Endocrine Progenitors Research Articles

β-Cell-Specific Deletion of HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) Reductase Causes Overt Diabetes due to Reduction of β-Cell Mass and Impaired Insulin Secretion.

Inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), statins, which are used to prevent cardiovascular diseases, are associated with a modest increase in the risk of new-onset diabetes. To investigate the role of HMGCR in the development of β-cells and glucose homeostasis, we deleted Hmgcr in a β-cell-specific manner by using the Cre-loxP technique. Mice lacking Hmgcr in β-cells (β-KO) exhibited hypoinsulinemic hyperglycemia as early as postnatal day 9 (P9) due to decreases in both β-cell mass and insulin secretion. Ki67-positive cells were reduced in β-KO mice at P9; thus, β-cell mass reduction was caused by proliferation disorder immediately after birth. The mRNA expression of neurogenin3 (Ngn3), which is transiently expressed in endocrine progenitors of the embryonic pancreas, was maintained despite a striking reduction in the expression of β-cell-associated genes, such as insulin, pancreatic and duodenal homeobox 1 (Pdx1), and MAF BZIP transcription factor A (Mafa) in the islets from β-KO mice. Histological analyses revealed dysmorphic islets with markedly reduced numbers of β-cells, some of which were also positive for glucagon. In conclusion, HMGCR plays critical roles not only in insulin secretion but also in the development of β-cells in mice.

Dapagliflozin promotes beta cell regeneration by inducing pancreatic endocrine cell phenotype conversion in type 2 diabetic mice

BackgroundClinical trials and animal studies have shown that sodium-glucose co-transporter type 2 (SGLT2) inhibitors improve pancreatic beta cell function. Our study aimed to investigate the effect of dapagliflozin on islet morphology and cell phenotype, and explore the origin and possible reason of the regenerated beta cells. MethodsTwo diabetic mouse models, db/db mice and pancreatic alpha cell lineage-tracing (glucagon-β-gal) mice whose diabetes was induced by high fat diet combined with streptozotocin, were used. Mice were treated by daily intragastric administration of dapagliflozin (1 mg/kg) or vehicle for 6 weeks. The plasma insulin, glucagon and glucagon-like peptide-1 (GLP-1) were determined by using ELISA. The evaluation of islet morphology and cell phenotype was performed with immunofluorescence. Primary rodent islets and αTC1.9, a mouse alpha cell line, were incubated with dapagliflozin (0.25–25 μmol/L) or vehicle in the presence or absence of GLP-1 receptor antagonist for 24 h in regular or high glucose medium. The expression of specific markers and hormone levels were determined. ResultsTreatment with dapagliflozin significantly decreased blood glucose in the two diabetic models and upregulated plasma insulin and GLP-1 levels in db/db mice. The dapagliflozin treatment increased islet and beta cell numbers in the two diabetic mice. The beta cell proliferation as indicated by C-peptide and BrdU double-positive cells was boosted by dapagliflozin. The alpha to beta cell conversion, as evaluated by glucagon and insulin double-positive cells and confirmed by using alpha cell lineage-tracing, was facilitated by dapagliflozin. After the dapagliflozin treatment, some insulin-positive cells were located in the duct compartment or even co-localized with duct cell markers, suggestive of duct-derived beta cell neogenesis. In cultured primary rodent islets and αTC1.9 cells, dapagliflozin upregulated the expression of pancreatic endocrine progenitor and beta cell specific markers (including Pdx1) under high glucose condition. Moreover, dapagliflozin upregulated the expression of Pcsk1 (which encodes prohormone convertase 1/3, an important enzyme for processing proglucagon to GLP-1), and increased GLP-1 content and secretion in αTC1.9 cells. Importantly, the dapagliflozin-induced upregulation of Pdx1 expression was attenuated by GLP-1 receptor antagonist. ConclusionsExcept for glucose-lowering effect, dapagliflozin has extra protective effects on beta cells in type 2 diabetes. Dapagliflozin enhances beta cell self-replication, induces alpha to beta cell conversion, and promotes duct-derived beta cell neogenesis. The promoting effects of dapagliflozin on beta cell regeneration may be partially mediated via GLP-1 secreted from alpha cells.

2051-P: Human Autoantibodies to SerpinB13 Are Both Protective and Functional in Type 1 Diabetes

Our laboratory has identified a novel autoantibody (AA) to serpinB13, a protease inhibitor of cathepsin L, and has found an association of this AA with protection from type 1 diabetes (T1D) in subjects enrolled in the Diabetes Prevention Trial for Type 1 Diabetes (DPT-1). Our additional studies with a monoclonal antibody (mAb) to serpinB13 confirmed these observations in mouse, and implicated this mAb in a protease-mediated cleavage of the Notch 1 receptor as the mechanism of development of pancreatic Ngn3+ endocrine progenitors and resistance to severe diabetes. Whether human AA to serpinB13 plays a similar active role in protection against T1D remains unclear. To examine the potential functionality of human serpinB13 AA, we dialyzed DPT-1 serum samples that were either positive or negative for serpinB13 AA and used them to stimulate mouse embryonic pancreas explants (E12.5) during a 48-hour in vitro culture. Using a quantitative IF microscopy approach, we found that pancreas explants exposed to serpinB13 AA positive serum samples had 50% more Ngn3+ cells compared with pancreas explants incubated with serpinB13 AA negative samples (p=0.0029). This effect was dependent on serpinB13AA rather than other serum factors as positive serum samples immunodepleted of this AA were no longer able to stimulate development of additional Ngn3+ cells (p=0.0016). These data demonstrate that serpinB13 AA is actively involved in regulating the pathways that control development of endocrine cell lineage in the pancreas. Consequently, to our knowledge, serpinB13 AA is the first example of an autoantibody that is not solely a biomarker, but also an active player responsible for improved outcomes in T1D. To examine the role of embryonic serpinB13 AA in human diabetes in future, we aim to measure serpinB13 AA in pregnant women with T1D, or at significant risk for this disease, and assess its impact on the development of diabetes in their offspring after birth. Disclosure Y. Kryvalap: None. J. Czyzyk: None. Funding American Diabetes Association (I-17-ICTS-083 to J.C.); National Institutes of Health (DK10873901A1)

Purification of Live Stem-Cell-Derived Islet Lineage Intermediates.

Stem-cell-derived tissues offer platforms to study organ development, model physiology during health and disease, and test novel therapies. We describe methods to isolate cells at successive stages during in vitro differentiation of human stem cells into the pancreatic endocrine lineage. Using flow cytometry, we purify live lineage intermediates in numbers not available by fetal biopsy. These include pancreatic and endocrine progenitors, isolated based on known surface markers. We further report a strategy that leverages intracellular zinc content and DPP4/CD26 expression to separate monohormonal insulin+ β cells from polyhormonal counterparts. These methods enable comprehensive molecular profiling during human islet lineage progression. © 2020 Wiley Periodicals LLC. Basic Protocol: In vitro isolation of human islet developmental intermediates.

2046-P: Retinoic Acid Signaling in Pancreatic Endocrine Cells Regulates Islet Cell Specification

Diabetes is characterized by β cell death and/or dysfunction. Currently, one promising treatment option for patients with diabetes involves replenishing the body with β cells to restore glucose homeostasis. Despite great progress using in vitro differentiation protocols to generate human β cells from stem cell populations, there are still challenges associated with generating fully functional β cells. To improve this therapeutic strategy, my studies are focusing on defining the role of cell signaling pathways in pancreatic islet cell specification. Genetic studies in fish, mice, and humans have demonstrated that retinoic acid (RA) is broadly required for pancreas development; however, the role of RA signaling in pancreatic endocrine cell specification remains unclear. By examining the role of RA using in vivo endocrine-specific inhibition of RA signaling at critical time points during pancreas development, we demonstrate that inhibition of RA signaling in the endocrine progenitor population results in the generation fewer β and increased Δ cell genes by embryonic day (e)16.5. These defects persist after birth and are accompanied by overt hyperglycemia in the adult. Whole transcriptome analysis further indicates that cross-talk between RA signaling and the Wnt pathway is important for islet cell specification. In addition, we show that RA signaling is also important for human β cell specification. Together, these data demonstrate the importance of RA signaling in the endocrine progenitor population and provide a framework to more clearly define the mechanisms by which RA signaling directs endocrine cell fate in mice and humans. Future studies on the role of RA and its interacting pathways during pancreas development will better inform strategies to treat diabetes using cell replacement therapies. Disclosure D. Lorberbaum: None. L. Sussel: None. Funding American Diabetes Association (1-18-PDF-107 to D.L.)

258-OR: DNA Methylation Regulates the Identity of Pancreatic Beta Cells

Diabetes is associated with the loss of beta-cell identity and function. Thus, understanding the mechanisms that simultaneously regulate beta-cell function and identity is critical for developing beta-cell replacement therapies. In this study, we show that the de novo DNA methyltransferase, Dnmt3a, orchestrates beta-cell identity and function. Previously, we have shown that loss of Dnmt3a in pancreatic beta-cells prevents the developmental metabolic reprogramming, resulting in loss of glucose stimulated insulin secretion (GSIS). To gain insights into the role of new DNA methylation patterns established in early pancreas development, we deleted Dnmt3a in the embryonic pancreatic progenitors in mice. RNA sequencing of Dnmt3aKO and control islets in adult life revealed that loss of Dnmt3a in progenitors leads to dysregulation of key genes involved in the maintenance of beta-cell identity and function. Notably, loss of Dnmt3a led to the upregulation of Tyrosine Hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, along with dysregulation of several genes involved in Dopamine signaling. Specifically, a large proportion of beta-cells in KO was marked by TH expression, compared to controls. Bisulfite sequencing analysis revealed that TH expression is repressed in beta-cells due to DNA methylation during differentiation of endocrine progenitors. In addition, the TH positive beta-cells displayed G2/M cell cycle arrest, along with elevated ER-stress markers. This alteration of cell identity was accompanied by impaired glucose tolerance and a GSIS profile resembling functionally immature beta-cells in the KO mice. The Dnmt3aKO mice did not show any significant change in beta-cell mass, while delta- and alpha- cell masses were increased at 7 month. Taken together, our results show that de novo methylation during early pancreatic development is critical for the establishment of beta-cell identity and function. Disclosure N. Parveen: None. J.K. Wang: None. S. Dhawan: None. Funding National Institutes of Health (R01DK120523)

M6A mRNA Methylation Controls Functional Maturation in Neonatal Murine β-Cells.

The N 6-methyladenosine (m6A) RNA modification is essential during embryonic development of various organs. However, its role in embryonic and early postnatal islet development remains unknown. Mice in which RNA methyltransferase-like 3/14 (Mettl3/14) were deleted in Ngn3+ endocrine progenitors (Mettl3/14 nKO ) developed hyperglycemia and hypoinsulinemia at 2 weeks after birth. We found that Mettl3/14 specifically regulated both functional maturation and mass expansion of neonatal β-cells before weaning. Transcriptome and m6A methylome analyses provided m6A-dependent mechanisms in regulating cell identity, insulin secretion, and proliferation in neonatal β-cells. Importantly, we found that Mettl3/14 were dispensable for β-cell differentiation but directly regulated essential transcription factor MafA expression at least partially via modulating its mRNA stability. Failure to maintain this modification impacted the ability to fulfill β-cell functional maturity. In both diabetic db/db mice and patients with type 2 diabetes (T2D), decreased Mettl3/14 expression in β-cells was observed, suggesting its possible role in T2D. Our study unraveled the essential role of Mettl3/14 in neonatal β-cell development and functional maturation, both of which determined functional β-cell mass and glycemic control in adulthood.

An islet maturation media to improve the development of young porcine islets during in vitro culture

ABSTRACT Background The use of pancreata from pre-weaned piglets has the potential to serve as an unlimited alternative source of islets for clinical xenotransplantation. As pre-weaned porcine islets (PPIs) are immature and require prolonged culture, we developed an islet maturation media (IMM) and evaluated its effect on improving the quantity and quality of PPIs over 14 days of culture. Methods PPIs were isolated from the pancreata of pre-weaned Yorkshire piglets (8–15 days old). Each independent islet isolation was divided for culture in either control Ham’s F-10 media (n = 5) or IMM (n = 5) for 14 days. On day 3, 7 and 14 of culture, islets were assessed for islet yield, isolation index, viability, insulin content, endocrine cellular composition, differentiation of beta cells, and insulin secretion during glucose stimulation. Results In comparison to control islets, culturing PPIs in IMM significantly increased islet yield. PPIs cultured in IMM also maintained a stable isolation index and viability throughout 14 days of culture. The insulin content, endocrine cellular composition, and differentiation of beta cells were significantly improved in PPIs cultured in IMM, which subsequently augmented their insulin secretory capacity in response to glucose challenge compared to control islets. Conclusions Culturing PPIs in IMM increases islet yield, isolation index, viability, insulin content, endocrine cellular composition, differentiation of endocrine progenitor cells toward beta cells, and insulin secretion. Due to the improved islet quantity and quality after in vitro culture, the use of IMM in the culture of PPIs will assist to advance the outcomes of clinical islet xenotransplantation.

Coregulator Sin3a Promotes Postnatal Murine β-Cell Fitness by Regulating Genes in Ca2+ Homeostasis, Cell Survival, Vesicle Biosynthesis, Glucose Metabolism, and Stress Response.

Swi-independent 3a and 3b (Sin3a and Sin3b) are paralogous transcriptional coregulators that direct cellular differentiation, survival, and function. Here, we report that mouse Sin3a and Sin3b are coproduced in most pancreatic cells during embryogenesis but become much more enriched in endocrine cells in adults, implying continued essential roles in mature endocrine cell function. Mice with loss of Sin3a in endocrine progenitors were normal during early postnatal stages but gradually developed diabetes before weaning. These physiological defects were preceded by the compromised survival, insulin-vesicle packaging, insulin secretion, and nutrient-induced Ca2+ influx of Sin3a-deficient β-cells. RNA sequencing coupled with candidate chromatin immunoprecipitation assays revealed several genes that could be directly regulated by Sin3a in β-cells, which modulate Ca2+/ion transport, cell survival, vesicle/membrane trafficking, glucose metabolism, and stress responses. Finally, mice with loss of both Sin3a and Sin3b in multipotent embryonic pancreatic progenitors had significantly reduced islet cell mass at birth, caused by decreased endocrine progenitor production and increased β-cell death. These findings highlight the stage-specific requirements for the presumed "general" coregulators Sin3a and Sin3b in islet β-cells, with Sin3a being dispensable for differentiation but required for postnatal function and survival.

The Ldb1 Transcriptional Co‐Regulator is Required for Establishment, Development, and Survival of the Pancreatic Endocrine Lineage

Pancreatic islets are comprised of hormone secreting cell types that are vital regulators of glucose metabolism. Specifically, the pancreatic β cell is indispensable for glucose control and its dysfunction is central to diabetes mellitus. β cell development is regulated by transcription factor (TF) cascades, mediating differentiation of progenitors into mature insulin producing β cells. Our prior studies show that TF, Islet1 (Isl1), interacts with a transcriptional co‐regulator, Ldb1, to regulate β cell maturation from embryonic day (E) 18.5 onward. However, Ldb1 is also expressed in early stages of pancreas development, before Isl1 is present (as early as E10.5). The earlier Ldb1+ cell types include Pdx1+ multipotent progenitor cells (MPCs) and endocrine progenitors expressing the TF Neurogenin3 (Ngn3). MPC progeny will populate the entire pancreas (endocrine and exocrine), while Ngn3+ endocrine progenitors (Isl1−) are fated to become islet cells. Our hypothesis is that Ldb1 has Isl1 independent roles in maintaining progenitor identity in these requisite populations during early pancreatic development. To test this we generated a whole pancreas knockout of Ldb1 (Ldb1Δpanc) and observed severe developmental and postnatal phenotypes. At E13.5, Ldb1Δpanc mice exhibit disorganized progenitor pools, suggesting early defects in endocrine identity. At E15.5, Ldb1Δpanc mice had a significant reduction of Ngn3+ progenitors and Pdx1HI immunoreactivity, a mark of presumptive β cells. Ldb1Δpanc mice die by postnatal day 7 (P7) with severe hyperglycemia and hypoinsulinemia due to drastic islet hormone cell reduction. Interestingly, total pancreatic mass remained unchanged in Ldb1Δpanc neonates, suggesting that Ldb1 impacts are islet specific in the pancreas. Considering these observations, we generated a new model of Ldb1 loss specifically in Ngn3+ islet progenitors, termed Ldb1Δendo. We confirmed loss of Ldb1 in endocrine clusters and observed postnatal hyperglycemia, with a reduction of islet cells in neonates, similar to that seen in Ldb1Δpanc mice. Chromatin immunoprecipitation in vivo and in vitro highlights that Ldb1 imparts control on Pdx1 through occupation of the Pdx1 Area I–II regulatory domains, and that Ngn3 control also occurs via direct occupation. We are now further assessing the developmental phenotype, examining markers of proliferation, apoptosis, and altered cell identity through lineage tracing in the Ldb1Δendo model. Concomitantly, our published work revealed that Ldb1 and Isl1 interact in complex with the single stranded DNA binding protein co‐regulator, SSBP3, which helps stabilize this transcriptional complex. To examine the functional relationship between SSBP3, Isl1, and Ldb1 in vivo, we developed a new SSBP3 floxed mouse model in order to generate pancreas wide loss of SSBP3 (SSBP3Δpanc). We observed preliminary reductions in key islet mRNAs along with hyperglycemia in neonates at P1. Our work provides insight into the transcriptional complexes dictating how islet progenitors adopt their cell fate. Enhancing our understanding of this differentiation process can contribute to therapeutic β and islet cell generation strategies to help the growing diabetic population.Support or Funding InformationNIH‐NIDDK R01DK111483‐01NIH‐F31DK120217NIH‐NIGMS T32GM008111

Islet-Resident Endocrine Progenitors: A New Hope for Beta Cell PROCReation?

The existence of an endocrine progenitor in the adult mouse pancreas has been controversial. Recently in Cell, Wang etal. (2020) use the cell-surface marker Procr to define a population of cells within the adult islet of Langerhans that is capable of generating all endocrine cells and is amenable to invitro expansion.

A Comprehensive Molecular Characterization of the Pancreatic Neuroendocrine Tumor Cell Lines BON-1 and QGP-1.

Experimental models of neuroendocrine tumor disease are scarce, with only a few existing neuroendocrine tumor cell lines of pancreatic origin (panNET). Their molecular characterization has so far focused on the neuroendocrine phenotype and cancer-related mutations, while a transcription-based assessment of their developmental origin and malignant potential is lacking. In this study, we performed immunoblotting and qPCR analysis of neuroendocrine, epithelial, developmental endocrine-related genes as well as next-generation sequencing (NGS) analysis of microRNAs (miRs) on three panNET cell lines, BON-1, QGP-1, and NT-3. All three lines displayed a neuroendocrine and epithelial phenotype; however, while insulinoma-derived NT-3 cells preferentially expressed markers of mature functional pancreatic β-cells (i.e., INS, MAFA), both BON-1 and QGP-1 displayed high expression of genes associated with immature or non-functional β/δ-cells genes (i.e., NEUROG3), or pancreatic endocrine progenitors (i.e., FOXA2). NGS-based identification of miRs in BON-1 and QGP-1 cells revealed the presence of all six members of the miR-17–92 cluster, which have been implicated in β-cell function and differentiation, but also have roles in cancer being both oncogenic or tumor suppressive. Notably, both BON-1 and QGP-1 cells expressed several miRs known to be negatively associated with epithelial–mesenchymal transition, invasion or metastasis. Moreover, both cell lines failed to exhibit migratory activity in vitro. Taken together, NT-3 cells resemble mature functional β-cells, while both BON-1 and QGP-1 are more similar to immature/non-functional pancreatic β/δ-cells or pancreatic endocrine progenitors. Based on the recent identification of three transcriptional subtypes in panNETs, NT-3 cells resemble the “islet/insulinoma tumors” (IT) subtype, while BON-1 and QGP-1 cells were tentatively classified as “metastasis-like/primary” (MLP). Our results provide a comprehensive characterization of three panNET cell lines and demonstrate their relevance as neuroendocrine tumor models.

Intermittent Fasting as Part of the Management for T2DM: from Animal Models to Human Clinical Studies.

Diet is a pillar of type 2 diabetes mellitus (T2DM) management. Intermittent fasting (IF) is postulated as a novel approach, able to improve glucose control and potentially capable of reversing some of the pathophysiological alterations of this condition. In this review, the molecular and clinical evidence of diets based on intermittent energy restriction (IER) in laboratory animal models and subjects with type 2 diabetes is discussed. The mechanisms through which IF are thought to improve glucose homeostasis and reverse β cell failure are also reviewed. Studies derived from murine models suggest that IER is associated with improvements in β cell function and insulin resistance. Two main mechanisms have been demonstrated, one derived from the autophagy-lysosome pathway and, the other from an increase in neurogenin3 (Ngn3) levels (a marker for endocrine progenitor cells like β cells during development). Notably, IER also promotes reconstruction of gut microbiota. In mice, all effects were independent of weight loss. By contrast, in human studies, outcomes are widely attributable to weight loss. The more consistent results are reductions in body weight, visceral fat, and glucose and insulin levels. Increases in HDL cholesterol levels are also frequently reported. The decrease in insulin levels observed in humans is in opposition with the increase reported in mice, suggesting that the main mechanism in humans is an improvement in peripheral insulin action. Recommending diets based on intermittent fasting in humans is based on the promising results found in animal models where an improvement in β cell function has been recorded. β cell function after IF has not been assessed in human subjects with T2DM. This review provides information regarding different protocols for the implementation of IF in diabetic persons and also provides important safety advice in order to avoid adverse effects. Clinical studies do not show an increased risk of hypoglycemia, and a recent case series reported reversal of T2DM.

Reversible expansion of pancreatic islet progenitors derived from human induced pluripotent stem cells.

Transplantation of pancreatic islets is an effective therapy for severe type 1 diabetes. As donor shortage is a major problem for this therapy, attempts have been made to produce a large number of pancreatic islets from human pluripotent stem cells (hPSCs). However, as the differentiation of hPSCs to pancreatic islets requires multiple and lengthy processes using various expensive cytokines, the process is variable, low efficiency and costly. Therefore, it would be beneficial if islet progenitors could be expanded. Neurogenin3 (NGN3)-expressing pancreatic endocrine progenitor (EP) cells derived from hPSCs exhibited the ability to differentiate into pancreatic islets while their cell cycle was arrested. By using a lentivirus vector, we introduced several growth-promoting genes into NGN3-expressing EP cells. We found that SV40LT expression induced proliferation of the EP cells but reduced the expression ofendocrine lineage-commitment factors, NGN3, NEUROD1 and NKX2.2, resulting in the suppression of islet differentiation. By using the Cre-loxP system, we removed SV40LT after the expansion, leading to re-expression of endocrine-lineage commitment genes and differentiation into functional pancreatic islets. Thus, our findings will pave a way to generate a large quantity of functional pancreatic islets through the expansion of EP cells from hPSCs.

Long-Term Expansion of Pancreatic Islet Organoids from Resident Procr+ Progenitors

It has generally proven challenging to produce functional β cells invitro. Here, we describe a previously unidentified protein C receptor positive (Procr+) cell population in adult mouse pancreas through single-cell RNA sequencing (scRNA-seq). The cells reside in islets, do not express differentiation markers, and feature epithelial-to-mesenchymal transition characteristics. By genetic lineage tracing, Procr+ islet cells undergo clonal expansion and generate all four endocrine cell types during adult homeostasis. Sorted Procr+ cells, representing ∼1% of islet cells, can robustly form islet-like organoids when cultured at clonal density. Exponential expansion can be maintained over long periods by serial passaging, while differentiation can be induced at any time point in culture. β cells dominate in differentiated islet organoids, while α, δ, and PP cells occur at lower frequencies. The organoids are glucose-responsive and insulin-secreting. Upon transplantation in diabetic mice, these organoids reverse disease. These findings demonstrate that the adult mouse pancreatic islet contains a population of Procr+ endocrine progenitors.

ISX-9 manipulates endocrine progenitor fate revealing conserved intestinal lineages in mouse and human organoids

ObjectiveEnteroendocrine cells (EECs) survey the gut luminal environment and coordinate hormonal, immune and neuronal responses to it. They exhibit well-characterised physiological roles ranging from the control of local gut function to whole body metabolism, but little is known regarding the regulatory networks controlling their differentiation, especially in the human gut. The small molecule isoxazole-9 (ISX-9) has been shown to stimulate neuronal and pancreatic beta-cell differentiation, both closely related to EEC differentiation. Our aim was to use ISX-9 as a tool to explore EEC differentiation. MethodsWe investigated the effects of ISX-9 on EEC differentiation in mouse and human intestinal organoids, using real-time quantitative polymerase chain reaction (RT-qPCR), fluorescent-activated cell sorting, immunostaining and single-cell RNA sequencing. ResultsISX-9 increased the number of neurogenin3-RFP (Ngn3)-positive endocrine progenitor cells and upregulated NeuroD1 and Pax4, transcription factors that play roles in mouse EEC specification. Single-cell analysis showed induction of Pax4 expression in a developmentally late Ngn3+ population of cells and potentiation of genes associated with progenitors biased toward serotonin-producing enterochromaffin (EC) cells. Further, we observed enrichment of organoids with functional EC cells that was partly dependent on stimulation of calcium signalling in a population of cells residing outside the crypt base. Inducible Pax4 overexpression, in ileal organoids, uncovered its importance as a component of early human endocrine specification and highlighted the potential existence of two major endocrine lineages, the early appearing enterochromaffin lineage and the later developing peptidergic lineage which contains classical gut hormone cell types. ConclusionOur data provide proof-of-concept for the controlled manipulation of specific endocrine lineages with small molecules, whilst also shedding new light on human EEC differentiation and its similarity to the mouse. Given their diverse roles, understanding endocrine lineage plasticity and its control could have multiple therapeutic implications.

Insulin-Deficient Diabetic Condition Upregulates the Insulin-Secreting Capacity of Human Induced Pluripotent Stem Cell-Derived Pancreatic Endocrine Progenitor Cells After Implantation in Mice.

The host environment is a crucial factor for considering the transplant of stem cell-derived immature pancreatic cells in patients with type 1 diabetes. Here, we investigated the effect of insulin (INS)-deficient diabetes on the fate of immature pancreatic endocrine cell grafts and the underlying mechanisms. Human induced pluripotent stem cell-derived pancreatic endocrine progenitor cells (EPCs), which contained a high proportion of chromogranin A+ NK6 homeobox 1+ cells and very few INS+ cells, were used. When the EPCs were implanted under the kidney capsule in immunodeficient mice, INS-deficient diabetes accelerated increase in plasma human C-peptide, a marker of graft-derived INS secretion. The acceleration was suppressed by INS infusion but not affected by partial attenuation of hyperglycemia by dapagliflozin, an INS-independent glucose-lowering agent. Immunohistochemical analyses indicated that the grafts from diabetic mice contained more endocrine cells including proliferative INS-producing cells compared with that from nondiabetic mice, despite no difference in whole graft mass between the two groups. These data suggest that INS-deficient diabetes upregulates the INS-secreting capacity of EPC grafts by increasing the number of endocrine cells including INS-producing cells without changing the graft mass. These findings provide useful insights into postoperative diabetic care for cell therapy using stem cell-derived pancreatic cells.

Adaptive Landscape Shaped by Core Endogenous Network Coordinates Complex Early Progenitor Fate Commitments in Embryonic Pancreas

The classical development hierarchy of pancreatic cell fate commitments describes that multipotent progenitors (MPs) first bifurcate into tip cells and trunk cells, and then these cells give rise to acinar cells and endocrine/ductal cells separately. However, lineage tracings reveal that pancreatic progenitors are highly heterogeneous in tip and trunk domains in embryonic pancreas. The progenitor fate commitments from multipotency to unipotency during early pancreas development is insufficiently characterized. In pursuing a mechanistic understanding of the complexity in progenitor fate commitments, we construct a core endogenous network for pancreatic lineage decisions based on genetic regulations and quantified its intrinsic dynamic properties using dynamic modeling. The dynamics reveal a developmental landscape with high complexity that has not been clarified. Not only well-characterized pancreatic cells are reproduced, but also previously unrecognized progenitors—tip progenitor (TiP), trunk progenitor (TrP), later endocrine progenitor (LEP), and acinar progenitors (AciP/AciP2) are predicted. Further analyses show that TrP and LEP mediate endocrine lineage maturation, while TiP, AciP, AciP2 and TrP mediate acinar and ductal lineage maturation. The predicted cell fate commitments are validated by analyzing single-cell RNA sequencing (scRNA-seq) data. Significantly, this is the first time that a redefined hierarchy with detailed early pancreatic progenitor fate commitment is obtained.

Long-Term Liraglutide Administration Induces Pancreas Neogenesis in Adult T2DM Mice

In vivo beta-cell neogenesis may be one way to treat diabetes. We aimed to investigate the effect of glucagon-like peptide-1 (GLP-1) on beta-cell neogenesis in type 2 diabetes mellitus (T2DM). Male C57BL/6J mice, 6 wk old, were randomly divided into three groups: Control, T2DM, and T2DM + Lira. T2DM was induced using high-fat diet and intraperitoneal injection of streptozotocin (40 mg/kg/d for 3 d). At 8 wk after streptozotocin injection, T2DM + Lira group was injected intraperitoneally with GLP-1 analog liraglutide (0.8 mg/kg/d) for 4 wk. Apparently for the first time, we report the appearance of a primitive bud connected to pancreas in all adult mice from each group. The primitive bud was characterized by scattered single monohormonal cells expressing insulin, GLP-1, somatostatin, or pancreatic polypeptide, and four-hormonal cells, but no acinar cells and ductal epithelial cells. Monohormonal cells in it were small, newborn, immature cells that rapidly proliferated and expressed cell markers indicative of immaturity. In parallel, Ngn3+ endocrine progenitors and Nestin+ cells existed in the primitive bud. Liraglutide facilitated neogenesis and rapid growth of acinar cells, pancreatic ducts, and blood vessels in the primitive bud. Meanwhile, scattered hormonal cells aggregated into cell clusters and grew into larger islets; polyhormonal cells differentiated into monohormonal cells. Extensive growth of exocrine and endocrine glands resulted in the neogenesis of immature pancreatic lobes in adult mice of T2DM + Lira group. Contrary to predominant acinar cells in mature pancreatic lobes, there were still a substantial number of mesenchymal cells around acinar cells in immature pancreatic lobes, which resulted in the loose appearance. Our results suggest that adult mice preserve the capacity of pancreatic neogenesis from the primitive bud, which liraglutide facilitates in adult T2DM mice. To our knowledge, this is the first time such a phenomenon has been reported.

Retinoic acid signaling within pancreatic endocrine progenitors regulates mouse and human β 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 β 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.