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Cellular identity during development is under the control of transcription factors that form gene regulatory networks. However, the transcription factors and gene regulatory networks underlying cellular identity in the human adult pancreas remain largely unexplored. Here, we integrate multiple single-cell RNA-sequencing datasets of the human adult pancreas, totaling 7393 cells, and comprehensively reconstruct gene regulatory networks. We show that a network of 142 transcription factors forms distinct regulatory modules that characterize pancreatic cell types. We present evidence that our approach identifies regulators of cell identity and cell states in the human adult pancreas. We predict that HEYL, BHLHE41 and JUND are active in acinar, beta and alpha cells, respectively, and show that these proteins are present in the human adult pancreas as well as in human induced pluripotent stem cell (hiPSC)-derived islet cells. Using single-cell transcriptomics, we found that JUND represses beta cell genes in hiPSC-alpha cells. BHLHE41 depletion induced apoptosis in primary pancreatic islets. The comprehensive gene regulatory network atlas can be explored interactively online. We anticipate our analysis to be the starting point for a more sophisticated dissection of how transcription factors regulate cell identity and cell states in the human adult pancreas., Miriam Cnop and Vincent Pasque share last and corresponding authorship, SCOPUS: ar.j, info:eu-repo/semantics/published

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In vitro differentiation of human induced pluripotent stem cells (iPSCs) into beta cells represents an important cell source for diabetes research. Here, we fully characterized iPSC-derived beta cell function in vitro and in vivo in humanized mice. Using a 7-stage protocol, human iPSCs were differentiated into islet-like aggregates with a yield of insulin-positive beta cells comparable to that of human islets. The last three stages of differentiation were conducted with two different 3D culture systems, rotating suspension or static microwells. In the latter, homogeneously small-sized islet-like aggregates were obtained, while in rotating suspension size was heterogeneous and aggregates often clumped. In vitro function was assessed by glucose-stimulated insulin secretion, NAD(P)H and calcium fluctuations. Stage 7 aggregates slightly increased insulin release in response to glucose in vitro. Aggregates were transplanted under the kidney capsule of NOD-SCID mice to allow for further in vivo beta cell maturation. In transplanted mice, grafts showed glucose-responsiveness and maintained normoglycemia after streptozotocin injection. In situ kidney perfusion assays showed modulation of human insulin secretion in response to different secretagogues. In conclusion, iPSCs differentiated with equal efficiency into beta cells in microwells compared to rotating suspension, but the former had a higher experimental success rate. In vitro differentiation generated aggregates lacking fully mature beta cell function. In vivo, beta cells acquired the functional characteristics typical of human islets. With this technology an unlimited supply of islet-like organoids can be generated from human iPSCs that will be instrumental to study beta cell biology and dysfunction in diabetes.

In vitro differentiation of human induced pluripotent stem cells (iPSCs) into beta cells represents an important cell source for diabetes research. Here, we fully characterized iPSC-derived beta cell function in vitro and in vivo in humanized mice. Using a 7-stage protocol, human iPSCs were differentiated into islet-like aggregates with a yield of insulin-positive beta cells comparable to that of human islets. The last three stages of differentiation were conducted with two different 3D culture systems, rotating suspension or static microwells. In the latter, homogeneously small-sized islet-like aggregates were obtained, while in rotating suspension size was heterogeneous and aggregates often clumped. In vitro function was assessed by glucose-stimulated insulin secretion, NAD(P)H and calcium fluctuations. Stage 7 aggregates slightly increased insulin release in response to glucose in vitro .Aggregates were transplanted under the kidney capsule of NOD-SCID mice to allow for further in vivo beta cell maturation. In transplanted mice, grafts showed glucose-responsiveness and maintained normoglycemia after streptozotocin injection. In situ kidney perfusion assays showed modulation of human insulin secretion in response to different secretagogues. In conclusion, iPSCs differentiated with equal efficiency into beta cells in microwells compared to rotating suspension, but the former had a higher experimental success rate. In vitro differentiation generated aggregates lacking fully mature beta cell function. In vivo ,beta cells acquired the functional characteristics typical of human islets. With this technology an unlimited supply of islet-like organoids can be generated from human iPSCs that will be instrumental to study beta cell biology and dysfunction in diabetes., SCOPUS: ar.j, info:eu-repo/semantics/published

DNAJC3, also known as P58IPK, is an Hsp40 family member that interacts with and inhibits PKR-like ER-localized eIF2α kinase (PERK). Dnajc3 deficiency in mice causes pancreatic β-cell loss and diabetes. Loss-of-function mutations in DNAJC3 cause early-onset diabetes and multisystemic neurodegeneration. The aim of our study was to investigate the genetic cause of early-onset syndromic diabetes in two unrelated patients, and elucidate the mechanisms of β-cell failure in this syndrome., info:eu-repo/semantics/published

Diabetes mellitus is a pandemic metabolic disorder that results from either the autoimmune destruction or the dysfunction of insulin-producing pancreatic beta cells. A promising cure is beta cell replacement through the transplantation of islets of Langerhans. However, donor shortage hinders the widespread implementation of this therapy. Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, represent an attractive alternative beta cell source for transplantation. Although major advances over the past two decades have led to the generation of stem cell-derived beta-like cells that share many features with genuine beta cells, producing fully mature beta cells remains challenging. Here, we review the current status of beta cell differentiation protocols and highlight specific challenges that are associated with producing mature beta cells. We address the challenges and opportunities that are offered by monogenic forms of diabetes. Finally, we discuss the remaining hurdles for clinical application of stem cell-derived beta cells and the status of ongoing clinical trials., SCOPUS: re.j, info:eu-repo/semantics/published

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