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Immediate early response 3 interacting-protein 1 (IER3IP1) is an endoplasmic reticulum resident protein, highly expressed in pancreatic cells and the developing brain cortex. Homozygous mutations in IER3IP1 have been found in individuals with microcephaly and neonatal diabetes, yet the underlying mechanism causing beta cell failure remains unclear. Here, we utilized differentiation of genome edited-stem cells into pancreatic islet cells to elucidate the molecular basis of IER3IP1 neonatal diabetes. Using CRISPR-Cas9, we generated two distinct IER3IP1-mutant human embryonic stem cell lines: a homozygous knock-in model of a patient mutation (IER3IP1V21G), and a knockout model (IER3IP1-/-). While these mutant stem cell lines differentiated normally into definitive endoderm and pancreatic progenitors, we observed that IER3IP1-KO stem cell derived-islets (SC-islets) presented a significant decrease in beta cell numbers and elevated ER stress. Retention Using Selective Hooks (RUSH) assay revealed three-fold reduction in ER-to-Golgi trafficking of proinsulin in IER3IP1 mutant beta cells. Additionally, IER3IP1 mutant SC-islets implanted into immunocompromised mice displayed defective human insulin secretion, indicating the deleterious impact of IER3IP1 mutations on beta cell function. Our study provides valuable insights into the role of IER3IP1 in human beta cell biology and establishes a useful model to investigate ER-to-Golgi trafficking defects within beta cells., (© 2024 by the American Diabetes Association.)

Aims/hypothesis: Stem cell-derived islets (SC-islets) are being used as cell replacement therapy for insulin-dependent diabetes. Non-invasive long-term monitoring methods for SC-islet grafts, which are needed to detect misguided differentiation in vivo and to optimise their therapeutic effectiveness, are lacking. Positron emission tomography (PET) has been used to monitor transplanted primary islets. We therefore aimed to apply PET as a non-invasive monitoring method for SC-islet grafts., Methods: We implanted different doses of human SC-islets, SC-islets derived using an older protocol or a state-of-the-art protocol and SC-islets genetically rendered hyper- or hypoactive into mouse calf muscle to yield different kinds of grafts. We followed the grafts with PET using two tracers, glucagon-like peptide 1 receptor-binding [ 18 F]F-dibenzocyclooctyne-exendin-4 ([ 18 F]exendin) and the dopamine precursor 6-[ 18 F]fluoro-L-3,4-dihydroxyphenylalanine ([ 18 F]FDOPA), for 5 months, followed by histological assessment of graft size and composition. Additionally, we implanted a kidney subcapsular cohort with different SC-islet doses to assess the connection between C-peptide and stem cell-derived beta cell (SC-beta cell) mass., Results: Small but pure and large but impure grafts were derived from SC-islets. PET imaging allowed detection of SC-islet grafts even <1 mm 3 in size, [ 18 F]exendin having a better detection rate than [ 18 F]FDOPA (69% vs 44%, <1 mm 3 ; 96% vs 85%, >1 mm 3 ). Graft volume quantified with [ 18 F]exendin (r 2 =0.91) and [ 18 F]FDOPA (r 2 =0.86) strongly correlated with actual graft volume. [ 18 F]exendin PET delineated large cystic structures and its uptake correlated with graft SC-beta cell proportion (r 2 =0.68). The performance of neither tracer was affected by SC-islet graft hyper- or hypoactivity. C-peptide measurements under fasted or glucose-stimulated conditions did not correlate with SC-islet graft volume or SC-beta cell mass, with C-peptide under hypoglycaemia having a weak correlation with SC-beta cell mass (r 2 =0.52)., Conclusions/interpretation: [ 18 F]exendin and [ 18 F]FDOPA PET enable non-invasive assessment of SC-islet graft size and aspects of graft composition. These methods could be leveraged for optimising SC-islet cell replacement therapy in diabetes., (© 2024. The Author(s).)

Stem and progenitor cells hold great promise for regenerative medicine and gene therapy approaches. However, transplantation of living cells entails a fundamental risk of unwanted growth, potentially exacerbated by CRISPR-Cas9 or other genetic manipulations. Here, we describe a safety system to control cell proliferation while allowing robust and efficient cell manufacture, without any added genetic elements. Inactivating TYMS, a key nucleotide metabolism enzyme, in several cell lines resulted in cells that proliferate only when supplemented with exogenous thymidine. Under supplementation, TYMS -/- -pluripotent stem cells proliferate, produce teratomas, and successfully differentiate into potentially therapeutic cell types such as pancreatic β cells. Our results suggest that supplementation with exogenous thymidine affects stem cell proliferation, but not the function of stem cell-derived cells. After differentiation, postmitotic cells do not require thymidine in vitro or in vivo, as shown by the production of functional human insulin in mice up to 5 months after implantation of stem cell-derived pancreatic tissue., Competing Interests: Declaration of interests R.S.-M. and K.W. are marked as inventors in a patent application by the University of Helsinki., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims/hypothesis: Regulatory factor X 6 (RFX6) is crucial for pancreatic endocrine development and differentiation. The RFX6 variant p.His293LeufsTer7 is significantly enriched in the Finnish population, with almost 1:250 individuals as a carrier. Importantly, the FinnGen study indicates a high predisposition for heterozygous carriers to develop type 2 and gestational diabetes. However, the precise mechanism of this predisposition remains unknown., Methods: To understand the role of this variant in beta cell development and function, we used CRISPR technology to generate allelic series of pluripotent stem cells. We created two isogenic stem cell models: a human embryonic stem cell model; and a patient-derived stem cell model. Both were differentiated into pancreatic islet lineages (stem-cell-derived islets, SC-islets), followed by implantation in immunocompromised NOD-SCID-Gamma mice., Results: Stem cell models of the homozygous variant RFX6 -/- predictably failed to generate insulin-secreting pancreatic beta cells, mirroring the phenotype observed in Mitchell-Riley syndrome. Notably, at the pancreatic endocrine stage, there was an upregulation of precursor markers NEUROG3 and SOX9, accompanied by increased apoptosis. Intriguingly, heterozygous RFX6 +/- SC-islets exhibited RFX6 haploinsufficiency (54.2% reduction in protein expression), associated with reduced beta cell maturation markers, altered calcium signalling and impaired insulin secretion (62% and 54% reduction in basal and high glucose conditions, respectively). However, RFX6 haploinsufficiency did not have an impact on beta cell number or insulin content. The reduced insulin secretion persisted after in vivo implantation in mice, aligning with the increased risk of variant carriers to develop diabetes., Conclusions/interpretation: Our allelic series isogenic SC-islet models represent a powerful tool to elucidate specific aetiologies of diabetes in humans, enabling the sensitive detection of aberrations in both beta cell development and function. We highlight the critical role of RFX6 in augmenting and maintaining the pancreatic progenitor pool, with an endocrine roadblock and increased cell death upon its loss. We demonstrate that RFX6 haploinsufficiency does not affect beta cell number or insulin content but does impair function, predisposing heterozygous carriers of loss-of-function variants to diabetes., Data Availability: Ultra-deep bulk RNA-seq data for pancreatic differentiation stages 3, 5 and 7 of H1 RFX6 genotypes are deposited in the Gene Expression Omnibus database with accession code GSE234289. Original western blot images are deposited at Mendeley ( https://data.mendeley.com/datasets/g75drr3mgw/2 )., (© 2024. The Author(s).)

Pluripotent stem cell-derived islets (SC-islets) have emerged as a new source for β-cell replacement therapy. The function of human islet transplants is hampered by excessive cell death posttransplantation; contributing factors include inflammatory reactions, insufficient revascularization, and islet amyloid formation. However, there is a gap in knowledge of the engraftment process of SC-islets. In this experimental study, we investigated the engraftment capability of SC-islets at 3 months posttransplantation and observed that cell apoptosis rates were lower but vascular density was similar in SC-islets compared with human islets. Whereas the human islet transplant vascular structures were a mixture of remnant donor endothelium and ingrowing blood vessels, the SC-islets contained ingrowing blood vessels only. Oxygenation in the SC-islet grafts was twice as high as that in the corresponding grafts of human islets, suggesting better vascular functionality. Similar to the blood vessel ingrowth, reinnervation of the SC-islets was four- to fivefold higher than that of the human islets. Both SC-islets and human islets contained amyloid at 1 and 3 months posttransplantation. We conclude that the vascular and neural engraftment of SC-islets are superior to those of human islets, but grafts of both origins develop amyloid, with potential long-term consequences., (© 2024 by the American Diabetes Association.)

MAPK activating death domain (MADD) is a multifunctional protein regulating small GTPases RAB3 and RAB27, MAPK signaling, and cell survival. Polymorphisms in the MADD locus are associated with glycemic traits, but patients with biallelic variants in MADD manifest a complex syndrome affecting nervous, endocrine, exocrine, and hematological systems. We identified a homozygous splice site variant in MADD in 2 siblings with developmental delay, diabetes, congenital hypogonadotropic hypogonadism, and growth hormone deficiency. This variant led to skipping of exon 30 and in-frame deletion of 36 amino acids. To elucidate how this mutation causes pleiotropic endocrine phenotypes, we generated relevant cellular models with deletion of MADD exon 30 (dex30). We observed reduced numbers of β cells, decreased insulin content, and increased proinsulin-to-insulin ratio in dex30 human embryonic stem cell-derived pancreatic islets. Concordantly, dex30 led to decreased insulin expression in human β cell line EndoC-βH1. Furthermore, dex30 resulted in decreased luteinizing hormone expression in mouse pituitary gonadotrope cell line LβT2 but did not affect ontogeny of stem cell-derived GnRH neurons. Protein-protein interactions of wild-type and dex30 MADD revealed changes affecting multiple signaling pathways, while the GDP/GTP exchange activity of dex30 MADD remained intact. Our results suggest MADD-specific processes regulate hormone expression in pancreatic β cells and pituitary gonadotropes.

Argininosuccinate lyase deficiency (ASLD) is a recessive metabolic disorder caused by variants in ASL. In an essential step in urea synthesis, ASL breaks down argininosuccinate (ASA), a pathognomonic ASLD biomarker. The severe disease forms lead to hyperammonemia, neurological injury, and even early death. The current treatments are unsatisfactory, involving a strict low-protein diet, arginine supplementation, nitrogen scavenging, and in some cases, liver transplantation. An unmet need exists for improved, efficient therapies. Here, we show the potential of a lipid nanoparticle-mediated CRISPR approach using adenine base editors (ABEs) for ASLD treatment. To model ASLD, we first generated human-induced pluripotent stem cells (hiPSCs) from biopsies of individuals homozygous for the Finnish founder variant (c.1153C>T [p.Arg385Cys]) and edited this variant using the ABE. We then differentiated the hiPSCs into hepatocyte-like cells that showed a 1,000-fold decrease in ASA levels compared to those of isogenic non-edited cells. Lastly, we tested three different FDA-approved lipid nanoparticle formulations to deliver the ABE-encoding RNA and the sgRNA targeting the ASL variant. This approach efficiently edited the ASL variant in fibroblasts with no apparent cell toxicity and minimal off-target effects. Further, the treatment resulted in a significant decrease in ASA, to levels of healthy donors, indicating restoration of the urea cycle. Our work describes a highly efficient approach to editing the disease-causing ASL variant and restoring the function of the urea cycle. This method relies on RNA delivered by lipid nanoparticles, which is compatible with clinical applications, improves its safety profile, and allows for scalable production., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Primary defects in folding of mutant proinsulin can cause dominant-negative proinsulin accumulation in the endoplasmic reticulum (ER), impaired anterograde proinsulin trafficking, perturbed ER homeostasis, diminished insulin production, and β-cell dysfunction. Conversely, if primary impairment of ER-to-Golgi trafficking (which also perturbs ER homeostasis) drives misfolding of nonmutant proinsulin-this might suggest bi-directional entry into a common pathological phenotype (proinsulin misfolding, perturbed ER homeostasis, and deficient ER export of proinsulin) that can culminate in diminished insulin storage and diabetes. Here, we've challenged β-cells with conditions that impair ER-to-Golgi trafficking, and devised an accurate means to assess the relative abundance of distinct folded/misfolded forms of proinsulin using a novel nonreducing SDS-PAGE/immunoblotting protocol. We confirm abundant proinsulin misfolding upon introduction of a diabetogenic INS mutation, or in the islets of db/db mice. Whereas blockade of proinsulin trafficking in Golgi/post-Golgi compartments results in intracellular accumulation of properly-folded proinsulin (bearing native disulfide bonds), impairment of ER-to-Golgi trafficking (regardless whether such impairment is achieved by genetic or pharmacologic means) results in decreased native proinsulin with more misfolded proinsulin. Remarkably, reversible ER-to-Golgi transport defects (such as treatment with brefeldin A or cellular energy depletion) upon reversal quickly restore the ER folding environment, resulting in the disappearance of pre-existing misfolded proinsulin while preserving proinsulin bearing native disulfide bonds. Thus, proper homeostatic balance of ER-to-Golgi trafficking is linked to a more favorable proinsulin folding (as well as trafficking) outcome., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Identifying genes linked to extreme phenotypes in humans has the potential to highlight biological processes not shared with all other mammals. Here, we report the identification of homozygous loss-of-function variants in the primate-specific gene ZNF808 as a cause of pancreatic agenesis. ZNF808 is a member of the KRAB zinc finger protein family, a large and rapidly evolving group of epigenetic silencers which target transposable elements. We show that loss of ZNF808 in vitro results in aberrant activation of regulatory potential contained in the primate-specific transposable elements it represses during early pancreas development. This leads to inappropriate specification of cell fate with induction of genes associated with liver identity. Our results highlight the essential role of ZNF808 in pancreatic development in humans and the contribution of primate-specific regions of the human genome to congenital developmental disease., (© 2023. The Author(s).)

Pancreatic islets regulate blood glucose homeostasis through the controlled release of insulin; however, current metabolic models of glucose-sensitive insulin secretion are incomplete. A comprehensive understanding of islet metabolism is integral to studies of endocrine cell development as well as diabetic islet dysfunction. Human pluripotent stem cell-derived islets (SC-islets) are a developmentally relevant model of human islet function that have great potential in providing a cure for type 1 diabetes. Using multiple 13 C-labeled metabolic fuels, we demonstrate that SC-islets show numerous divergent patterns of metabolite trafficking in proposed insulin release pathways compared with primary human islets but are still reliant on mitochondrial aerobic metabolism to derive function. Furthermore, reductive tricarboxylic acid cycle activity and glycolytic metabolite cycling occur in SC-islets, suggesting that non-canonical coupling factors are also present. In aggregate, we show that many facets of SC-islet metabolism overlap with those of primary islets, albeit with a retained immature signature., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The paired-like homeobox transcription factor LEUTX is expressed in human preimplantation embryos between the 4- and 8-cell stages, and then silenced in somatic tissues. To characterize the function of LEUTX, we performed a multiomic characterization of LEUTX using two proteomics methods and three genome-wide sequencing approaches. Our results show that LEUTX stably interacts with the EP300 and CBP histone acetyltransferases through its 9 amino acid transactivation domain (9aaTAD), as mutation of this domain abolishes the interactions. LEUTX targets genomic cis-regulatory sequences that overlap with repetitive elements, and through these elements it is suggested to regulate the expression of its downstream genes. We find LEUTX to be a transcriptional activator, upregulating several genes linked to preimplantation development as well as 8-cell-like markers, such as DPPA3 and ZNF280A. Our results support a role for LEUTX in preimplantation development as an enhancer binding protein and as a potent transcriptional activator., Competing Interests: Y.M. is an inventor on a patent related to NET-CAGE technology. Other authors declare no competing interest., (© 2023 The Authors.)

Type 1 diabetes (T1D) is a disease of both autoimmunity and β-cells. The β-cells play an active role in their own demise by mounting defense mechanisms that are insufficient at best, and that can become even deleterious in the long term. This complex crosstalk is important to understanding the physiological defense mechanisms at play in healthy conditions, their alterations in the T1D setting, and therapeutic agents that may boost such mechanisms. Robust protocols to develop stem-cell-derived islets (SC-islets) from human pluripotent stem cells (hPSCs), and islet-reactive cytotoxic CD8 + T-cells from peripheral blood mononuclear cells offer unprecedented opportunities to study this crosstalk. Challenges to develop in vitro β-cell killing models include the cluster morphology of SC-islets, the relatively weak cytotoxicity of most autoimmune T-cells and the variable behavior of in vitro expanded CD8 + T-cells. These challenges may however be highly rewarding in light of the opportunities offered by such models. Herein, we discuss these opportunities including: the β-cell/immune crosstalk in an islet microenvironment; the features that make β-cells more sensitive to autoimmunity; therapeutic agents that may modulate β-cell vulnerability; and the possibility to perform analyses in an autologous setting, i.e., by generating T-cell effectors and SC-islets from the same donor., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor LR declared a shared affiliation with the authors CH and RM at the time of review., (Copyright © 2023 Halliez, Ibrahim, Otonkoski and Mallone.)

We present here a robust and reliable protocol by which to differentiate pancreatic islet-like aggregates (SC-islets) from human pluripotent stem cells. The 7-stage protocol mimics developmental patterning factors that induce endocrine lineage formation and spans monolayer, microwell, and aggregate suspension culture. The SC-islets demonstrate dynamic glucose-sensitive insulin secretion and an endocrine cell composition similar to those of primary human islets. SC-islets generated using this optimized protocol are suitable for in vitro modeling of islet cell pathophysiology and therapeutic applications. For complete details on the use and execution of this protocol, please refer to Balboa et al. (2022)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Nuclear receptor subfamily 5 group A member 1 (NR5A1) encodes steroidogenic factor 1 (SF1), a key regulatory factor that determines gonadal development and coordinates endocrine functions. Here, we have established a stem cell-based model of human gonadal development and applied it to evaluate the effects of NR5A1 during the transition from bipotential gonad to testicular cells. We combined directed differentiation of human induced pluripotent stem cells (46,XY) with activation of endogenous NR5A1 expression by conditionally-inducible CRISPR activation. The resulting male gonadal-like cells expressed several Sertoli cell transcripts, secreted anti-Müllerian hormone and responded to follicle-stimulating hormone by producing sex steroid intermediates. These characteristics were not induced without NR5A1 activation. A total of 2691 differentially expressed genetic elements, including both coding and non-coding RNAs, were detected immediately following activation of NR5A1 expression. Of those, we identified novel gonad-related putative NR5A1 targets, such as SCARA5, which we validated also by immunocytochemistry. In addition, NR5A1 activation was associated with dynamic expression of multiple gonad- and infertility-related differentially expressed genes. In conclusion, by combining targeted differentiation and endogenous activation of NR5A1 we have for the first time, been able to examine in detail the effects of NR5A1 in early human gonadal cells. The model and results obtained provide a useful resource for future investigations exploring the causative reasons for gonadal dysgenesis and infertility in humans., Competing Interests: Declaration of competing interest none., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of insulin producing pancreatic β-cells. One of the genes associated with T1D is TYK2, which encodes a Janus kinase with critical roles in type-Ι interferon (IFN-Ι) mediated intracellular signalling. To study the role of TYK2 in β-cell development and response to IFNα, we generated TYK2 knockout human iPSCs and directed them into the pancreatic endocrine lineage. Here we show that loss of TYK2 compromises the emergence of endocrine precursors by regulating KRAS expression, while mature stem cell-islets (SC-islets) function is not affected. In the SC-islets, the loss or inhibition of TYK2 prevents IFNα-induced antigen processing and presentation, including MHC Class Ι and Class ΙΙ expression, enhancing their survival against CD8 + T-cell cytotoxicity. These results identify an unsuspected role for TYK2 in β-cell development and support TYK2 inhibition in adult β-cells as a potent therapeutic target to halt T1D progression., (© 2022. The Author(s).)

Characterization of gene expression in pancreatic islets and its alteration in type 2 diabetes (T2D) are vital in understanding islet function and T2D pathogenesis. We leveraged RNA sequencing and genome-wide genotyping in islets from 188 donors to create the Islet Gene View (IGW) platform to make this information easily accessible to the scientific community. Expression data were related to islet phenotypes, diabetes status, other islet-expressed genes, islet hormone-encoding genes and for expression in insulin target tissues. The IGW web application produces output graphs for a particular gene of interest. In IGW, 284 differentially expressed genes (DEGs) were identified in T2D donor islets compared with controls. Forty percent of DEGs showed cell-type enrichment and a large proportion significantly co-expressed with islet hormone-encoding genes; glucagon (&lt;i&gt;GCG&lt;/i&gt;, 56%), amylin (&lt;i&gt;IAPP&lt;/i&gt;, 52%), insulin (&lt;i&gt;INS&lt;/i&gt;, 44%), and somatostatin (&lt;i&gt;SST&lt;/i&gt;, 24%). Inhibition of two DEGs, &lt;i&gt;UNC5D&lt;/i&gt; and &lt;i&gt;SERPINE2&lt;/i&gt;, impaired glucose-stimulated insulin secretion and impacted cell survival in a human β-cell model. The exploratory use of IGW could help designing more comprehensive functional follow-up studies and serve to identify therapeutic targets in T2D., (© 2022 Asplund et al.)

Embryonic genome activation (EGA) is critical for embryonic development. However, our understanding of the regulatory mechanisms of human EGA is still incomplete. Human embryonic stem cells (hESCs) are an established model for studying developmental processes, but they resemble epiblast and are sub-optimal for modeling EGA. DUX4 regulates human EGA by inducing cleavage-stage-specific genes, while it also induces cell death. We report here that a short-pulsed expression of DUX4 in primed hESCs activates an EGA-like gene expression program in up to 17% of the cells, retaining cell viability. These DUX4-induced cells resembled eight-cell stage blastomeres and were named induced blastomere-like (iBM) cells. The iBM cells showed marked reduction of POU5F1 protein, as previously observed in mouse two-cell-like cells. Finally, the iBM cells were successfully enriched using an antibody against NaPi2b (SLC34A2), which is expressed in human blastomeres. The iBM cells provide an improved model system to study human EGA transcriptome., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes., (© 2022. The Author(s).)

The ability to maintain normoglycaemia, through glucose-sensitive insulin release, is a key aspect of postnatal beta cell function. However, terminally differentiated beta cell identity does not necessarily imply functional maturity. Beta cell maturation is therefore a continuation of beta cell development, albeit a process that occurs postnatally in mammals. Although many important features have been identified in the study of beta cell maturation, as of yet no unified mechanistic model of beta cell functional maturity exists. Here, we review recent findings about the underlying mechanisms of beta cell functional maturation. These findings include systemic hormonal and nutritional triggers that operate through energy-sensing machinery shifts within beta cells, resulting in primed metabolic states that allow for appropriate glucose trafficking and, ultimately, insulin release. We also draw attention to the expansive synergistic nature of these pathways and emphasise that beta cell maturation is dependent on overlapping regulatory and metabolic networks., (© 2022. The Author(s).)

Type 1 diabetes (T1D) results from autoimmune destruction of β-cells in the pancreas. Protein tyrosine phosphatases (PTPs) are candidate genes for T1D and play a key role in autoimmune disease development and β-cell dysfunction. Here, we assessed the global protein and individual PTP profiles in the pancreas from nonobese mice with early-onset diabetes (NOD) mice treated with an anti-CD3 monoclonal antibody and interleukin-1 receptor antagonist. The treatment reversed hyperglycemia, and we observed enhanced expression of PTPN2, a PTP family member and T1D candidate gene, and endoplasmic reticulum (ER) chaperones in the pancreatic islets. To address the functional role of PTPN2 in β-cells, we generated PTPN2-deficient human stem cell-derived β-like and EndoC-βH1 cells. Mechanistically, we demonstrated that PTPN2 inactivation in β-cells exacerbates type I and type II interferon signaling networks and the potential progression toward autoimmunity. Moreover, we established the capacity of PTPN2 to positively modulate the Ca2+-dependent unfolded protein response and ER stress outcome in β-cells. Adenovirus-induced overexpression of PTPN2 partially protected from ER stress-induced β-cell death. Our results postulate PTPN2 as a key protective factor in β-cells during inflammation and ER stress in autoimmune diabetes., (© 2022 by the American Diabetes Association.)

Double homeobox 4 ( DUX4 ) is expressed at the early pre-implantation stage in human embryos. Here we show that induced human DUX4 expression substantially alters the chromatin accessibility of non-coding DNA and activates thousands of newly identified transcribed enhancer-like regions, preferentially located within ERVL-MaLR repeat elements. CRISPR activation of transcribed enhancers by C-terminal DUX4 motifs results in the increased expression of target embryonic genome activation (EGA) genes ZSCAN4 and KHDC1P1 . We show that DUX4 is markedly enriched in human zygotes, followed by intense nuclear DUX4 localization preceding and coinciding with minor EGA. DUX4 knockdown in human zygotes led to changes in the EGA transcriptome but did not terminate the embryos. We also show that the DUX4 protein interacts with the Mediator complex via the C-terminal KIX binding motif. Our findings contribute to the understanding of DUX4 as a regulator of the non-coding genome., Competing Interests: Y.T. and A.K. are employees of K.K.DNAFORM. Y. T. and Y. M. are inventors on a patent related to NET-CAGE technology., (© 2022 The Authors.)

Congenital hyperinsulinism (CHI) is a severe inherited neonatal disorder characterized by inappropriate insulin secretion caused by genetic defects of the pancreatic beta cells. Several open questions remain in CHI research, such as the optimal treatment for the most common type of CHI, caused by mutations in the genes encoding ATP-sensitive potassium channels, and the molecular mechanisms of newly identified CHI genes. Answering these questions requires robust preclinical models, particularly since primary patient material is extremely scarce and accurate animal models are not available. In this short review, we explain why pluripotent stem cell derived islets present an attractive solution to these issues and outline the current progress in stem-cell based modeling of CHI. Stem cell derived islets enable the study of molecular mechanisms of CHI and the discovery of novel antihypoglycemic drugs, while also providing a valuable model to study the biology of variable functional states of beta cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lithovius and Otonkoski.)

Conventional reprogramming methods rely on the ectopic expression of transcription factors to reprogram somatic cells into induced pluripotent stem cells (iPSCs). The forced expression of transcription factors may lead to off-target gene activation and heterogeneous reprogramming, resulting in the emergence of alternative cell types and aberrant iPSCs. Activation of endogenous pluripotency factors by CRISPR activation (CRISPRa) can reduce this heterogeneity. Here, we describe a high-efficiency reprogramming of human somatic cells into iPSCs using optimized CRISPRa. Efficient reprogramming was dependent on the additional targeting of the embryo genome activation-enriched Alu-motif and the miR-302/367 locus. Single-cell transcriptome analysis revealed that the optimized CRISPRa reprogrammed cells more directly and specifically into the pluripotent state when compared to the conventional reprogramming method. These findings support the use of CRISPRa for high-quality pluripotent reprogramming of human cells., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Metabolic characteristics of adult stem cells are distinct from their differentiated progeny, and cellular metabolism is emerging as a potential driver of cell fate conversions 1-4 . How these metabolic features are established remains unclear. Here we identified inherited metabolism imposed by functionally distinct mitochondrial age-classes as a fate determinant in asymmetric division of epithelial stem-like cells. While chronologically old mitochondria support oxidative respiration, the electron transport chain of new organelles is proteomically immature and they respire less. After cell division, selectively segregated mitochondrial age-classes elicit a metabolic bias in progeny cells, with oxidative energy metabolism promoting differentiation in cells that inherit old mitochondria. Cells that inherit newly synthesized mitochondria with low levels of Rieske iron-sulfur polypeptide 1 have a higher pentose phosphate pathway activity, which promotes de novo purine biosynthesis and redox balance, and is required to maintain stemness during early fate determination after division. Our results demonstrate that fate decisions are susceptible to intrinsic metabolic bias imposed by selectively inherited mitochondria., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Surgery with curative intent can be offered to congenital hyperinsulinism (CHI) patients, provided that the lesion is focal. Radiolabeled exendin-4 specifically binds the glucagonlike peptide 1 receptor on pancreatic β-cells. In this study, we compared the performance of 18 F-DOPA PET/CT, the current standard imaging method for CHI, and PET/CT with the new tracer 68 Ga-NODAGA-exendin-4 in the preoperative detection of focal CHI. Methods: Nineteen CHI patients underwent both 18 F-DOPA PET/CT and 68 Ga-NODAGA-exendin-4 PET/CT before surgery. The images were evaluated in 3 settings: a standard clinical reading, a masked expert reading, and a joint reading. The target (lesion)-to-nontarget (normal pancreas) ratio was determined using SUV max Image quality was rated by pediatric surgeons in a questionnaire. Results: Fourteen of 19 patients having focal lesions underwent surgery. On the basis of clinical readings, the sensitivity of 68 Ga-NODAGA-exendin-4 PET/CT (100%; 95% CI, 77%-100%) was higher than that of 18 F-DOPA PET/CT (71%; 95% CI, 42%-92%). Interobserver agreement between readings was higher for 68 Ga-NODAGA-exendin-4 than for 18 F-DOPA PET/CT (Fleiss κ = 0.91 vs. 0.56). 68 Ga-NODAGA-exendin-4 PET/CT provided significantly ( P = 0.021) higher target-to-nontarget ratios (2.02 ± 0.65) than did 18 F-DOPA PET/CT (1.40 ± 0.40). On a 5-point scale, pediatric surgeons rated 68 Ga-NODAGA-exendin-4 PET/CT as superior to 18 F-DOPA PET/CT. Conclusion: For the detection of focal CHI, 68 Ga-NODAGA-exendin-4 PET/CT has higher clinical sensitivity and better interobserver correlation than 18 F-DOPA PET/CT. Better contrast and image quality make 68 Ga-NODAGA-exendin-4 PET/CT superior to 18 F-DOPA PET/CT in surgeons' intraoperative quest for lesion localization., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)

Human induced pluripotent stem cells (hiPSCs) allow in vitro study of genetic diseases and hold potential for personalized stem cell therapy. Gene editing, precisely modifying specifically targeted loci, represents a valuable tool for different hiPSC applications. This is especially useful in monogenic diseases to dissect the function of unknown mutations or to create genetically corrected, patient-derived hiPSCs. Here we describe a highly efficient method for simultaneous base editing and reprogramming of fibroblasts employing a CRISPR-Cas9 adenine base editor. As a proof of concept, we apply this approach to generate gene-edited hiPSCs from skin biopsies of four patients carrying a Finnish-founder pathogenic point mutation in either NOTCH3 or LDLR genes. We also show LDLR activity restoration after the gene correction. Overall, this method yields tens of gene-edited hiPSC monoclonal lines with unprecedented efficiency and robustness while considerably reducing the cell culture time and thus the risk for in vitro alterations., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-resident protein that plays a crucial role in attenuating ER stress responses. Although MANF is indispensable for the survival and function of mouse β-cells, its precise role in human β-cell development and function is unknown. In this study, we show that lack of MANF in humans results in diabetes due to increased ER stress, leading to impaired β-cell function. We identified two patients from different families with childhood diabetes and a neurodevelopmental disorder associated with homozygous loss-of-function mutations in the MANF gene. To study the role of MANF in human β-cell development and function, we knocked out the MANF gene in human embryonic stem cells and differentiated them into pancreatic endocrine cells. Loss of MANF induced mild ER stress and impaired insulin-processing capacity of β-cells in vitro. Upon implantation to immunocompromised mice, the MANF knockout grafts presented elevated ER stress and functional failure, particularly in recipients with diabetes. By describing a new form of monogenic neurodevelopmental diabetes syndrome caused by disturbed ER function, we highlight the importance of adequate ER stress regulation for proper human β-cell function and demonstrate the crucial role of MANF in this process., (© 2021 by the American Diabetes Association.)

Context: The management of congenital hyperinsulinism (CHI) has improved., Objective: To examine the treatment and long-term outcome of Finnish patients with persistent and transient CHI (P-CHI and T-CHI)., Design: A population-based retrospective study of CHI patients treated from 1972 to 2015., Patients: 106 patients with P-CHI and 132 patients with T-CHI (in total, 42 diagnosed before and 196 after year 2000) with median follow-up durations of 12.5 and 6.2 years, respectively., Main Outcome Measures: Recovery, diabetes, pancreatic exocrine dysfunction, neurodevelopment., Results: The overall incidence of CHI (n = 238) was 1:11 300 live births (1972-2015). From 2000 to 2015, the incidence of P-CHI (n = 69) was 1:13 500 and of T-CHI (n = 127) 1:7400 live births. In the 21st century P-CHI group, hyperinsulinemic medication was initiated and normoglycemia achieved faster relative to earlier. Of the 74 medically treated P-CHI patients, 68% had discontinued medication. Thirteen (12%) P-CHI patients had partial pancreatic resection and 19 (18%) underwent near-total pancreatectomy. Of these, 0% and 84% developed diabetes and 23% and 58% had clinical pancreatic exocrine dysfunction, respectively. Mild neurological difficulties (21% vs 16%, respectively) and intellectual disability (9% vs 5%, respectively) were as common in the P-CHI and T-CHI groups. However, the 21st century P-CHI patients had significantly more frequent normal neurodevelopment and significantly more infrequent diabetes and pancreatic exocrine dysfunction compared with those diagnosed earlier., Conclusions: Our results demonstrated improved treatment and long-term outcome in the 21st century P-CHI patients relative to earlier., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Aims/hypothesis: Congenital hyperinsulinism caused by mutations in the K ATP -channel-encoding genes (K ATP HI) is a potentially life-threatening disorder of the pancreatic beta cells. No optimal medical treatment is available for patients with diazoxide-unresponsive diffuse K ATP HI. Therefore, we aimed to create a model of K ATP HI using patient induced pluripotent stem cell (iPSC)-derived islets., Methods: We derived iPSCs from a patient carrying a homozygous ABCC8 V187D mutation, which inactivates the sulfonylurea receptor 1 (SUR1) subunit of the K ATP -channel. CRISPR-Cas9 mutation-corrected iPSCs were used as controls. Both were differentiated to stem cell-derived islet-like clusters (SC-islets) and implanted into NOD-SCID gamma mice., Results: SUR1-mutant and -corrected iPSC lines both differentiated towards the endocrine lineage, but SUR1-mutant stem cells generated 32% more beta-like cells (SC-beta cells) (64.6% vs 49.0%, p = 0.02) and 26% fewer alpha-like cells (16.1% vs 21.8% p = 0.01). SUR1-mutant SC-beta cells were 61% more proliferative (1.23% vs 0.76%, p = 0.006), and this phenotype could be induced in SUR1-corrected cells with pharmacological K ATP -channel inactivation. The SUR1-mutant SC-islets secreted 3.2-fold more insulin in low glucose conditions (0.0174% vs 0.0054%/min, p = 0.0021) and did not respond to K ATP -channel-acting drugs in vitro. Mice carrying grafts of SUR1-mutant SC-islets presented with 38% lower fasting blood glucose (4.8 vs 7.7 mmol/l, p = 0.009) and their grafts failed to efficiently shut down insulin secretion during induced hypoglycaemia. Explanted SUR1-mutant grafts displayed an increase in SC-beta cell proportion and SC-beta cell nucleomegaly, which was independent of proliferation., Conclusions/interpretation: We have created a model recapitulating the known pathophysiology of K ATP HI both in vitro and in vivo. We have also identified a novel role for K ATP -channel activity during human islet development. This model will enable further studies for the improved understanding and clinical management of K ATP HI without the need for primary patient tissue.

Neonatal diabetes is caused by single gene mutations reducing pancreatic β cell number or impairing β cell function. Understanding the genetic basis of rare diabetes subtypes highlights fundamental biological processes in β cells. We identified 6 patients from 5 families with homozygous mutations in the YIPF5 gene, which is involved in trafficking between the endoplasmic reticulum (ER) and the Golgi. All patients had neonatal/early-onset diabetes, severe microcephaly, and epilepsy. YIPF5 is expressed during human brain development, in adult brain and pancreatic islets. We used 3 human β cell models (YIPF5 silencing in EndoC-βH1 cells, YIPF5 knockout and mutation knockin in embryonic stem cells, and patient-derived induced pluripotent stem cells) to investigate the mechanism through which YIPF5 loss of function affects β cells. Loss of YIPF5 function in stem cell-derived islet cells resulted in proinsulin retention in the ER, marked ER stress, and β cell failure. Partial YIPF5 silencing in EndoC-βH1 cells and a patient mutation in stem cells increased the β cell sensitivity to ER stress-induced apoptosis. We report recessive YIPF5 mutations as the genetic cause of a congenital syndrome of microcephaly, epilepsy, and neonatal/early-onset diabetes, highlighting a critical role of YIPF5 in β cells and neurons. We believe this is the first report of mutations disrupting the ER-to-Golgi trafficking, resulting in diabetes.

CRISPR activation (CRISPRa) has revealed great potential as a tool to modulate the expression of targeted cellular genes. Here, we successfully applied the CRISPRa system to trigger the Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation in latently infected cells by selectively activating ORF50 gene directly from the virus genome. We found that a nuclease-deficient Cas9 (dCas9) fused to a destabilization domain (DD) and 12 copies of the VP16 activation domain (VP192) triggered a more efficient KSHV lytic cycle and virus production when guided to two different sites on the ORF50 promoter, instead of only a single site. To our surprise, the virus reactivation induced by binding of the stable DD-dCas9-VP192 on the ORF50 promoter was even more efficient than reactivation induced by ectopic expression of ORF50. This suggests that recruitment of additional transcriptional activators to the ORF50 promoter, in addition to ORF50 itself, are needed for the efficient virus production. Further, we show that CRISPRa can be applied to selectively express the early lytic gene, ORF57, without disturbing the viral latency. Therefore, CRISPRa-based systems can be utilized to facilitate virus-host interaction studies by controlling the expression of not only cellular but also of specific KSHV genes.

Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and axonal neuropathy and in Parkinson's disease. They form a complex of unknown function. Here we address the importance of these two proteins in human motor neurons. We show that gene edited human induced pluripotent stem cells (iPSC) lacking either CHCHD2 or CHCHD10 are viable and can be differentiated into functional motor neurons that fire spontaneous and evoked action potentials. Mitochondria in knockout iPSC and motor neurons sustain ultrastructure but show increased proton leakage and respiration, and reciprocal compensatory increases in CHCHD2 or CHCHD10. Knockout motor neurons have largely overlapping transcriptome profiles compared to isogenic control line, in particular for synaptic gene expression. Our results show that the absence of either CHCHD2 or CHCHD10 alters mitochondrial respiration in human motor neurons, inducing similar compensatory responses. Thus, pathogenic mechanisms may involve loss of synaptic function resulting from defective energy metabolism., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Context: Major advances have been made in the genetics and classification of congenital hyperinsulinism (CHI)., Objective: To examine the genetics and clinical characteristics of patients with persistent and transient CHI., Design: A cross-sectional study with the register data and targeted sequencing of 104 genes affecting glucose metabolism., Patients: Genetic and phenotypic data were collected from 153 patients with persistent (n = 95) and transient (n = 58) CHI diagnosed between 1972 and 2015. Of these, 86 patients with persistent and 58 with transient CHI participated in the analysis of the selected 104 genes affecting glucose metabolism, including 10 CHI-associated genes, and 9 patients with persistent CHI were included because of their previously confirmed genetic diagnosis., Main Outcome Measures: Targeted next-generation sequencing results and genotype-phenotype associations., Results: Five novel and 21 previously reported pathogenic or likely pathogenic variants in ABCC8, KCNJ11, GLUD1, GCK, HNF4A, and SLC16A1 genes were found in 68% (n = 65) and 0% of the patients with persistent and transient CHI, respectively. KATP channel mutations explained 82% of the mutation positive cases., Conclusions: The genetic variants found in this nationwide CHI cohort are in agreement with previous studies, mutations in the KATP channel genes being the major causes of the disease. Pathogenic CHI-associated variants were not identified in patients who were both diazoxide responsive and able to discontinue medication within the first 4 months. Therefore, our results support the notion that genetic testing should be focused on patients with inadequate response or prolonged need for medication., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A rare loss-of-function allele p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8), which is enriched in Western Finland, protects against type 2 diabetes (T2D). We recruited relatives of the identified carriers and showed that protection was associated with better insulin secretion due to enhanced glucose responsiveness and proinsulin conversion, particularly when compared with individuals matched for the genotype of a common T2D-risk allele in SLC30A8, p.Arg325. In genome-edited human induced pluripotent stem cell (iPSC)-derived β-like cells, we establish that the p.Arg138* allele results in reduced SLC30A8 expression due to haploinsufficiency. In human β cells, loss of SLC30A8 leads to increased glucose responsiveness and reduced K ATP channel function similar to isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aimed at maintaining insulin secretion capacity in T2D.

Background: Murine boundary cap-derived neural crest stem cells (NCSCs) are capable of enhancing islet function by stimulating beta cell proliferation as well as increasing the neural and vascular density in the islets both in vitro and in vivo . This study aimed to isolate NCSC-like cells from human bone marrow. Methods: CD271 magnetic cell separation and culture techniques were used to purify a NCSC-enriched population of human bone marrow. Analyses of the CD271+ and CD271- fractions in terms of protein expression were performed, and the capacity of the CD271+ bone marrow cells to form 3-dimensional spheres when grown under non-adherent conditions was also investigated. Moreover, the NCSC characteristics of the CD271+ cells were evaluated by their ability to migrate toward human islets as well as human islet-like cell clusters (ICC) derived from pluripotent stem cells. Results: The CD271+ bone marrow population fulfilled the criterion of being multipotent stem cells, having the potential to differentiate into glial cells, neurons as well as myofibroblasts in vitro . They had the capacity to form 3-dimensional spheres as well as an ability to migrate toward human islets, further supporting their NCSC identity. Additionally, we demonstrated similar migration features toward stem cell-derived ICC. Conclusion: The results support the NCSC identity of the CD271-enriched human bone marrow population. It remains to investigate whether the human bone marrow-derived NCSCs have the ability to improve transplantation efficacy of not only human islets but stem cell-derived ICC as well.

The appearance of type 1 diabetes (T1D)-associated autoantibodies is the first and only measurable parameter to predict progression toward T1D in genetically susceptible individuals. However, autoantibodies indicate an active autoimmune reaction, wherein the immune tolerance is already broken. Therefore, there is a clear and urgent need for new biomarkers that predict the onset of the autoimmune reaction preceding autoantibody positivity or reflect progressive β-cell destruction. Here we report the mRNA sequencing-based analysis of 306 samples including fractionated samples of CD4 + and CD8 + T cells as well as CD4 - CD8 - cell fractions and unfractionated peripheral blood mononuclear cell samples longitudinally collected from seven children who developed β-cell autoimmunity (case subjects) at a young age and matched control subjects. We identified transcripts, including interleukin 32 ( IL32 ), that were upregulated before T1D-associated autoantibodies appeared. Single-cell RNA sequencing studies revealed that high IL32 in case samples was contributed mainly by activated T cells and NK cells. Further, we showed that IL32 expression can be induced by a virus and cytokines in pancreatic islets and β-cells, respectively. The results provide a basis for early detection of aberrations in the immune system function before T1D and suggest a potential role for IL32 in the pathogenesis of T1D., (© 2019 by the American Diabetes Association.)

Understanding the molecular mechanisms behind beta cell dysfunction is essential for the development of effective and specific approaches for diabetes care and prevention. Physiological human beta cell models are needed for this work. We review the possibilities and limitations of currently available human beta cell models and how they can be dramatically enhanced using genome-editing technologies. In addition to the gold standard, primary isolated islets, other models now include immortalised human beta cell lines and pluripotent stem cell-derived islet-like cells. The scarcity of human primary islet samples limits their use, but valuable gene expression and functional data from large collections of human islets have been made available to the scientific community. The possibilities for studying beta cell physiology using immortalised human beta cell lines and stem cell-derived islets are rapidly evolving. However, the functional immaturity of these cells is still a significant limitation. CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) has enabled precise engineering of specific genetic variants, targeted transcriptional modulation and genome-wide genetic screening. These approaches can now be exploited to gain understanding of the mechanisms behind coding and non-coding diabetes-associated genetic variants, allowing more precise evaluation of their contribution to diabetes pathogenesis. Despite all the progress, genome editing in primary pancreatic islets remains difficult to achieve, an important limitation requiring further technological development.

The original version of this Article contained an error in Fig. 7. In panel b, the survival curves were shifted relative to the y axis. This error has been corrected in both the PDF and HTML versions of the Article.

Elevated MYC expression sensitizes tumor cells to apoptosis but the therapeutic potential of this mechanism remains unclear. We find, in a model of MYC-driven breast cancer, that pharmacological activation of AMPK strongly synergizes with BCL-2/BCL-X L inhibitors to activate apoptosis. We demonstrate the translational potential of an AMPK and BCL-2/BCL-X L co-targeting strategy in ex vivo and in vivo models of MYC-high breast cancer. Metformin combined with navitoclax or venetoclax efficiently inhibited tumor growth, conferred survival benefits and induced tumor infiltration by immune cells. However, withdrawal of the drugs allowed tumor re-growth with presentation of PD-1+/CD8+ T cell infiltrates, suggesting immune escape. A two-step treatment regimen, beginning with neoadjuvant metformin+venetoclax to induce apoptosis and followed by adjuvant metformin+venetoclax+anti-PD-1 treatment to overcome immune escape, led to durable antitumor responses even after drug withdrawal. We demonstrate that pharmacological reactivation of MYC-dependent apoptosis is a powerful antitumor strategy involving both tumor cell depletion and immunosurveillance.

Pancreatic β-cells are the only source of insulin. Disturbances in β-cell development or function may thus result in insulin deficiency or excess, presenting as hyper- or hypoglycemia. It is increasingly evident that common forms of diabetes (types 1 and 2) are pathogenically heterogeneous. Development of efficient therapies is dependent on reliable disease models. Although animal models are remarkably useful research tools, they present limitations because of species differences. As an alternative, human pluripotent stem cell technologies offer multiple possibilities for the study of human diseases in vitro. In the last decade, advances in the derivation of induced pluripotent stem cells from diabetic patients, combined with β-cell differentiation protocols, have resulted in the generation of useful disease models for diabetes. First disease models have been focusing on monogenic diabetes. The development of genome editing technologies, more advanced differentiation protocols and humanized mouse models based on transplanted cells have opened new horizons for the modeling of more complex forms of β-cell dysfunction. We present here the incremental progress made in the modeling of diabetes using pluripotent stem cells. We discuss the current challenges and opportunities of these approaches to dissect β-cell pathology and devise new pharmacological and cell replacement therapies. Stem Cells 2019;37:33-41., (© 2018 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Global lack of mesencephalic astrocyte-derived neurotropic factor (MANF) leads to progressive postnatal loss of β-cell mass and insulin-dependent diabetes in mice. Similar to Manf -/- mice, embryonic ablation of MANF specifically from the pancreas results in diabetes. In this study, we assessed the importance of MANF for the postnatal expansion of pancreatic β-cell mass and for adult β-cell maintenance in mice. Detailed analysis of Pdx-1Cre +/- :: Manf fl/fl mice revealed mosaic MANF expression in postnatal pancreata and a significant correlation between the number of MANF-positive β-cells and β-cell mass in individual mice. In vitro, recombinant MANF induced β-cell proliferation in islets from aged mice and protected from hyperglycemia-induced endoplasmic reticulum (ER) stress. Consequently, excision of MANF from β-cells of adult MIP-1Cre ERT ::Manf fl/fl mice resulted in reduced β-cell mass and diabetes caused largely by β-cell ER stress and apoptosis, possibly accompanied by β-cell dedifferentiation and reduced rates of β-cell proliferation. Thus, MANF expression in adult mouse β-cells is needed for their maintenance in vivo. We also revealed a mechanistic link between ER stress and inflammatory signaling pathways leading to β-cell death in the absence of MANF. Hence, MANF might be a potential target for regenerative therapy in diabetes., (© 2018 by the American Diabetes Association.)

Insulin gene mutations are a leading cause of neonatal diabetes. They can lead to proinsulin misfolding and its retention in endoplasmic reticulum (ER). This results in increased ER-stress suggested to trigger beta-cell apoptosis. In humans, the mechanisms underlying beta-cell failure remain unclear. Here we show that misfolded proinsulin impairs developing beta-cell proliferation without increasing apoptosis. We generated induced pluripotent stem cells (iPSCs) from people carrying insulin ( INS ) mutations, engineered isogenic CRISPR-Cas9 mutation-corrected lines and differentiated them to beta-like cells. Single-cell RNA-sequencing analysis showed increased ER-stress and reduced proliferation in INS-mutant beta-like cells compared with corrected controls. Upon transplantation into mice, INS-mutant grafts presented reduced insulin secretion and aggravated ER-stress. Cell size, mTORC1 signaling, and respiratory chain subunits expression were all reduced in INS -mutant beta-like cells, yet apoptosis was not increased at any stage. Our results demonstrate that neonatal diabetes-associated INS-mutations lead to defective beta-cell mass expansion, contributing to diabetes development., Competing Interests: DB, JS, DB, MS, PL, EG, SE, JU, HG, HH, JP, KW, TO No competing interests declared, (© 2018, Balboa et al.)

Transfer RNAs (tRNAs) are non-coding RNA molecules essential for protein synthesis. Post-transcriptionally they are heavily modified to improve their function, folding and stability. Intronic polymorphisms in CDKAL1, a tRNA methylthiotransferase, are associated with increased type 2 diabetes risk. Loss-of-function mutations in TRMT10A, a tRNA methyltransferase, are a monogenic cause of early onset diabetes and microcephaly. Here we confirm the role of TRMT10A as a guanosine 9 tRNA methyltransferase, and identify tRNAGln and tRNAiMeth as two of its targets. Using RNA interference and induced pluripotent stem cell-derived pancreatic β-like cells from healthy controls and TRMT10A-deficient patients we demonstrate that TRMT10A deficiency induces oxidative stress and triggers the intrinsic pathway of apoptosis in β-cells. We show that tRNA guanosine 9 hypomethylation leads to tRNAGln fragmentation and that 5'-tRNAGln fragments mediate TRMT10A deficiency-induced β-cell death. This study unmasks tRNA hypomethylation and fragmentation as a hitherto unknown mechanism of pancreatic β-cell demise relevant to monogenic and polygenic forms of diabetes.

Aims/hypothesis: There is a great need to identify factors that could protect pancreatic beta cells against apoptosis or stimulate their replication and thus prevent or reverse the development of diabetes. One potential candidate is mesencephalic astrocyte-derived neurotrophic factor (MANF), an endoplasmic reticulum (ER) stress inducible protein. Manf knockout mice used as a model of diabetes develop the condition because of increased apoptosis and reduced proliferation of beta cells, apparently related to ER stress. Given this novel association between MANF and beta cell death, we studied the potential of MANF to protect human beta cells against experimentally induced ER stress., Methods: Primary human islets were challenged with proinflammatory cytokines, with or without MANF. Cell viability was analysed and global transcriptomic analysis performed. Results were further validated using the human beta cell line EndoC-βH1., Results: There was increased expression and secretion of MANF in human beta cells in response to cytokines. Addition of recombinant human MANF reduced cytokine-induced cell death by 38% in human islets (p < 0.05). MANF knockdown in EndoC-βH1 cells led to increased ER stress after cytokine challenge. Mechanistic studies showed that the protective effect of MANF was associated with repression of the NF-κB signalling pathway and amelioration of ER stress. MANF also increased the proliferation of primary human beta cells twofold when TGF-β signalling was inhibited (p < 0.01)., Conclusions/interpretation: Our studies show that exogenous MANF protein can provide protection to human beta cells against death induced by inflammatory stress. The antiapoptotic and mitogenic properties of MANF make it a potential therapeutic agent for beta cell protection.

CRISPR-Cas9-based gene activation (CRISPRa) is an attractive tool for cellular reprogramming applications due to its high multiplexing capacity and direct targeting of endogenous loci. Here we present the reprogramming of primary human skin fibroblasts into induced pluripotent stem cells (iPSCs) using CRISPRa, targeting endogenous OCT4, SOX2, KLF4, MYC, and LIN28A promoters. The low basal reprogramming efficiency can be improved by an order of magnitude by additionally targeting a conserved Alu-motif enriched near genes involved in embryo genome activation (EEA-motif). This effect is mediated in part by more efficient activation of NANOG and REX1. These data demonstrate that human somatic cells can be reprogrammed into iPSCs using only CRISPRa. Furthermore, the results unravel the involvement of EEA-motif-associated mechanisms in cellular reprogramming.

The generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming holds great potential for modeling human diseases. However, the reprogramming process remains very inefficient and a better understanding of its basic biology is required. The mesenchymal-to-epithelial transition (MET) has been recognized as a crucial step for the successful reprogramming of fibroblasts into iPSCs. It has been reported that the p53 tumor suppressor gene acts as a barrier of this process, while its homolog p63 acts as an enabling factor. In this regard, the information concerning the role of the third homolog, p73, during cell reprogramming is limited. Here, we derive total Trp73 knockout mouse embryonic fibroblasts, with or without Trp53, and examine their reprogramming capacity. We show that p73 is required for effective reprogramming by the Yamanaka factors, even in the absence of p53. Lack of p73 affects the early stages of reprogramming, impairing the MET and resulting in altered maturation and stabilization phases. Accordingly, the obtained p73-deficient iPSCs have a defective epithelial phenotype and alterations in the expression of pluripotency markers. We demonstrate that p73 deficiency impairs the MET, at least in part, by hindering BMP pathway activation. We report that p73 is a positive modulator of the BMP circuit, enhancing its activation by DNp73 repression of the Smad6 promoter. Collectively, these findings provide mechanistic insight into the MET process, proposing p73 as an enhancer of MET during cellular reprogramming.

OCT4 is a crucial transcription factor in the pluripotent stem cell gene regulatory network and an essential factor for pluripotent reprogramming. We engineered the previously reported HEL24.3 hiPSC to generate an OCT4 reporter cell line by knocking-in a T2A nuclear EmGFP reporter cassette before the OCT4 gene STOP codon sequence. To enhance targeted insertion, homologous recombination was stimulated using targeted cutting at the OCT4 STOP codon with CRISPR/SpCas9. This HEL24.3-OCT4-nEmGFP cell line faithfully reports endogenous OCT4 expression, serving as a useful tool to examine temporal changes in OCT4 expression in live cells during hiPSC culture, differentiation and somatic cell reprogramming., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Cell-type-specific functions and identity are tightly regulated by interactions between the cell cytoskeleton and the extracellular matrix (ECM). Human pluripotent stem cells (hPSCs) have ultimate differentiation capacity and exceptionally low-strength ECM contact, yet the organization and function of adhesion sites and associated actin cytoskeleton remain poorly defined. We imaged hPSCs at the cell-ECM interface with total internal reflection fluorescence microscopy and discovered that adhesions at the colony edge were exceptionally large and connected by thick ventral stress fibers. The actin fence encircling the colony was found to exert extensive Rho-ROCK-myosin-dependent mechanical stress to enforce colony morphology, compaction, and pluripotency and to define mitotic spindle orientation. Remarkably, differentiation altered adhesion organization and signaling characterized by a switch from ventral to dorsal stress fibers, reduced mechanical stress, and increased integrin activity and cell-ECM adhesion strength. Thus, pluripotency appears to be linked to unique colony organization and adhesion structure., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

SOX2 is an important transcription factor involved in pluripotency maintenance, pluripotent reprogramming and differentiation towards neural lineages. Here we engineered the previously described HEL24.3 hiPSC to generate a SOX2 reporter by knocking-in a T2A fused nuclear tdTomato reporter cassette before the STOP codon of the SOX2 gene coding sequence. CRISPR/SaCas9-mediated stimulation of homologous recombination was utilized to facilitate faithful targeted insertion. This line accurately reports the expression of endogenous SOX2 and therefore constitutes a useful tool to study the SOX2 expression dynamics upon hiPSC culture, differentiation and somatic cell reprogramming., (Copyright © 2017 The Authors. Published by Elsevier B.V. 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.)