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.

SARS-CoV-2 infection of the pancreas promotes thrombofibrosis and is associated with new-onset diabetes.

Evidence suggests an association between severe acute respiratory syndrome-cornavirus-2 (SARS-CoV-2) infection and the occurrence of new-onset diabetes. We examined pancreatic expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), the cell entry factors for SARS-CoV-2, using publicly available single-cell RNA sequencing data sets, and pancreatic tissue from control male and female nonhuman primates (NHPs) and humans. We also examined SARS-CoV-2 immunolocalization in pancreatic cells of SARS-CoV-2-infected NHPs and patients who had died from coronavirus disease 2019 (COVID-19). We report expression of ACE2 in pancreatic islet, ductal, and endothelial cells in NHPs and humans. In pancreata from SARS-CoV-2-infected NHPs and COVID-19 patients, SARS-CoV-2 infected ductal, endothelial, and islet cells. These pancreata also exhibited generalized fibrosis associated with multiple vascular thrombi. Two out of 8 NHPs developed new-onset diabetes following SARS-CoV-2 infection. Two out of 5 COVID-19 patients exhibited new-onset diabetes at admission. These results suggest that SARS-CoV-2 infection of the pancreas may promote acute and especially chronic pancreatic dysfunction that could potentially lead to new-onset diabetes.

Temporal single-cell regeneration studies: the greatest thing since sliced pancreas?

The application of single-cell analytic techniques to the study of stem/progenitor cell niches supports the emerging view that pancreatic cell lineages are in a state of flux between differentiation stages. For all their value, however, such analyses merely offer a snapshot of the cellular palette of the tissue at any given time point. Conclusions about potential developmental/regeneration paths are solely based on bioinformatics inferences. In this context, the advent of new techniques for the long-term culture and lineage tracing of human pancreatic slices offers a virtual window into the native organ and presents the field with a unique opportunity to serially resolve pancreatic regeneration dynamics at the single-cell level.

Long-term culture of human pancreatic slices as a model to study real-time islet regeneration.

The culture of live pancreatic tissue slices is a powerful tool for the interrogation of physiology and pathology in an in vitro setting that retains near-intact cytoarchitecture. However, current culture conditions for human pancreatic slices (HPSs) have only been tested for short-term applications, which are not permissive for the long-term, longitudinal study of pancreatic endocrine regeneration. Using a culture system designed to mimic the physiological oxygenation of the pancreas, we demonstrate high viability and preserved endocrine and exocrine function in HPS for at least 10 days after sectioning. This extended lifespan allowed us to dynamically lineage trace and quantify the formation of insulin-producing cells in HPS from both non-diabetic and type 2 diabetic donors. This technology is expected to be of great impact for the conduct of real-time regeneration/developmental studies in the human pancreas.

Publisher Correction: Long-term culture of human pancreatic slices as a model to study real-time islet regeneration.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Endocrine Significance of SARS-CoV-2's Reliance on ACE2.

The current COVID-19 pandemic is the most disruptive event in the past 50 years, with a global impact on health care and world economies. It is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a coronavirus that uses angiotensin-converting enzyme 2 (ACE2) as an entry point to the cells. ACE2 is a transmembrane carboxypeptidase and member of the renin-angiotensin system. This mini-review summarizes the main findings regarding ACE2 expression and function in endocrine tissues. We discuss rapidly evolving knowledge on the potential role of ACE2 and SARS coronaviruses in endocrinology and the development of diabetes mellitus, hypogonadism, and pituitary and thyroid diseases.

Single-cell resolution analysis of the human pancreatic ductal progenitor cell niche.

We have described multipotent progenitor-like cells within the major pancreatic ducts (MPDs) of the human pancreas. They express PDX1, its surrogate surface marker P2RY1, and the bone morphogenetic protein (BMP) receptor 1A (BMPR1A)/activin-like kinase 3 (ALK3), but not carbonic anhydrase II (CAII). Here we report the single-cell RNA sequencing (scRNA-seq) of ALK3bright+-sorted ductal cells, a fraction that harbors BMP-responsive progenitor-like cells. Our analysis unveiled the existence of multiple subpopulations along two major axes, one that encompasses a gradient of ductal cell differentiation stages, and another featuring cells with transitional phenotypes toward acinar tissue. A third potential ducto-endocrine axis is revealed upon integration of the ALK3bright+ dataset with a single-cell whole-pancreas transcriptome. When transplanted into immunodeficient mice, P2RY1+/ALK3bright+ populations (enriched in PDX1+/ALK3+/CAII- cells) differentiate into all pancreatic lineages, including functional ß-cells. This process is accelerated when hosts are treated systemically with an ALK3 agonist. We found PDX1+/ALK3+/CAII- progenitor-like cells in the MPDs of types 1 and 2 diabetes donors, regardless of the duration of the disease. Our findings open the door to the pharmacological activation of progenitor cells in situ.

The Role of MicroRNAs in Diabetes-Related Oxidative Stress.

Cellular stress, combined with dysfunctional, inadequate mitochondrial phosphorylation, produces an excessive amount of reactive oxygen species (ROS) and an increased level of ROS in cells, which leads to oxidation and subsequent cellular damage. Because of its cell damaging action, an association between anomalous ROS production and disease such as Type 1 (T1D) and Type 2 (T2D) diabetes, as well as their complications, has been well established. However, there is a lack of understanding about genome-driven responses to ROS-mediated cellular stress. Over the last decade, multiple studies have suggested a link between oxidative stress and microRNAs (miRNAs). The miRNAs are small non-coding RNAs that mostly suppress expression of the target gene by interaction with its 3'untranslated region (3'UTR). In this paper, we review the recent progress in the field, focusing on the association between miRNAs and oxidative stress during the progression of diabetes.

A Double Fail-Safe Approach to Prevent Tumorigenesis and Select Pancreatic ß Cells from Human Embryonic Stem Cells.

The transplantation of human embryonic stem cell (hESC)-derived insulin-producing ß cells for the treatment of diabetes is finally approaching the clinical stage. However, even with state-of-the-art differentiation protocols, a significant percentage of undefined non-endocrine cell types are still generated. Most importantly, there is the potential for carry-over of non-differentiated cell types that may produce teratomas. We sought to modify hESCs so that their differentiated progeny could be selectively devoid of tumorigenic cells and enriched for cells of the desired phenotype (in this case, ß cells). Here we report the generation of a modified hESC line harboring two suicide gene cassettes, whose expression results in cell death in the presence of specific pro-drugs. We show the efficacy of this system at enriching for ß cells and eliminating tumorigenic ones both in vitro and in vivo. Our approach is innovative inasmuch as it allows for the preservation of the desired cells while eliminating those with the potential to develop teratomas.

Pancreatic Progenitors: There and Back Again.

Adult pancreatic regeneration is one of the most contentious topics in modern biology. The long-held view that the islets of Langerhans can be replenished throughout adult life through the reactivation of ductal progenitor cells has been replaced over the past decade by the now prevailing notion that regeneration does not involve progenitors and occurs only through the duplication of pre-existing mature cells. Here we dissect the limitations of lineage tracing (LT) to draw categorical conclusions about pancreatic regeneration, especially in view of emerging evidence that traditional lineages are less homogeneous and cell fates more dynamic than previously thought. This new evidence further suggests that the two competing hypotheses about regeneration are not mutually exclusive.

P2RY1/ALK3-Expressing Cells within the Adult Human Exocrine Pancreas Are BMP-7 Expandable and Exhibit Progenitor-like Characteristics.

Treatment of human pancreatic non-endocrine tissue with Bone Morphogenetic Protein 7 (BMP-7) leads to the formation of glucose-responsive ß-like cells. Here, we show that BMP-7 acts on extrainsular cells expressing PDX1 and the BMP receptor activin-like kinase 3 (ALK3/BMPR1A). In vitro lineage tracing indicates that ALK3+ cell populations are multipotent. PDX1+/ALK3+ cells are absent from islets but prominently represented in the major pancreatic ducts and pancreatic duct glands. We identified the purinergic receptor P2Y1 (P2RY1) as a surrogate surface marker for PDX1. Sorted P2RY1+/ALK3bright+ cells form BMP-7-expandable colonies characterized by NKX6.1 and PDX1 expression. Unlike the negative fraction controls, these colonies can be differentiated into multiple pancreatic lineages upon BMP-7 withdrawal. RNA-seq further corroborates the progenitor-like nature of P2RY1+/ALK3bright+ cells and their multilineage differentiation potential. Our studies confirm the existence of progenitor cells in the adult human pancreas and suggest a specific anatomical location within the ductal and glandular networks.

Immunomodulatory and therapeutic role of Cinnamomum verum extracts in collagen-induced arthritic BALB/c mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinnamomum verum (CV), also known as 'Dalchini', is the dry bark of the Cinnamomum verum (L.) plant, and has been used as a traditional Pakistani medicine to alleviate pain and inflammation in patients suffering from arthritic rheumatism. It contains alkaloids, triterpenes, Cinnamaldehyde and other volatile oils. The aim of the present study was to investigate the underlying biological effect of ethyl alcohol (EtOH) and methyl alcohol (MeOH) extracts from CV on collagen type-II induced arthritic (CIA) mice. MATERIALS AND METHODS: Gas chromatography mass spectrophotometry was used to perform fingerprinting identification of the EtOH and MeOH extracts. CIA mice model was established by subdermal injections of type-II bovine collagen (CII) on the 1st, 8th and 14th day of the experiment. Ethyl alcohol extract and methyl alcohol extract (1 mg/KgBW, 2 mg/KgBW and 4 mg/KgBW), was orally administered from the 15th day onwards for 2 weeks. Progression of oedema and joint inflammation was measured in the paws using a digital Vernier calliper every 3 days from day 1 till the end of the experiment. The oxidative scavenging ability of cinnamaldehyde was evaluated using a DPPH assay. Similarly, the nitrogen free radical (NOS) production of isolated lymphocytes was evaluated using Greiss's method. The spleen index was calculated and knee joint changes were observed by histopathological sectioning. Western blot analysis was performed on peripheral blood derived serum for CII, CAPN1, TNFα and NFATc3. RESULTS: Extracts were shown to be enriched in trans-cinnamaldehyde and its analogues. Extracts showed good ameliorative effects (p < 0.05) after day 2 of treatment. A greater therapeutic role was observed for the 4 mg/kgBW dosage of the methanolic extract (p < 0.01). Swelling in the spleen was greatly reduced along with the generation of free radicals by lymphocytes, post treatment. There was also an inhibitory role by the extracts on NFATc3 (p < 0.05), TNF-Alpha (p < 0.05), CAII (p < 0.05) and mCalpain (p < 0.05) all proteins involved in RA. CONCLUSION: In this study, it has been demonstrated that administration of CV has a therapeutic potential on CIA. The data suggest that CV could have a potential role in the treatment of RA patients.

Polymorphisms in DNA repair genes XRCC1 and OGG1 lead to the progression of rheumatoid arthritis in Pakistani patients.

This study points at the elucidation of a possible association of Rheumatoid arthritis (RA) with Ser326Cys in OGG1 Arg194Trp and Arg399Gln polymorphisms of XRCC1 using a sample size of 100 patients and 100 controls from a Pakistani population. This association was determined using Random Fragment Length Polymorphism Analysis as well as the DAS scoring system. In RA, oxidative damage due to free radical production leads to destructive proliferative synovitis showing cellular transformations of synoviocytes into a tumorigenic state. XRCC1 and OGG1 genes, which are part of the DNA Break Excision Repair pathway, manifest various polymorphisms which may cause a variation in the response to inflammation by changing DNA repair potential. Our results showed a significant association between the DAS28 score values as well as the genotypic state of the RA patients. It was seen that the score was significantly higher in GG genotypes thereby corroborating the role of the polymorphism XRCC1 Arg399Gln. Using a Pearson's correlation test it was found to be <0.000003. It has been shown by the results in this research that an increased risk of DNA damage exists when the polymorphic genotypes studied, exist in a RA patient.