Differentiation Of Pancreatic Endocrine Cells Research Articles

Identification and removal of unexpected proliferative off-target cells emerging after iPSC-derived pancreatic islet cell implantation

Differentiation of pancreatic endocrine cells from human pluripotent stem cells (PSCs) has been thoroughly investigated for application in cell therapy against diabetes. In the context of induced pancreatic endocrine cell implantation, previous studies have reported graft enlargement resulting from off-target pancreatic lineage cells. However, there is currently no documented evidence of proliferative off-target cells beyond the pancreatic lineage in existing studies. Here, we show that the implantation of seven-stage induced PSC-derived pancreatic islet cells (s7-iPICs) leads to the emergence of unexpected off-target cells with proliferative capacity via in vivo maturation. These cells display characteristics of both mesenchymal stem cells (MSCs) and smooth muscle cells (SMCs), termed proliferative MSC- and SMC-like cells (PMSCs). The frequency of PMSC emergence was found to be high when 108 s7-iPICs were used. Given that clinical applications involve the use of a greater number of induced cells than 108, it is challenging to ensure the safety of clinical applications unless PMSCs are adequately addressed. Accordingly, we developed a detection system and removal methods for PMSCs. To detect PMSCs without implantation, we implemented a 4-wk-extended culture system and demonstrated that putative PMSCs could be reduced by compound treatment, particularly with the taxane docetaxel. When docetaxel-treated s7-iPICs were implanted, the PMSCs were no longer observed. This study provides useful insights into the identification and resolution of safety issues, which are particularly important in the field of cell-based medicine using PSCs.

Insulin regulates human pancreatic endocrine cell differentiation in vitro

ObjectiveThe consequences of mutations in genes associated with monogenic forms of diabetes on human pancreas development cannot be studied in a time-resolved fashion in vivo. More specifically, if recessive mutations in the insulin gene influence human pancreatic endocrine lineage formation is still an unresolved question. MethodsTo model the extremely reduced insulin levels in patients with recessive insulin gene mutations, we generated a novel knock-in H2B-Cherry reporter human induced pluripotent stem cell (iPSC) line expressing no insulin upon differentiation to stem cell-derived (SC-) β cells in vitro. Differentiation of iPSCs into the pancreatic and endocrine lineage, combined with immunostaining, Western blotting and proteomics analysis phenotypically characterized the insulin gene deficiency in SC-islets. Furthermore, we leveraged FACS analysis and confocal microscopy to explore the impact of insulin shortage on human endocrine cell induction, composition, differentiation and proliferation. ResultsInterestingly, insulin-deficient SC-islets exhibited low insulin receptor (IR) signaling when stimulated with glucose but displayed increased IR sensitivity upon treatment with exogenous insulin. Furthermore, insulin shortage did not alter neurogenin-3 (NGN3)-mediated endocrine lineage induction. Nevertheless, lack of insulin skewed the SC-islet cell composition with an increased number in SC-β cell formation at the expense of SC-α cells. Finally, insulin deficiency reduced the rate of SC-β cell proliferation but had no impact on the expansion of SC-α cells. ConclusionsUsing iPSC disease modelling, we provide first evidence of insulin function in human pancreatic endocrine lineage formation. These findings help to better understand the phenotypic impact of recessive insulin gene mutations during pancreas development and shed light on insulin gene function beside its physiological role in blood glucose regulation.

Decoding pancreatic endocrine cell differentiation and β cell regeneration in zebrafish.

In contrast to mice, zebrafish have an exceptional yet elusive ability to replenish lost β cells in adulthood. Understanding this framework would provide mechanistic insights for β cell regeneration, which may be extrapolated to humans. Here, we characterize a krt4-expressing ductal cell type, which is distinct from the putative Notch-responsive cells, showing neogenic competence and giving rise to the majority of endocrine cells during postembryonic development. Furthermore, we demonstrate a marked ductal remodeling process featuring a Notch-responsive to krt4+ luminal duct transformation during late development, indicating several origins of krt4+ ductal cells displaying similar transcriptional patterns. Single-cell transcriptomics upon a series of time points during β cell regeneration unveil a previously unrecognized dlb+ transitional endocrine precursor cell, distinct regulons, and a differentiation trajectory involving cellular shuffling through differentiation and dedifferentiation dynamics. These results establish a model of zebrafish pancreatic endocrinogenesis and highlight key values of zebrafish for translational studies of β cell regeneration.

Characterization of cell-fate decision landscapes by estimating transcription factor dynamics

Time-specific modulation of gene expression during differentiation by transcription factors promotes cell diversity. However, estimating their dynamic regulatory activity at the single-cell level and in a high-throughput manner remains challenging. We present FateCompass, an integrative approach that utilizes single-cell transcriptomics data to identify lineage-specific transcription factors throughout differentiation. By combining a probabilistic framework with RNA velocities or differentiation potential, we estimate transition probabilities, while a linear model of gene regulation is employed to compute transcription factor activities. Considering dynamic changes and correlations of expression and activities, FateCompass identifies lineage-specific regulators. Our validation using in silico data and application to pancreatic endocrine cell differentiation datasets highlight both known and potentially novel lineage-specific regulators. Notably, we uncovered undescribed transcription factors of an enterochromaffin-like population during invitro differentiation toward ß-like cells. FateCompass provides a valuable framework for hypothesis generation, advancing our understanding of the gene regulatory networks driving cell-fate decisions.

A case of early-onset diabetes with impaired insulin secretion carrying a PAX6 gene Gln135* mutation.

SummaryA paired homeodomain transcription factor, PAX6 (paired-box 6), is essential for the development and differentiation of pancreatic endocrine cells as well as ocular cells. Despite the impairment of insulin secretion observed in PAX6-deficient mice, evidence implicating causal association between PAX6 gene mutations and monogenic forms of human diabetes is limited. We herein describe a 33-year-old Japanese woman with congenital aniridia who was referred to our hospital because of her uncontrolled diabetes with elevated hemoglobin A1c (13.1%) and blood glucose (32.5 mmol/L) levels. Our biochemical analysis revealed that her insulin secretory capacity was modestly impaired as represented by decreased 24-h urinary C-peptide levels (38.0 μg/day), primarily explaining her diabetes. Intriguingly, there was a trend toward a reduction in her serum glucagon levels as well. Based on the well-recognized association of PAX6 gene mutations with congenital aniridia, we screened the whole PAX6 coding sequence, leading to an identification of a heterozygous Gln135* mutation. We tested our idea that this mutation may at least in part explain the impaired insulin secretion observed in this patient. In cultured pancreatic β-cells, exogenous expression of the PAX6 Gln135* mutant produced a truncated protein that lacked the transcriptional activity to induce insulin gene expression. Our observation together with preceding reports support the recent attempt to include PAX6 in the growing list of genes causally responsible for monogenic diabetes. In addition, since most cases of congenital aniridia carry PAX6 mutations, we may need to pay more attention to blood glucose levels in these patients.Learning pointsPAX6 Gln135* mutation may be causally associated not only with congenital aniridia but also with diabetes.Blood glucose levels may deserve more attention in cases of congenital aniridia with PAX6 mutations.Our case supports the recent attempt to include PAX6 in the list of MODY genes, and Gln135* may be pathogenic.

Single-cell transcriptome and accessible chromatin dynamics during endocrine pancreas development

Delineating gene regulatory networks that orchestrate cell-type specification is a continuing challenge for developmental biologists. Single-cell analyses offer opportunities to address these challenges and accelerate discovery of rare cell lineage relationships and mechanisms underlying hierarchical lineage decisions. Here, we describe the molecular analysis of mouse pancreatic endocrine cell differentiation using single-cell transcriptomics, chromatin accessibility assays coupled to genetic labeling, and cytometry-based cell purification. We uncover transcription factor networks that delineate β-, α-, and δ-cell lineages. Through genomic footprint analysis, we identify transcription factor-regulatory DNA interactions governing pancreatic cell development at unprecedented resolution. Our analysis suggests that the transcription factor Neurog3 may act as a pioneer transcription factor to specify the pancreatic endocrine lineage. These findings could improve protocols to generate replacement endocrine cells from renewable sources, like stem cells, for diabetes therapy.

Characterization and reduction of non-endocrine cells accompanying islet-like endocrine cells differentiated from human iPSC

The differentiation of pancreatic endocrine cells from human pluripotent stem cells has been thoroughly investigated for their application in cell therapy against diabetes. Although non-endocrine cells are inevitable contaminating by-products of the differentiation process, a comprehensive profile of such cells is lacking. Therefore, we characterized non-endocrine cells in iPSC-derived pancreatic islet cells (iPIC) using single-cell transcriptomic analysis. We found that non-endocrine cells consist of (1) heterogeneous proliferating cells, and (2) cells with not only pancreatic traits but also liver or intestinal traits marked by FGB or AGR2. Non-endocrine cells specifically expressed FGFR2, PLK1, and LDHB. We demonstrated that inhibition of pathways involving these genes selectively reduced the number of non-endocrine cells in the differentiation process. These findings provide useful insights into cell purification approaches and contribute to the improvement of the mass production of endocrine cells for stem cell-derived cell therapy for diabetes.

177-OR: The Swi/Snf Chromatin Remodeling Complex Controls the Specification, Expansion, and Differentiation of Pancreatic Endocrine Progenitor Cells

A key feature of type 1 and type 2 diabetes is insufficient functional β-cell mass. Strategies to augment functional β-cell mass include directed differentiation of embryonic stem cells towards a β-cell fate, which requires extensive knowledge of the transcriptional programs governing endocrine cell differentiation in vivo. Pdx1, an essential transcription factor involved in pancreas formation, endocrine progenitor cell expansion and β-cell function, dynamically recruits coregulators to drive gene expression programs in those cells vital for its activity. Pdx1 binds to numerous coregulators in the β-cell, including the ATP-dependent Swi/Snf chromatin remodeling complex. Previous work by our group revealed a critical role for Pdx1:Swi/Snf in both the developing pancreas and mature β-cell. Here we interrogate the role of Swi/Snf on governing Pdx1-dependent gene expression programs and chromatin accessibility in pancreatic endocrine cell development. We developed a mouse model to eliminate the activity of the two core ATPase subunits of Swi/Snf, Brg1 and Brm from endocrine progenitor cells using Neurogenin3-Cre (Δendo). Conditional knockout of Brg1 (Brg1ΔendoBrm+/-) from endocrine progenitor cells leads to severe glucose intolerance, ad-libitum fed hyperglycemia, reduced serum insulin, compromised glucose stimulated insulin secretion from isolated islets and reduced islet mass in 4-week old animals. Whereas Brg1Δendo/+Brm-/- mice are normal, remarkably, no DKOΔendo (double knockout (DKO): targeted both Brg1 and Brm) mice have been found at weaning, suggesting loss of all Swi/Snf activity from the endocrine progenitor cell pool is postnatally lethal. Future experiments will be directed at determining how Swi/Snf controls Pdx1-dependent gene expression programs and chromatin accessibility in endocrine progenitor cells that are critical for islet cell expansion, differentiation and maturation. Disclosure J. Spaeth: None. R. K. Davidson: None. N. Casey: None. Funding National Institutes of Health (R03DK127129, K01DK115633)

The SNAG Domain of Insm1 Regulates Pancreatic Endocrine Cell Differentiation and Represses β- to δ-Cell Transdifferentiation.

The allocation and specification of pancreatic endocrine lineages are tightly regulated by transcription factors. Disturbances in differentiation of these lineages contribute to the development of various metabolic diseases, including diabetes. The insulinoma-associated protein 1 (Insm1), which encodes a protein containing one SNAG domain and five zinc fingers, plays essential roles in pancreatic endocrine cell differentiation and in mature β-cell function. In the current study, we compared the differentiation of pancreatic endocrine cells between Insm1 null and Insm1 SNAG domain mutants (Insm1delSNAG) to explore the specific function of the SNAG domain of Insm1. We show that the δ-cell number is increased in Insm1delSNAG but not in Insm1 null mutants as compared with the control mice. We also show a less severe reduction of the β-cell number in Insm1delSNAG as that in Insm1 null mutants. In addition, similar deficits are observed in α-, PP, and ε-cells in Insm1delSNAG and Insm1 null mutants. We further identified that the increased δ-cell number is due to β- to δ-cell transdifferentiation. Mechanistically, the SNAG domain of Insm1 interacts with Lsd1, the demethylase of H3K4me1/2. Mutation in the SNAG domain of Insm1 results in impaired recruitment of Lsd1 and increased H3K4me1/2 levels at hematopoietically expressed homeobox (Hhex) loci that are bound by Insm1, thereby promoting the transcriptional activity of the δ-cell-specific gene Hhex Our study has identified a novel function of the SNAG domain of Insm1 in the regulation of pancreatic endocrine cell differentiation, particularly in the repression of β- to δ-cell transdifferentiation.

The effect of short-term necrostatin-1 treatment on the differentiation of human induced pluripotent stem beta cells

: Despite ongoing effort over the past decade, current protocols have been unable to generate mature functional beta cells from human induced pluripotent stem cells (hiPSCs). Based on our previous findings that short-term necrostatin-1 (Nec-1) treatment enhanced pancreatic endocrine cell differentiation and insulin secretion capacity of young porcine islets, we assessed whether short-term treatment of Nec-1 can improve the differentiation and function of hiPS beta cells. After 3 days of culture, hiPS beta cells, cultured in either control differentiation media (n=3) or supplemented with Nec-1 (100 µM, n=3), were evaluated for viability, cellular composition, GLUT2 expression in beta cells, and glucose-stimulated insulin expression. While the viability and levels of beta-, alpha-, and GLUT2-positive beta cells were unaffected, the level of insulin- and glucagon-positive bi-hormonal cells was significantly lower in Nec-1 treated hiPS beta cells. Short-term Nec-1 treatment also did not affect the insulin secretion ability of hiPS beta cells. The addition of Nec-1 to the differentiation media of hiPS beta cells reduced the number of bi-hormonal cells after short-term culture, suggesting that future studies should evaluate different concentrations and treatment duration of Nec-1.

A Network of microRNAs Acts to Promote Cell Cycle Exit and Differentiation of Human Pancreatic Endocrine Cells.

SummaryPancreatic endocrine cell differentiation is orchestrated by the action of transcription factors that operate in a gene regulatory network to activate endocrine lineage genes and repress lineage-inappropriate genes. MicroRNAs (miRNAs) are important modulators of gene expression, yet their role in endocrine cell differentiation has not been systematically explored. Here we characterize miRNA-regulatory networks active in human endocrine cell differentiation by combining small RNA sequencing, miRNA over-expression, and network modeling approaches. Our analysis identified Let-7g, Let-7a, miR-200a, miR-127, and miR-375 as endocrine-enriched miRNAs that drive endocrine cell differentiation-associated gene expression changes. These miRNAs are predicted to target different transcription factors, which converge on genes involved in cell cycle regulation. When expressed in human embryonic stem cell-derived pancreatic progenitors, these miRNAs induce cell cycle exit and promote endocrine cell differentiation. Our study delineates the role of miRNAs in human endocrine cell differentiation and identifies miRNAs that could facilitate endocrine cell reprogramming.

Necrostatin-1 supplementation enhances young porcine islet maturation and in vitro function.

Necroptosis has been demonstrated to be a primary mechanism of islet cell death. This study evaluated whether the supplementation of necrostatin-1 (Nec-1), a potent inhibitor of necroptosis, to islet culture media could improve the recovery, maturation, and function of pre-weaned porcine islets (PPIs). PPIs were isolated from pre-weaned Yorkshire piglets (8-15days old) and either cultured in control islet culture media (n=6) or supplemented with Nec-1 (100µM, n=5). On days 3 and 7 of culture, islets were assessed for recovery, insulin content, viability, cellular composition, GLUT2 expression in beta cells, differentiation of pancreatic endocrine progenitor cells, function, and oxygen consumption rate. Nec-1 supplementation induced a 2-fold increase in the insulin content of PPIs on day 7 of culture. When compared to untreated islets, Nec-1 treatment doubled the beta- and alpha-cell composition and accelerated the development of delta cells. Additionally, beta cells of Nec-1-treated islets had a significant upregulation in GLUT2 expression. The enhanced development of major endocrine cells and GLUT2 expression after Nec-1 treatment subsequently led to a significant increase in the amount of insulin secreted in response to in vitro glucose challenge. Islet recovery, viability, and oxygen consumption rate were unaffected by Nec-1. This study underlines the importance of necroptosis in islet cell death after isolation and demonstrates the novel effects of Nec-1 to increase islet insulin content, enhance pancreatic endocrine cell development, facilitate GLUT2 upregulation in beta cells, and augment insulin secretion. Nec-1 supplementation to culture media significantly improves islet quality prior to xenotransplantation.

105-OR: Lineage Determination of Endocrine Progenitors in Murine and Human Pancreatic Development

Generation of functional beta cells for regenerative medicine would benefit from an increased understanding of pancreatic endocrine cell differentiation. During pancreatic development, expression of Ngn3 defines the progenitor cells that will differentiate into one of five endocrine lineages, including the beta cell. Here, we identify and further characterize a distinct endocrine progenitor population defined by differential expression of a transcription factor named Fev in murine and human endocrine cell development. Using single-cell RNA-sequencing and genetic lineage tracing, we build a transcriptomic profile of murine Fev+ progenitors that is distinct from that of Ngn3+ progenitors and reconstruct lineage relationships among Ngn3+, Fev+, and differentiated, hormone+ cells. We further identify candidate transcriptional regulators along the differentiation trajectory of this Fev+ progenitor population toward the alpha or beta cell lineages. Translating our findings in murine pancreatic development to that of human, we identify an analogous FEV+ population and putative gene regulators of endocrine cell fate in human fetal pancreas. In a stepwise differentiation platform designed to generate beta cells from human embryonic stem cells (hESCs), we also uncover robust FEV expression in endocrine progenitors prior to their differentiation into insulin-producing beta cells. Single-cell RNA-sequencing and lineage reconstruction of hESC-derived cells at the differentiated beta cell stage reveal FEV+ cells that may represent endocrine progenitors that have failed to acquire a beta cell identity; this failure of FEV+ endocrine cell differentiation may facilitate in uncovering the required molecular signals and genetic circuitry that push endocrine progenitors toward a beta cell fate. This work establishes a new model of pancreatic endocrine cell differentiation and identifies a Fev+ endocrine progenitor as a new cell stage in endocrine lineage determination. Disclosure D.M. Wong: None. L. Byrnes: None. J.O. Bouza: None. G. Peixoto: None. R. McMullen: None. J.B. Sneddon: Advisory Panel; Spouse/Partner; Akouos. Advisory Panel; Self; Encellin. Advisory Panel; Spouse/Partner; Encellin. Consultant; Self; Semma Therapeutics, Inc. Funding Nora Eccles Treadwell Foundation; University of California, San Francisco

Pancreatic development: one cell at a(pseudo)time.

Gaining a comprehensive understanding of the principles guiding lineage differentiation during organogenesis is a challenge. In this issue, Yu et al (2019) tackle this venture by taking advantage of single‐cell transcriptomic analyses and multiple mouse genetic tools. Their work provides new insights into pancreatic endocrine and exocrine cell differentiation landscapes and identifies new pathways regulating pancreatic lineage allocation in vivo .

Cooperative function of Pdx1 and Oc1 in multipotent pancreatic progenitors impacts postnatal islet maturation and adaptability.

The transcription factors pancreatic and duodenal homeobox 1 (Pdx1) and onecut1 (Oc1) are coexpressed in multipotent pancreatic progenitors (MPCs), but their expression patterns diverge in hormone-expressing cells, with Oc1 expression being extinguished in the endocrine lineage and Pdx1 being maintained at high levels in β-cells. We previously demonstrated that cooperative function of these two factors in MPCs is necessary for proper specification and differentiation of pancreatic endocrine cells. In those studies, we observed a persistent decrease in expression of the β-cell maturity factor MafA. We therefore hypothesized that Pdx1 and Oc1 cooperativity in MPCs impacts postnatal β-cell maturation and function. Here our model of Pdx1-Oc1 double heterozygosity was used to investigate the impact of haploinsufficiency for both of these factors on postnatal β-cell maturation, function, and adaptability. Examining mice at postnatal day (P) 14, we observed alterations in pancreatic insulin content in both Pdx1 heterozygotes and double heterozygotes. Gene expression analysis at this age revealed significantly decreased expression of many genes important for glucose-stimulated insulin secretion (e.g., Glut2, Pcsk1/2, Abcc8) exclusively in double heterozygotes. Analysis of P14 islets revealed an increase in the number of mixed islets in double heterozygotes. We predicted that double-heterozygous β-cells would have an impaired ability to respond to stress. Indeed, we observed that β-cell proliferation fails to increase in double heterozygotes in response to either high-fat diet or placental lactogen. We thus report here the importance of cooperation between regulatory factors early in development for postnatal islet maturation and adaptability.

Ultrastructure of endocrine pancreatic granules during pancreatic differentiation in the grass snake, Natrix natrix L. (Lepidosauria, Serpentes).

We used transmission electron microscopy to study the pancreatic main endocrine cell types in the embryos of the grass snake Natrix natrix L. with focus on the morphology of their secretory granules. The embryonic endocrine part of the pancreas in the grass snake contains four main types of cells (A, B, D, and PP), which is similar to other vertebrates. The B granules contained a moderately electron-dense crystalline-like core that was polygonal in shape and an electron-dense outer zone. The A granules had a spherical electron-dense eccentrically located core and a moderately electron-dense outer zone. The D granules were filled with a moderately electron-dense non-homogeneous content. The PP granules had a spherical electron-dense core with an electron translucent outer zone. Within the main types of granules (A, B, D, PP), different morphological subtypes were recognized that indicated their maturity, which may be related to the different content of these granules during the process of maturation. The sequence of pancreatic endocrine cell differentiation in grass snake embryos differs from that in many vertebrates. In the grass snake embryos, the B and D cells differentiated earlier than A and PP cells. The different sequence of endocrine cell differentiation in snakes and other vertebrates has been related to phylogenetic position and nutrition during early developmental stages.

Target Cells for Stem Cell Factor in the Adult Islets of Langerhans, Simultaneously Synthesizing Glucagon and Insulin

C-kit—a stem cell receptor, or CD117, is one of the markers of human pancreatic endocrinocyte progenitor cells. Its important role in the proliferation and differentiation of pancreatic endocrine cells during human prenatal development has already been proven. However, the presence and role of c-kit-positive cells in adult pancreatic islets remains unclear. The purpose of our study was to establish the presence of c-kit-positive cells in the adult pancreatic islets and to assess whether they retain the ability to proliferate during a lifetime. We studied pancreatic autopsies of adults aged 50 to 70 years, which were obtained in pathology departments of Kazan hospitals. Samples of the pancreas were embedded in paraffin using standard techniques. Paraffin sections of the pancreas were stained immunohistochemically with commercial antibodies against c-kit, insulin, glucagon, and Ki-67. Few c-kit-positive cells simultaneously secreting insulin or glucagon were detected in all samples studied. These data suggest that such cells are in a state of differentiation into endocrinocytes, but no proliferation of c-kit-positive cells was observed.

Reconstructing human pancreatic differentiation by mapping specific cell populations during development.

Information remains scarce on human development compared to animal models. Here, we reconstructed human fetal pancreatic differentiation using cell surface markers. We demonstrate that at 7weeks of development, the glycoprotein 2 (GP2) marks a multipotent cell population that will differentiate into the acinar, ductal or endocrine lineages. Development towards the acinar lineage is paralleled by an increase in GP2 expression. Conversely, a subset of the GP2+ population undergoes endocrine differentiation by down-regulating GP2 and CD142 and turning on NEUROG3, a marker of endocrine differentiation. Endocrine maturation progresses by up-regulating SUSD2 and lowering ECAD levels. Finally, in vitro differentiation of pancreatic endocrine cells derived from human pluripotent stem cells mimics key in vivo events. Our work paves the way to extend our understanding of the origin of mature human pancreatic cell types and how such lineage decisions are regulated.

Multi-site Neurogenin3 Phosphorylation Controls Pancreatic Endocrine Differentiation

SummaryThe proneural transcription factor Neurogenin3 (Ngn3) plays a critical role in pancreatic endocrine cell differentiation, although regulation of Ngn3 protein is largely unexplored. Here we demonstrate that Ngn3 protein undergoes cyclin-dependent kinase (Cdk)-mediated phosphorylation on multiple serine-proline sites. Replacing wild-type protein with a phosphomutant form of Ngn3 increases α cell generation, the earliest endocrine cell type to be formed in the developing pancreas. Moreover, un(der)phosphorylated Ngn3 maintains insulin expression in adult β cells in the presence of elevated c-Myc and enhances endocrine specification during ductal reprogramming. Mechanistically, preventing multi-site phosphorylation enhances both Ngn3 stability and DNA binding, promoting the increased expression of target genes that drive differentiation. Therefore, multi-site phosphorylation of Ngn3 controls its ability to promote pancreatic endocrine differentiation and to maintain β cell function in the presence of pro-proliferation cues and could be manipulated to promote and maintain endocrine differentiation in vitro and in vivo.

An inhibitor of fibroblast growth factor receptor-1 (FGFR1) promotes late-stage terminal differentiation from NGN3+ pancreatic endocrine progenitors.

Human induced pluripotent stem cells (hiPSCs) provide a potential resource for regenerative medicine. To identify the signalling pathway(s) contributing to the development of functional β cells, we established a tracing model consisting of dual knock-in hiPSCs (INS-Venus/NGN3-mCherry) (hIveNry) expressing the fluorescent proteins Venus and mCherry under the control of intrinsic insulin (INS) and neurogenin 3 (NGN3) promoters, respectively. hIveNry iPSCs differentiated into NGN3- and mCherry-positive endocrine progenitors and then into Venus-positive β cells expressing INS, PDX1, NKX6.1, and glucokinase (GCK). Using these cells, we conducted high-throughput screening of chemicals and identified a specific kinase inhibitor of fibroblast growth factor receptor 1 (FGFR1) that acted in a stage-dependent manner to promote the terminal differentiation of pancreatic endocrine cells, including β cells, from the intermediate stage of pancreatic endocrine progenitors while blocking the early development of pancreatic progenitors. This FGFR1 inhibitor augmented the expression of functional β cell markers (SLC30A8 and ABCC8) and improved glucose-stimulated INS secretion. Our findings indicate that the hIveNry model could provide further insights into the mechanisms of hiPS-derived β cell differentiation controlled by FGFR1-mediated regulatory pathways in a temporal-dependent fashion.