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Understanding the origin of pancreatic β cells has profound implications for regenerative therapies in diabetes. For over a century, it was widely held that adult pancreatic duct cells act as endocrine progenitors, but lineage-tracing experiments challenged this dogma. Gribben et al. recently used two existing lineage-tracing models and single-cell RNA sequencing to conclude that adult pancreatic ducts contain endocrine progenitors that differentiate to insulin-expressing β cells at a physiologically important rate. We now offer an alternative interpretation of these experiments. Our data indicate that the two Cre lines that were used directly label adult islet somatostatin-producing ∂ cells, which precludes their use to assess whether β cells originate from duct cells. Furthermore, many labeled ∂ cells, which have an elongated neuron-like shape, were likely misclassified as β cells because insulin-somatostatin coimmunolocalizations were not used. We conclude that most evidence so far indicates that endocrine and exocrine lineage borders are rarely crossed in the adult pancreas., Competing Interests: Declaration of interests D.A.M. serves on the advisory board for Cell Stem Cell., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Sequence variants in cis-acting enhancers are important for polygenic disease, but their role in Mendelian disease is poorly understood. Redundancy between enhancers that regulate the same gene is thought to mitigate the pathogenic impact of enhancer mutations. Recent findings, however, have shown that loss-of-function mutations in a single enhancer near PTF1A cause pancreas agenesis and neonatal diabetes. Using mouse and human genetic models, we show that this enhancer activates an entire PTF1A enhancer cluster in early pancreatic multipotent progenitors. This leading role, therefore, precludes functional redundancy. We further demonstrate that transient expression of PTF1A in multipotent progenitors sets in motion an epigenetic cascade that is required for duct and endocrine differentiation. These findings shed insights into the genome regulatory mechanisms that drive pancreas differentiation. Furthermore, they reveal an enhancer that acts as a regulatory master key and is thus vulnerable to pathogenic loss-of-function mutations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Summary Homeostatic regulation of the intestinal enteroendocrine lineage hierarchy is a poorly understood process. We resolved transcriptional changes during enteroendocrine differentiation in real time at single-cell level using a novel knockin allele of Neurog3 , the master regulator gene briefly expressed at the onset of enteroendocrine specification. A bi-fluorescent reporter, Neurog3Chrono, measures time from the onset of enteroendocrine differentiation and enables precise positioning of single-cell transcriptomes along an absolute time axis. This approach yielded a definitive description of the enteroendocrine hierarchy and its sub-lineages, uncovered differential kinetics between sub-lineages, and revealed time-dependent hormonal plasticity in enterochromaffin and L cells. The time-resolved map of transcriptional changes predicted multiple novel molecular regulators. Nine of these were validated by conditional knockout in mice or CRISPR modification in intestinal organoids. Six novel candidate regulators (Sox4, Rfx6, Tox3, Myt1, Runx1t1, and Zcchc12) yielded specific enteroendocrine phenotypes. Our time-resolved single-cell transcriptional map presents a rich resource to unravel enteroendocrine differentiation. Graphical Abstract Highlights • Neurog3Chrono arranges enteroendocrine single-cell transcriptomes on real-time axis • Enteroendocrine cells show hormonal plasticity in the course of their maturation • A time-resolved map characterizes fate specification of all enteroendocrine lineages • Individual knockout of 6 identified regulators gives robust enteroendocrine phenotypes The hierarchical lineage of intestinal enteroendocrine cells is defined at a spatiotemporal single-cell manner and validated using organoid and in vivo models. [ABSTRACT FROM AUTHOR]

Generation of functional β cells from pluripotent sources would accelerate diagnostic and therapeutic applications for diabetes research and therapy. However, it has been challenging to generate competent β cells with dynamic insulin-secretory capacity to glucose and incretin stimulations. We introduced transcription factors, critical for β-cell development and function, in differentiating human induced pluripotent stem cells (PSCs) and assessed the impact on the functionality of derived β-cell (psBC) progeny. A perifusion system revealed stepwise transduction of the PDX1, NEUROG3, and MAFA triad (PNM) enabled in vitro generation of psBCs with glucose and GLP-1 responsiveness within 3 weeks. PNM transduction upregulated genes associated with glucose sensing, insulin secretion, and β-cell maturation. In recipient diabetic mice, PNM-transduced psBCs showed glucose-responsive insulin secretion as early as 1 week post transplantation. Thus, enhanced pre-emptive β-cell specification of PSCs by PNM drives generation of glucose- and incretin-responsive psBCs in vitro, offering a competent tissue-primed biotherapy., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Human embryonic stem cells (hESCs) are a potential unlimited source of insulin-producing β cells for diabetes treatment. A greater understanding of how β cells form during embryonic development will improve current hESC differentiation protocols. All pancreatic endocrine cells, including β cells, are derived from Neurog3-expressing endocrine progenitors. This study characterizes the single-cell transcriptomes of 6,905 mouse embryonic day (E) 15.5 and 6,626 E18.5 pancreatic cells isolated from Neurog3-Cre; Rosa26 mT/mG embryos, allowing for enrichment of endocrine progenitors (yellow; tdTomato + EGFP) and endocrine cells (green; EGFP). Using a NEUROG3-2A-eGFP CyT49 hESC reporter line (N5-5), 4,462 hESC-derived GFP+ cells were sequenced. Differential expression analysis revealed enrichment of markers that are consistent with progenitor, endocrine, or previously undescribed cell-state populations. This study characterizes the single-cell transcriptomes of mouse and hESC-derived endocrine progenitors and serves as a resource (https://lynnlab.shinyapps.io/embryonic_pancreas) for improving the formation of functional β-like cells from hESCs., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Direct lineage reprogramming can convert readily available cells in the body into desired cell types for cell replacement therapy. This is usually achieved through forced activation or repression of lineage-defining factors or pathways. In particular, reprogramming toward the pancreatic β cell fate has been of great interest in the search for new diabetes therapies. It has been suggested that cells from various endodermal lineages can be converted to β-like cells. However, it is unclear how closely induced cells resemble endogenous pancreatic β cells and whether different cell types have the same reprogramming potential. Here, we report in vivo reprogramming of pancreatic ductal cells through intra-ductal delivery of an adenoviral vector expressing the transcription factors Pdx1, Neurog3, and Mafa. Induced β-like cells are mono-hormonal, express genes essential for β cell function, and correct hyperglycemia in both chemically and genetically induced diabetes models. Compared with intrahepatic ducts and hepatocytes treated with the same vector, pancreatic ducts demonstrated more rapid activation of β cell transcripts and repression of donor cell markers. This approach could be readily adapted to humans through a commonly performed procedure, endoscopic retrograde cholangiopancreatography (ERCP), and provides potential for cell replacement therapy in type 1 diabetes patients., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The production of insulin-producing β cells from human embryonic stem cells (hESCs) in vitro represents a promising strategy for a cell-based therapy for type 1 diabetes mellitus. To explore the cellular heterogeneity and temporal progression of endocrine progenitors and their progeny, we performed single-cell qPCR on more than 500 cells across several stages of in vitro differentiation of hESCs and compared them with human islets. We reveal distinct subpopulations along the endocrine differentiation path and an early lineage bifurcation toward either polyhormonal cells or β-like cells. We uncover several similarities and differences with mouse development and reveal that cells can take multiple paths to the same differentiation state, a principle that could be relevant to other systems. Notably, activation of the key β-cell transcription factor NKX6.1 can be initiated before or after endocrine commitment. The single-cell temporal resolution we provide can be used to improve the production of functional β cells., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Activating germline mutations in STAT3 were recently identified as a cause of neonatal diabetes mellitus associated with beta-cell autoimmunity. We have investigated the effect of an activating mutation, STAT3 K392R , on pancreatic development using induced pluripotent stem cells (iPSCs) derived from a patient with neonatal diabetes and pancreatic hypoplasia. Early pancreatic endoderm differentiated similarly from STAT3 K392R and healthy-control cells, but in later stages, NEUROG3 expression was upregulated prematurely in STAT3 K392R cells together with insulin (INS) and glucagon (GCG). RNA sequencing (RNA-seq) showed robust NEUROG3 downstream targets upregulation. STAT3 mutation correction with CRISPR/Cas9 reversed completely the disease phenotype. STAT3 K392R -activating properties were not explained fully by altered DNA-binding affinity or increased phosphorylation. Instead, reporter assays demonstrated NEUROG3 promoter activation by STAT3 in pancreatic cells. Furthermore, proteomic and immunocytochemical analyses revealed increased nuclear translocation of STAT3 K392R . Collectively, our results demonstrate that the STAT3 K392R mutation causes premature endocrine differentiation through direct induction of NEUROG3 expression., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)