in males. The mechanisms that drive sex differences in ß cell failure is unknown. Studying sex differences in islet regulation and function represent a unique avenue to understand the sex-specific heterogeneity in ß cell failure in diabetes. Here, we examined sex and race differences in human pancreatic islets from up to 52 donors with and without T2D (including 37 donors from the Human Pancreas Analysis Program [HPAP] dataset) using an orthogonal series of experiments including single cell RNA-seq (scRNA-seq), single nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq), dynamic hormone secretion, and bioenergetics. In cultured islets from nondiabetic (ND) donors, in the absence of the in vivo hormonal environment, sex differences in islet cell type gene accessibility and expression predominantly involved sex chromosomes. Of particular interest were sex differences in the X-linked KDM6A and Y-linked KDM5D chromatin remodelers in female and male islet cells respectively. Islets from T2D donors exhibited similar sex differences in differentially expressed genes (DEGs) from sex chromosomes. However, in contrast to islets from ND donors, islets from T2D donors exhibited major sex differences in DEGs from autosomes. Comparing ß cells from T2D and ND donors revealed that females had more DEGs from autosomes compared to male ß cells. Gene set enrichment analysis of female ß cell DEGs showed a suppression of oxidative phosphorylation and electron transport chain pathways, while male ß cell had suppressed insulin secretion pathways. Thus, although sex-specific differences in gene accessibility and expression of cultured ND human islets predominantly affect sex chromosome genes, major differences in autosomal gene expression between sexes appear during the transition to T2D and which highlight mitochondrial failure in female ß cells.

An Integrated Map of Cell Type-Specific Gene Expression in Pancreatic Islets.

Pancreatic islets consist of multiple cell types that produce hormones required for glucose homeostasis, and islet dysfunction is a major factor in type 1 and type 2 diabetes. Numerous studies have assessed transcription across individual cell types using single-cell assays; however, there is no canonical reference of gene expression in islet cell types that is also easily accessible for researchers to query and use in bioinformatics pipelines. Here we present an integrated map of islet cell type-specific gene expression from 192,203 cells from single-cell RNA sequencing of 65 donors without diabetes, donors who were type 1 diabetes autoantibody positive, donors with type 1 diabetes, and donors with type 2 diabetes from the Human Pancreas Analysis Program. We identified 10 distinct cell types, annotated subpopulations of several cell types, and defined cell type-specific marker genes. We tested differential expression within each cell type across disease states and identified 1,701 genes with significant changes in expression, with most changes observed in ß-cells from donors with type 1 diabetes. To facilitate user interaction, we provide several single-cell visualization and reference mapping tools, as well as the open-access analytical pipelines used to create this reference. The results will serve as a valuable resource to investigators studying islet biology.

An integrated map of cell type-specific gene expression in pancreatic islets.

Pancreatic islets are comprised of multiple endocrine cell types that produce hormones required for glucose homeostasis, and islet dysfunction is a major factor in the development of type 1 and type 2 diabetes (T1D, T2D). Numerous studies have generated gene expression profiles in individual islet cell types using single cell assays. However, there is no canonical reference of gene expression in islet cell types in both health and disease that is also easily accessible for researchers to access, query, and use in bioinformatics pipelines. Here we present an integrated reference map of islet cell type-specific gene expression from 192,203 cells derived from single cell RNA-seq assays of 65 non-diabetic, T1D autoantibody positive (Aab+), T1D, and T2D donors from the Human Pancreas Analysis Program. We identified 10 endocrine and non-endocrine cell types as well as sub-populations of several cell types, and defined sets of marker genes for each cell type and sub-population. We tested for differential expression within each cell type in T1D Aab+, T1D, and T2D states, and identified 1,701 genes with significant changes in expression in any cell type. Most changes were observed in beta cells in T1D, and, by comparison, there were almost no genes with changes in T1D Aab+. To facilitate user interaction with this reference, we provide the data using several single cell visualization and reference mapping tools as well as open-access analytical pipelines used to create this reference. The results will serve as a valuable resource to investigators studying islet biology and diabetes.

Native Zinc Catalyzes Selective and Traceless Release of Small Molecules in ß-Cells.

The loss of insulin-producing ß-cells is the central pathological event in type 1 and 2 diabetes, which has led to efforts to identify molecules to promote ß-cell proliferation, protection, and imaging. However, the lack of ß-cell specificity of these molecules jeopardizes their therapeutic potential. A general platform for selective release of small-molecule cargoes in ß-cells over other islet cells ex vivo or other cell-types in an organismal context will be immensely valuable in advancing diabetes research and therapeutic development. Here, we leverage the unusually high Zn(II) concentration in ß-cells to develop a Zn(II)-based prodrug system to selectively and tracelessly deliver bioactive small molecules and fluorophores to ß-cells. The Zn(II)-targeting mechanism enriches the inactive cargo in ß-cells as compared to other pancreatic cells; importantly, Zn(II)-mediated hydrolysis triggers cargo activation. This prodrug system, with modular components that allow for fine-tuning selectivity, should enable the safer and more effective targeting of ß-cells.

Human Multipotent Stromal Cell Secreted Effectors Accelerate Islet Regeneration.

Human multipotent stromal cells (hMSC) can induce islet regeneration after transplantation via the secretion of proteins that establish an islet regenerative niche. However, the identity of hMSC-secreted signals and the mechanisms by which pancreatic islet regeneration is induced remain unknown. Recently, mammalian pancreatic α-cells have been shown to possess considerable plasticity, and differentiate into ß-like cells after near complete ß-cell loss or overexpression of key transcriptional regulators. These studies have generated new excitement that islet regeneration during diabetes may be possible if we can identify clinically applicable stimuli to modulate these key regulatory pathways. Herein, we demonstrate that intrapancreatic-injection of concentrated hMSC-conditioned media (CM) stimulated islet regeneration without requiring cell transfer. hMSC CM-injection significantly reduced hyperglycemia, increased circulating serum insulin concentration, and improved glucose tolerance in streptozotocin-treated mice. The rate and extent of endogenous ß-cell mass recovery was dependent on total protein dose administered and was further augmented by the activation of Wnt-signaling using GSK3-inhibition during CM generation. Intrapancreatic hMSC CM-injection immediately set in motion a cascade of regenerative events that included the emergence of proliferating insulin+ clusters adjacent to ducts, NKX6.1 expression in glucagon+ cells at days 1-4 suggesting the acquisition of ß-cell phenotype by α-cells, and accelerated ß-cell maturation with increased MAFA-expression for >1 month postinjection. Discovery and validation of islet regenerative hMSC-secreted protein may lead to the development of cell-free regenerative therapies able to tip the balance in favor of ß-cell regeneration versus destruction during diabetes. Stem Cells 2019;37:516-528.

Quantitative Proteomics Evaluation of Human Multipotent Stromal Cell for ß Cell Regeneration.

Human multipotent stromal cells (hMSCs) are one of the most versatile cell types used in regenerative medicine due to their ability to respond to injury. In the context of diabetes, it has been previously shown that the regenerative capacity of hMSCs is donor specific after transplantation into streptozotocin (STZ)-treated immunodeficient mice. However, in vivo transplantation models to determine regenerative potency of hMSCs are lengthy, costly, and low throughput. Therefore, a high-throughput quantitative proteomics assay was developed to screen ß cell regenerative potency of donor-derived hMSC lines. Using proteomics, we identified 16 proteins within hMSC conditioned media that effectively identify ß cell regenerative hMSCs. This protein signature was validated using human islet culture assay, ELISA, and the potency was confirmed by recovery of hyperglycemia in STZ-treated mice. Herein, we demonstrated that quantitative proteomics can determine sample-specific protein signatures that can be used to classify previously uncharacterized hMSC lines for ß cell regenerative clinical applications.

BMS 493 Modulates Retinoic Acid-Induced Differentiation During Expansion of Human Hematopoietic Progenitor Cells for Islet Regeneration.

Cellular therapies are emerging as a novel treatment strategy for diabetes. Thus, the induction of endogenous islet regeneration in situ represents a feasible goal for diabetes therapy. Umbilical cord blood-derived hematopoietic progenitor cells (HPCs), isolated by high aldehyde dehydrogenase activity (ALDHhi), have previously been shown to reduce hyperglycemia after intrapancreatic (iPan) transplantation into streptozotocin (STZ)-treated nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. However, these cells are rare and require ex vivo expansion to reach clinically applicable numbers for human therapy. Therefore, we investigated whether BMS 493, an inverse retinoic acid receptor agonist, could prevent retinoic acid-induced differentiation and preserve islet regenerative functions during expansion. After 6-day expansion, BMS 493-treated cells showed a twofold increase in the number of ALDHhi cells available for transplantation compared with untreated controls. Newly expanded ALDHhi cells showed increased numbers of CD34 and CD133-positive cells, as well as a reduction in CD38 expression, a marker of hematopoietic cell differentiation. BMS 493-treated cells showed similar hematopoietic colony-forming capacity compared with untreated cells, with ALDHhi subpopulations producing more colonies than low aldehyde dehydrogenase activity subpopulations for expanded cells. To determine if the secreted proteins of these cells could augment the survival and/or proliferation of ß-cells in vitro, conditioned media (CM) from cells expanded with or without BMS 493 was added to human islet cultures. The total number of proliferating ß-cells was increased after 3- or 7-day culture with CM generated from BMS 493-treated cells. In contrast to freshly isolated ALDHhi cells, 6-day expansion with or without BMS 493 generated progeny that were unable to reduce hyperglycemia after iPan transplantation into STZ-treated NOD/SCID mice. Further strategies to reduce retinoic acid differentiation during HPC expansion is required to expand ALDHhi cells without the loss of islet regenerative functions.