RFX3 in human pancreatic islet development has not been explored. This study aims to investigate the function of RFX3 in human pancreatic islet development using human islet organoids derived from iPSCs, hypothesizing that RFX3 regulates human islet cell differentiation. We generated RFX3 knockout ( RFX3 KO) iPSC lines using CRISPR/Cas9 and differentiated them into pancreatic islet organoids. Various techniques were employed to assess gene expression, cell markers, apoptosis, proliferation, and glucose-stimulated insulin secretion. Single-cell RNA sequencing (scRNA-seq) datasets from hESC-derived pancreatic islets were re-analyzed to investigate RFX3 expression in specific cell populations at various developmental stages. Furthermore, bulk RNA sequencing was conducted to further assess transcriptomic changes. RFX3 was found to be highly expressed in pancreatic endocrine cell populations within pancreatic progenitors (PPs), endocrine progenitors (EPs), and mature islet stages derived from iPSCs. scRNA-seq further confirmed RFX3 expression across different endocrine cell clusters during differentiation. RFX3 loss disrupted pancreatic endocrine gene regulation, reduced hormone-secreting islet cells, and impaired beta-cell function and insulin secretion. Despite a significant reduction in pancreatic islet hormones, the pan-endocrine marker CHGA remained unchanged at both EP and islet stages, likely due to an increase in enterochromaffin cells (ECs). This was supported by our findings of high EC marker expression in RFX3 KO EPs and islets. In addition, RFX3 loss led to smaller islet organoids, elevated TXNIP levels, and increased apoptosis in EPs and islets. These findings underscore the crucial role of RFX3 in regulating human islet cell differentiation and its role in suppressing enterochromaffin cell specification. These insights into RFX3 function have implications for understanding islet biology and potential diabetes susceptibility. ### Competing Interest Statement S.H. is a co-founder and shareholder of Sequantrix GmbH and has research funding from by Novo Nordisk and Askbio. The authors declare that there are no relationships or activities that might bias, or be perceived to bias, their work.

The role of regulatory factor X 3 (RFX3) in human pancreatic islet development has not been explored. This study aims to investigate the function of RFX3 in human pancreatic islet development using human islet organoids derived from induced pluripotent stem cells (iPSCs), hypothesising that RFX3 regulates human islet cell differentiation. We generated RFX3 knockout (RFX3 KO) iPSC lines using CRISPR/Cas9 and differentiated them into pancreatic islet organoids. Various techniques were employed to assess gene expression, cell markers, apoptosis, proliferation and glucose-stimulated insulin secretion. Single-cell RNA-seq datasets from human embryonic stem cell-derived pancreatic islet differentiation were re-analysed to investigate RFX3 expression in specific cell populations at various developmental stages. Furthermore, bulk RNA-seq was conducted to further assess transcriptomic changes. RFX3 overexpression was implemented to reverse dysregulated gene expression. RFX3 was found to be highly expressed in pancreatic endocrine cell populations within pancreatic progenitors (PPs), endocrine progenitors (EPs) and mature islet stages derived from iPSCs. Single-cell RNA-seq further confirmed RFX3 expression across different endocrine cell clusters during differentiation. The loss of RFX3 disrupted pancreatic endocrine gene regulation, reduced the number of hormone-secreting islet cells and impaired beta cell function and insulin secretion. Despite a significant reduction in the expression levels of pancreatic islet hormones, the pan-endocrine marker chromogranin A remained unchanged at both EP and islet stages, likely due to an increase in the abundance of enterochromaffin cells (ECs). This was supported by our findings of high EC marker expression levels in RFX3 KO EPs and islets. In addition, RFX3 loss led to smaller islet organoids, elevated thioredoxin-interacting protein levels and increased apoptosis in EPs and islets. Furthermore, RFX3 overexpression rescued the expression of dysregulated genes in RFX3 KO at the PP and EP stages. These findings underscore the crucial role of RFX3 in regulating human islet cell differentiation and its role in suppressing EC specification. These insights into RFX3 function have implications for understanding islet biology and potential diabetes susceptibility. The RNA-seq datasets have been submitted to the Zenodo repository and can be accessed via the following links: DOI https://doi.org/10.5281/zenodo.13647651 (PPs); and DOI https://doi.org/10.5281/zenodo.13762055 (SC-islets).

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 ).

RFX6 is essential for pancreatic development and insulin secretion, while its role in diabetes pathogenesis is unclear. Here, RFX6 expression was detected in PDX1+ cells in the hESC-derived posterior foregut (PF). However, in the pancreatic progenitors (PPs), RFX6 did not co-localize with PDX1 and NKX6.1, but instead with NEUROG3, NKX2.2, and islet hormones in the endocrine progenitor (EPs) and islets. Single-cell analysis revealed high RFX6 expression 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 it did not affect PPs co-expressing PDX1 and NKX6.1. RNA sequencing 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 (CAT) expression, implying a protective role for RFX6. Overexpression of RFX6 reversed defective phenotypes in PPs and EPs. 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.### Competing Interest StatementSH is a co-founder and shareholder of Sequantrix GmbH and has research funding from by Novo Nordisk and Askbio. The authors declare that there are no relationships or activities that might bias, or be perceived to bias, their work.

Targeting tumour metabolism through glucose transporters is an attractive approach. However, the role these transporters play through interaction with other signalling proteins is not yet defined. The glucose transporter SLC2A3 (GLUT3) is a member of the solute carrier transporter proteins. GLUT3 has a high affinity for D-glucose and regulates glucose uptake in the neurons, as well as other tissues. Herein, we show that GLUT3 is involved in the uptake of arsenite, and its level is regulated by peroxiredoxin 1 (PRDX1). In the absence of PRDX1, GLUT3 mRNA and protein expression levels are low, but they are increased upon arsenite treatment, correlating with an increased uptake of glucose. The downregulation of GLUT3 by siRNA or deletion of the gene by CRISPR cas-9 confers resistance to arsenite. Additionally, the overexpression of GLUT3 sensitises the cells to arsenite. We further show that GLUT3 interacts with PRDX1, and it forms nuclear foci, which are redistributed upon arsenite exposure, as revealed by immunofluorescence analysis. We propose that GLUT3 plays a role in mediating the uptake of arsenite into cells, and its homeostatic and redox states are tightly regulated by PRDX1. As such, GLUT3 and PRDX1 are likely to be novel targets for arsenite-based cancer therapy.

Human pluripotent stem cells (hPSCs) are a valuable tool for the study of the cellular and molecular mechanisms that underlie different types of diabetes. However, one pitfall of hPSCs is that genomic aberrations can develop during the reprogramming process as a result of gene editing or simply during extended cell culture (1). Some of these aberrations, such as trisomy 1, hamper embryonic development and lead to elimination of the fetus (2). Such detrimental aberrations acquired in hPSCs can cause genetic imbalance, thus affect-ing cellular identity and function. Therefore, given the fatal phenotypes observed in their in vivo manifestations, the use of hPSC lines that acquire such fatal genomic aberrations in disease modeling studies must be prevented. In the article by Carrasco et al. (3), the authors used induced pluripotent stem cells (iPSCs) that carry abnormal chromosomal content to investigate molecular mechanisms underly-ing maturity-onset diabetes of the young type 1 (MODY1). They generated iPSCs from a patient with MODY1 (here termed MODY1-iPSCs) carrying a mutation in the HNF4A gene. They also corrected this mutation in MODY1-iPSCs, using gene editing, to serve as isogenic controls and then differ-entiated them into pancreatic b -cells to evaluate the effect of mutation on b -cell development and function (3). The differ-ences observed by the authors using both two-dimensional andthree-dimensional differentiation approaches variedbased on the effect of the cellular environment on biological pro-cesses being switched on. Prior to differentiation, they used karyotyping analysis to examine the integrity of the

Background The genetic factors associated with insulin resistance (IR) are not well understood. Clinical studies on first-degree relatives of type 2 diabetic (T2D) patients, which have the highest genetic predisposition to T2D, have given insights into the role of IR in T2D pathogenesis. Induced pluripotent stem cells (iPSCs) are excellent tools for disease modeling as they can retain the genetic imprint of the disease. Therefore, in this study, we aimed to investigate the genetic perturbations associated with insulin resistance (IR) in the offspring of T2D parents using patient-specific iPSCs. Methods We generated iPSCs from IR individuals (IR-iPSCs) that were offspring of T2D parents as well as from insulin-sensitive (IS-iPSCs) individuals. We then performed transcriptomics to identify key dysregulated gene networks in the IR-iPSCs in comparison to IS-iPSCs and functionally validated them. Results Transcriptomics on IR-iPSCs revealed dysregulated gene networks and biological processes indicating that they carry the genetic defects associated with IR that may lead to T2D. The IR-iPSCs had increased lactate secretion and a higher phosphorylation of AKT upon stimulation with insulin. IR-iPSCs have increased cellular oxidative stress indicated by a high production of reactive oxygen species and higher susceptibility to H 2 O 2 -induced apoptosis. Conclusions IR-iPSCs generated from offspring of diabetic patients confirm that oxidative stress and increased lactate secretion, associated with IR, are inherited in this population, and may place them at a high risk of T2D. Overall, our IR-iPSC model can be employed for T2D modeling and drug screening studies that target genetic perturbations associated with IR in individuals with a high risk for T2D.

Although genome profiling provides important genetic and phenotypic details for applying precision medicine to diabetes, it is imperative to integrate in vitro human cell models, accurately recapitulating the genetic alterations associated with diabetes. The absence of the appropriate preclinical human models and the unavailability of genetically relevant cells substantially limit the progress in developing personalized treatment for diabetes. Human pluripotent stem cells (hPSCs) provide a scalable source for generating diabetes-relevant cells carrying the genetic signatures of the patients. Remarkably, allogenic hPSC-derived pancreatic progenitors and β cells are being used in clinical trials with promising preliminary results. Autologous hiPSC therapy options exist for those with monogenic and type 2 diabetes; however, encapsulation or immunosuppression must be accompanied with in the case of type 1 diabetes. Furthermore, genome-wide association studies-identified candidate variants can be introduced in hPSCs for deciphering the associated molecular defects. The hPSC-based disease models serve as excellent resources for drug development facilitating personalized treatment. Indeed, hPSC-based diabetes models have successfully provided valuable knowledge by modeling different types of diabetes, which are discussed in this review. Herein, we also evaluate their strengths and shortcomings in dissecting the underlying pathogenic molecular mechanisms and discuss strategies for improving hPSC-based disease modeling investigations.

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling, however, with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell's physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The molecular link between SARS-CoV-2 infection and susceptibility is not well understood. Nonetheless, a bi-directional relationship between SARS-CoV-2 and diabetes has been proposed. The angiotensin-converting enzyme 2 (ACE2) is considered as the primary protein facilitating SARS-CoV and SARS-CoV-2 attachment and entry into the host cells. Studies suggested that ACE2 is expressed in the endocrine cells of the pancreas including beta cells, in addition to the lungs and other organs; however, its expression in the islets, particularly beta cells, has been met with some contradiction. Importantly, ACE2 plays a crucial role in glucose homoeostasis and insulin secretion by regulating beta cell physiology. Given the ability of SARS-CoV-2 to infect human pluripotent stem cell-derived pancreatic cells in vitro and the presence of SARS-CoV-2 in pancreatic samples from COVID-19 patients strongly hints that SARS-CoV-2 can invade the pancreas and directly cause pancreatic injury and diabetes. However, more studies are required to dissect the underpinning molecular mechanisms triggered in SARS-CoV-2-infected islets that lead to aggravation of diabetes. Regardless, it is important to understand the function of ACE2 in the pancreatic islets to design relevant therapeutic interventions in combatting the effects of SARS-CoV-2 on diabetes pathophysiology. Herein, we detail the function of ACE2 in pancreatic beta cells crucial for regulating insulin sensitivity, secretion, and glucose metabolism. Also, we discuss the potential role played by ACE2 in aiding SARS-COV-2 entry into the pancreas and the possibility of ACE2 cooperation with alternative entry factors as well as how that may be linked to diabetes pathogenesis.

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.

Psoriasis is characterized by hyperproliferation and defective differentiation of keratinocytes (KCs). Patients with psoriasis are at a high risk of developing diabetes and cardiovascular diseases. The debate on the genetic origin of psoriasis pathogenesis remains unresolved due to lack of suitable in vitro human models mimicking the disease phenotypes. In this study, we provide the first human induced pluripotent stem cell (iPSC) model for psoriasis carrying the genetic signature of the patients. iPSCs were generated from patients with psoriasis (PsO-iPSCs) and healthy donors (Ctr-iPSCs) and were efficiently differentiated into mature KCs. RNA sequencing of KCs derived from Ctr-iPSCs and PsO-iPSCs identified 361 commonly upregulated and 412 commonly downregulated genes. KCs derived from PsO-iPSCs showed dysregulated transcripts associated with psoriasis and KC differentiation, such as HLA-C, KLF4, chemokines, type I interferon-inducible genes, solute carrier family, IVL, DSG1, and HLA-DQA1, as well as transcripts associated with insulin resistance, such as IRS2, GDF15, GLUT10, and GLUT14. Our data suggest that the KC abnormalities are the main driver triggering psoriasis pathology and highlights the substantial contribution of genetic predisposition in the development of psoriasis and insulin resistance.

Diabetes mellitus (DM) is one of the most prevalent metabolic disorders. In order to replace the function of the destroyed pancreatic beta cells in diabetes, islet transplantation is the most widely practiced treatment. However, it has several limitations. As an alternative approach, human pluripotent stem cells (hPSCs) can provide an unlimited source of pancreatic cells that have the ability to secrete insulin in response to a high blood glucose level. However, the determination of the appropriate pancreatic lineage candidate for the purpose of cell therapy for the treatment of diabetes is still debated. While hPSC-derived beta cells are perceived as the ultimate candidate, their efficiency needs further improvement in order to obtain a sufficient number of glucose responsive beta cells for transplantation therapy. On the other hand, hPSC-derived pancreatic progenitors can be efficiently generated in vitro and can further mature into glucose responsive beta cells in vivo after transplantation. Herein, we discuss the advantages and predicted challenges associated with the use of each of the two pancreatic lineage products for diabetes cell therapy. Furthermore, we address the co-generation of functionally relevant islet cell subpopulations and structural properties contributing to the glucose responsiveness of beta cells, as well as the available encapsulation technology for these cells.

Diabetes is a metabolic disease caused by the loss or impaired function of insulin-producing pancreatic β-cells. Different therapeutic strategies aim to restore the endogenous production of insulin rather than the cornerstone insulin injections treatment. Human pluripotent stem cells (hPSCs) have been proposed as an unlimited source for cell-based therapy of diabetes through the directed differentiation into functional pancreatic β cells. Step-wise differentiation protocols based on developmental biology of pancreas, have led to the generation of insulin-producing β cells. However, the majority of the cells produced were poly-hormonal as they expressed other hormones in addition to insulin and have failed to respond when challenged with glucose. The coordinate expression of particular transcription factors (TF) in distinct stages governs the differentiation of hPSCs into insulin β cells. Pancreatic and duodenal homeobox protein (PDX1) is a crucial TF required for pancreas development. On the other hand, homeobox protein NKX6.1 is a potent bi-functional TF that is essential for β cells maturation, proliferation and insulin metabolism. The dual expression of PDX1 and NKX6.1 during multipotent progenitor cell (MPC) stage is vital for guiding the cells towards functional β cells lineage. However, cells expressing PDX1 but lack NKX6.1 expression tend to take the poly-hormonal path. This guided the differentiation protocols to focus on enriching MPC population co-expressing PDX1 and NKX6.1. The aim of this study was to further explore different MPC populations in terms of PDX1/NKX6.1 expression. We used two different differentiation protocols to differentiate hESCs and hiPSCs into MPCs. The mRNA and protein expressions of the generated MPCs were analyzed using immunocytochemistry, RT-PCR, and flow cytometry. Our results showed that hPSCs were successfully differentiated into the conventional (PDX1+/NKX6.1+) and (PDX1+/NKX6.1-) MPC populations. The efficiency of differentiating hPSCs into PDX1+/NKX6.1+ MPCs has varied between the two used protocols. Immunofluorescence staining has unveiled the generation of a novel population that expressed NKX6.1 independently of PDX1 (PDX1-/NKX6.1+) in both hESCs and hiPSCs. This is surprising considering that PDX1 was reported to bind to the promoter of NKX6.1 gene and is needed for NKX6.1 expression. Furthermore, using our optimized protocol, this uncharacterized subset of MPCs was enriched and found to exhibit a pattern of three-dimensional (3D) aggregates that were consistently (PDX1-/NKX6.1+) and surrounded by either (PDX1+/NKX6.1+) or (PDX1+/NKX6.1-) MPCs. To understand and characterize this unique population, we examined the expression of other TFs including endocrine precursors markers Chromogranin A (CHGA) and NKX2.2. CHGA was found to be expressed in the same areas that were positive for NKX6.1 and PDX1. However, the 3D structures that were PDX1-/NKX6.1+ did not co-express CHGA. On the contrary, few cells of these 3D aggregates co-expressed NKX2.2, suggesting that this population may have an undefined role in the development of MPCs into endocrine progenitors. These findings showcase a novel population of NKX6.1 expressing MPCs that did not require PDX1 expression at this stage. Moreover, this population may retain an alternative path towards pancreatic islet cells development that is independent of PDX1. A thorough characterization of this population is needed to explore the regulatory gene network controlling their lineage specification.

Scalable production of human pluripotent stem cell (hPSC)-derived β cells in vitro would greatly facilitate transplantation therapy and drug discovery for treating diabetes. Employing step-wise differentiation protocols, hPSCs can be differentiated through consecutive stages of endoderm, foregut, pancreatic and endocrine progenitors to ultimately give insulin secreting β cells. Pancreatic progenitors co-expressing the two key transcription factors (TFs), PDX1 and NKX6.1, are recognized as the indispensable precursors of functional, mono-hormonal β cells. Here, we established an optimized protocol for maximizing PDX1+/NKX6.1+ co-positive pancreatic progenitors from hESCs in monolayer culture and increasing their proliferative capacity. Our technique of dissociating densely formed endodermal cells and re-plating them in lower densities on fresh matrigel matrix followed by an augmented duration of retinoid and FGF10 signaling strikingly increased the expression of NKX6.1, which is exclusive only to β cells amongst all endocrine cells. This high induction of NKX6.1 resulted in an increased proportion of PDX1+/ NKX6.1+ population, generating up to >90% PDX1+/ NKX6.1+ co-positive progenitors in monolayer, higher than previously published protocols. In contrast to multiple studies showing negligible induction of NKX6.1 at lower densities, we provide evidence that higher folds of NKX6.1 can be induced in dissociated cells re-plated lower densities compared to aggregations in non-dissociated culture if the duration of retinoid and FGF signaling is prolonged. Our optimized protocol enhanced pancreatic differentiation efficiency by up-regulating pancreatic progenitor TFs such as PDX1, SOX9, HNF6 and FOXA2 and increased the mRNA levels of endocrine TFs such as NEUROG3, NKX2.2 and NEUROD1. Additionally, we show that manipulating cell-cell attachment following endoderm generation in vitro during pancreatic differentiation dramatically inhibited alternate hepatic fate specification by down-regulating hepatic markers like AFP and ALB expression in our optimized protocol in comparison to recently published protocols for generating pancreatic progenitors. Notably, cell cycle and BrdU incorporation assays revealed that our method increased the proliferative capacity of pancreatic progenitors throughout the differentiation stages by increasing the fraction of cells entering S phase of cell cycle and a comparative increase in Ki67 expression, the proliferation marker. As a result, we obtained >70% Ki67+ /SOX9+ pancreatic progenitors in monolayer confirming an increased self-replicating capacity of the generated PDX1+/ NKX6.1+ progenitors. Furthermore, using our optimized protocol for pancreatic differentiation, we were able to enrich a novel and uncharacterized NKX6.1+ /PDX1- population, devoid of Chromogranin A (CHGA) expression, which are therefore proposed to be more mature precursors of β cell. This population re-arranged themselves in embedded, highly compact three-dimensional structures that showed high expression of Ki67. Continuation of our optimized protocol into endocrine differentiation stage validated the ability of our PDX1+/ NKX6.1+ to generate NGN3+/ NKX6.1+ co-positive endocrine progenitors in vitro with a high expression of CHGA and NKX2.2. Therefore, here we show that manipulating the cellular density, cell-cell attachment and cues from extracellular matrix plays a major role in improving pancreatic differentiation efficiency and proliferation thereby providing a cost-effective method for generating pancreatic progenitors in vitro in adherent culture. Indeed, our novel method for maximizing PDX1+/ NKX6.1+ progenitors from hPSCs in monolayer culture could serve as a source of highly proliferative pancreatic progenitors aiding scalable production of functional β cells in vitro.

Insulin resistance (IR) is a precursor and accelerating factor for Type 2 diabetes (T2D), the greatest health challenge facing Qatar and the world today. Psoriasis, is an immune-mediated, chronic skin disorder that can aggregate in families, because of its strong genetic predisposition. It has been found that patients with psoriasis for more than two years, regardless the severity of the disease, are at a very high risk of developing insulin resistance and diabetes. Although several studies highlighted the link between IR and skin disorders, no reports studied the relationship between IR, T2D, and epidermal dysfunction using induced pluripotent stem cells (iPSCs). Therefore, our aim in this study was to generate patient-specific hiPSCs from IR Qatari patients (associated with psoriasis or T2D) and differentiate them into insulin target cells. Blood samples were collected from Qatari individuals with a family history of IR associated with T2D or psoriasis as well as from healthy individuals. The Ficoll-Paque density gradient method was used to separate the peripheral blood mononuclear cells (PBMCs). The isolated PBMCs were cultured in vitro for 5 days in StemPro-34 culture medium before transduction. PBMCs were reporgammed using Sendai viral vectors encoding four pluripotency factors including OCT4, SOX2, C-MYC, and KLF4. After 20-30 days of the transduction, several undifferentiated colonies of high morphological quality (defined border and high nuclear to cytoplasmic ratio) were manually picked up and transferred to new matrigel-coated plates to establish different clones. Several hiPSC clones were established from each individual (sample) and only three clones were maintained after extensive characterization of all clones. The generated hiPSC clones were characterized using different techniques including immunostaining, RT-PCR, Western blotting, alkaline phosphatase assay, embroid body (EB) formation, karyotyping, and hPSC ScoreCard assay. H1-human embryonic stem cell (H1-hiESC) line was used as a positive control in all experiments. Similar to hESCs, hiPSC clones expressed pluripotency markers, such as OCT4, SOX2, NANOG, KLF4, C-MYC, SSEA4, TRA-60, TRA-81, REX-1, DPPA4, and TERT at mRNA and protein levels. All hiPSC lines showed standard hESC morphology, normal karyotype and stained positive for alkalaine phosphatase. Only the hiPSC lines that showed similar characteristics as those of hESCs were maintained and expanded in culture. To confirm the pluripotent ability of the generated hiPSCs, we used EB technique to differentiate patient-specific hiPSCs into three germ layers in vitro. Immunostaining and RT-PCR analyses showed that these hiPSCs can differentiate into endodermal (SOX17+/FOXA2+), mesodermal (BRACHYURY+) and ecodermal (NESTIN+) lineages. To further validate the multilineage differentiation potential of the generated hiPSCs, we used the hPSC ScoreCard assay in vitro. These findings indicate that hiPSCs generated in this study are pluripotent, fully reprogrammed stem cells having the ability to differentiate into any cell type of the body. Thus, we will differentiate patient-specific hiPSCs into insulin target cells carrying the genetic background of the patients to identify signaling mechanism involved in the inherited form of IR and to understand the genetic link between IR, T2D, and psoriasis. To our knowledge, this is the first study to generate hiPSCs from diabetic and psoriatic patients in the MENA region.

The original article [1] contains a number of small errors which the authors would like to clarify.

The expression of a specific combination of transcription factors (TFs) in the multipotent progenitor cells (MPCs) is critical for determining pancreatic cell fate. NKX6.1 expression in PDX1+ MPCs is required for functional β cell generation. We have recently demonstrated the generation of a novel population of human pluripotent stem cell (hPSC)-derived MPCs that exclusively express NKX6.1, independently of PDX1 (PDX1−/NKX6.1+). Therefore, the aim of this study was to characterize this novel population to elucidate its role in pancreatic development.

The loss of functional β cells leads to development of diabetes. Several studies have shown that β cells are specified through several stages of progenitors during pancreas development, each stage defined by the expression of specific transcription factors (TFs). Understanding signalling pathways that control the differentiation and specification processes during embryogenesis will facilitate efforts to obtain functional β cells in vitro. Our current knowledge of the mechanisms involved in pancreatic β cell development and survival under normal or diabetic conditions has come largely from animal studies. However, there are marked differences in islet structure and physiological properties between humans and animals, and not all phenotypes of human diabetes can be recapitulated in animal models. Therefore, human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs) offer a great opportunity for increasing our understanding of the pathways regulating human pancreatic β-cell development and survival. Furthermore, hPSCs provide a renewable source of functional pancreatic β cells for cell replacement therapy as well as disease modelling. Herein, we discuss the signalling pathways involved in the development of pancreatic β cells during embryogenesis. Additionally, we describe how these pathways are manipulated in vitro to differentiate hPSCs into functional β cells. Finally, we highlight the progress that has been made for the applications of those cells in treating and modelling diabetes.