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Aims/hypothesis: Homozygous mutations in RFX6 lead to neonatal diabetes accompanied by a hypoplastic pancreas, whereas heterozygous mutations cause MODY. Recent studies have also shown RFX6 variants to be linked with type 2 diabetes. Despite RFX6's known function in islet development, its specific role in diabetes pathogenesis remains unclear. Here, we aimed to understand the mechanisms underlying the impairment of pancreatic islet development and subsequent hypoplasia due to loss-of-function mutations in RFX6. Methods: We examined regulatory factor X6 (RFX6) expression during human embryonic stem cell (hESC) differentiation into pancreatic islets and re-analysed a single-cell RNA-seq dataset to identify RFX6-specific cell populations during islet development. Furthermore, induced pluripotent stem cell (iPSC) lines lacking RFX6 were generated using CRISPR/Cas9. Various approaches were then employed to explore the consequences of RFX6 loss across different developmental stages. Subsequently, we evaluated transcriptional changes resulting from RFX6 loss through RNA-seq of pancreatic progenitors (PPs) and endocrine progenitors (EPs). Results: RFX6 expression was detected in PDX1+ cells in the hESC-derived posterior foregut (PF). However, in the PPs, RFX6 did not co-localise with pancreatic and duodenal homeobox 1 (PDX1) or NK homeobox 1 (NKX6.1) but instead co-localised with neurogenin 3, NK2 homeobox 2 and islet hormones in the EPs and islets. Single-cell analysis revealed high RFX6 expression levels in endocrine clusters across various hESC-derived pancreatic differentiation stages. Upon differentiating iPSCs lacking RFX6 into pancreatic islets, a significant decrease in PDX1 expression at the PF stage was observed, although this did not affect PPs co-expressing PDX1 and NKX6.1. RNA-seq analysis showed the downregulation of essential genes involved in pancreatic endocrine differentiation, insulin secretion and ion transport due to RFX6 deficiency. Furthermore, RFX6 deficiency resulted in the formation of smaller islet organoids due to increased cellular apoptosis, linked to reduced catalase expression, implying a protective role for RFX6. Overexpression of RFX6 reversed defective phenotypes in RFX6-knockout PPs, EPs and islets. Conclusions/interpretation: These findings suggest that pancreatic hypoplasia and reduced islet cell formation associated with RFX6 mutations are not due to alterations in PDX1+/NKX6.1+ PPs but instead result from cellular apoptosis and downregulation of pancreatic endocrine genes. Data availability: RNA-seq datasets have been deposited in the Zenodo repository with accession link (DOI: https://doi.org/10.5281/zenodo.10656891). [ABSTRACT FROM AUTHOR]

Recent studies reported that pancreatic β-cells are heterogeneous in terms of their transcriptional profiles and their abilities for insulin secretion. Sub-populations of pancreatic β-cells have been identified based on the functionality and expression of specific surface markers. Under diabetes condition, β-cell identity is altered leading to different β-cell sub-populations. Furthermore, cell–cell contact between β-cells and other endocrine cells within the islet play an important role in regulating insulin secretion. This highlights the significance of generating a cell product derived from stem cells containing β-cells along with other major islet cells for treating patients with diabetes, instead of transplanting a purified population of β-cells. Another key question is how close in terms of heterogeneity are the islet cells derived from stem cells? In this review, we summarize the heterogeneity in islet cells of the adult pancreas and those generated from stem cells. In addition, we highlight the significance of this heterogeneity in health and disease conditions and how this can be used to design a stem cell-derived product for diabetes cell therapy. [ABSTRACT FROM AUTHOR]

Aim: Beta cell replacement strategies are a promising alternative for diabetes treatment. Human pluripotent stem cells (hPSCs) serve as a scalable source for producing insulin‐secreting cells for transplantation therapy. We recently generated novel hPSC‐derived pancreatic progenitors, expressing high levels of the transcription factor NKX6.1, in the absence of PDX1 (PDX1−/NKX6.1+). Herein, our aim was to characterize this novel population and assess its ability to differentiate into insulin‐secreting beta cells in vitro. Materials and Methods: Three different hPSC lines were differentiated into PDX1−/NKX6.1+ progenitors, which were further differentiated into insulin‐secreting cells using two different protocols. The progenitors and beta cells were extensively characterized. Transcriptome analysis was performed at different stages and compared with the profiles of various pancreatic counterparts. Results: PDX1−/NKX6.1+ progenitors expressed high levels of nestin, a key marker of pancreatic islet‐derived progenitors, in the absence of E‐cadherin, similar to pancreatic mesenchymal stem cells. At progenitor stage, comparison of the two populations showed downregulation of pancreatic epithelial genes and upregulation of neuronal development genes in PDX1−/NKX6.1+ cells in comparison to the PDX1+/NKX6.1+ cells. Interestingly, on further differentiation, PDX1−/NKX6.1+ cells generated mono‐hormonal insulin+ cells and activated pancreatic key genes, such as PDX1. The transcriptome profile of PDX1−/NKX6.1+‐derived beta (3D‐beta) was closely similar to those of human pancreatic islets and purified hPSC‐derived beta cells. Also, the 3D‐beta cells secreted C‐peptide in response to increased glucose concentrations indicating their functionality. Conclusion: These findings provide a novel source of insulin‐secreting cells that can be used for beta cell therapy for diabetes. [ABSTRACT FROM AUTHOR]

The article focuses on the impact of genomic aberrations in human pluripotent stem cells (hPSCs) on diabetes research. Topics include induced pluripotent stem cells (iPSCs) with abnormal chromosomal content to study the molecular mechanisms of maturity-onset diabetes; and the need for comprehensive studies on the transcriptome and functional properties of pancreatic cells derived from hPSCs with chromosomal abnormalities before their application in disease modeling.

Background: Neonatal diabetes mellitus (NDM) is a rare condition that occurs within the first six months of life. Permanent NDM (PNDM) is caused by mutations in specific genes that are known for their expression at early and/or late stages of pancreatic beta‐ cell development, and are either involved in beta‐cell survival, insulin processing, regulation, and release. The native population in Qatar continues to practice consanguineous marriages that lead to a high level of homozygosity. To our knowledge, there is no previous report on the genomics of NDM among the Qatari population. The aims of the current study are to identify patients with NDM diagnosed between 2001 and 2016, and examine their clinical and genetic characteristics. Methods: To calculate the incidence of PNDM, all patients with PNDM diagnosed between 2001 and 2016 were compared to the total number of live births over the 16‐year‐period. Whole Genome Sequencing (WGS) was used to investigate the genetic etiology in the PNDM cohort. Results: PNDM was diagnosed in nine (n = 9) patients with an estimated incidence rate of 1:22,938 live births among the indigenous Qatari. Seven different mutations in six genes (PTF1A, GCK, SLC2A2, EIF2AK3, INS, and HNF1B) were identified. In the majority of cases, the genetic etiology was part of a previously identified autosomal recessive disorder. Two novel de novo mutations were identified in INS and HNF1B. Conclusion: Qatar has the second highest reported incidence of PNDM worldwide. A majority of PNDM cases present as rare familial autosomal recessive disorders. Pancreas associated transcription factor 1a (PTF1A) enhancer deletions are the most common cause of PNDM in Qatar, with only a few previous cases reported in the literature. [ABSTRACT FROM AUTHOR]