Altered Regulation of the Glucose Transporter GLUT3 in PRDX1 Null Cells Caused Hypersensitivity to Arsenite.

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.

iPSCs derived from insulin resistant offspring of type 2 diabetic patients show increased oxidative stress and lactate secretion.

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 H2O2 -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.

Toward Precision Medicine with Human Pluripotent Stem Cells for Diabetes.

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 ß 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.

Highly Efficient Differentiation of Human Pluripotent Stem Cells into Pancreatic Progenitors Co-expressing PDX1 and NKX6.1.

Diabetes is a complex metabolic disorder, with no available treatment. Islet transplantation is currently practiced beta cell replacement therapy option, however, with major limitations. Human pluripotent stem cells (hPSCs) can be used as a scalable source for generation of insulin-secreting cells as hPSCs have high proliferative capacity and can differentiate into any tissue type. In vitro stepwise protocols have been designed for differentiating hPSCs into pancreatic lineages that finally give rise to beta cells; however, these hPSC-derived beta cells are dissimilar to adult human beta cells in key aspects of gene expression and functionality. Alternatively, pancreatic progenitors, when transplanted in the body, have been shown to mature into functional insulin-secreting beta cells, capable of reversing hyperglycemia. These pancreatic progenitors require the co-expression of PDX1, a transcription factor (TF) regulating pancreatic development, and NKX6.1, another TF key for beta cell maturation and function, to produce glucose-responsive beta cells. Given the crucial role played by NKX6.1, we optimized an in vitro differentiation protocol to enhance the generation of pancreatic progenitors co-expressing PDX1 and NKX6.1 by modulating cell density, matrix availability, and cellular dissociation.

ACE2 function in the pancreatic islet: Implications for relationship between SARS-CoV-2 and diabetes.

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.

PDX1- /NKX6.1+ progenitors derived from human pluripotent stem cells as a novel source of insulin-secreting cells.

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.

Differentiation of Human Pluripotent Stem Cells into Pancreatic Beta-cell Precursors in a 2D Culture System.

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.

Stem Cell Therapy for Diabetes: Beta Cells versus Pancreatic Progenitors.

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.

Keratinocytes Derived from Patient-Specific Induced Pluripotent Stem Cells Recapitulate the Genetic Signature of Psoriasis Disease.

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.

Correction to: Enhanced differentiation of human pluripotent stem cells into pancreatic progenitors co-expressing PDX1 and NKX6.1.

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

Differentiation of human pluripotent stem cells into two distinct NKX6.1 populations of pancreatic progenitors.

BACKGROUND: 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 ß 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. METHODS: The hPSCs were exposed to two differentiation protocols to generate MPCs that were analyzed using different techniques. RESULTS: Based on the expression of PDX1 and NKX6.1, we generated three different populations of MPCs, two of them were NKX6.1+. One of these NKX6.1 populations coexpressed PDX1 (PDX1+/NKX6.1+) which is known to mature into functional ß cells, and an additional novel population did not express PDX1 (PDX1-/NKX6.1+) with an undefined role in pancreatic cell fate. This novel population was enriched using our recently established protocol, allowing their reorganization in three-dimensional (3D) structures. Since NKX6.1 induction in MPCs can direct them to endocrine and/or ductal cells in humans, we examined the coexpression of endocrine and ductal markers. We found that the expression of the pancreatic endocrine progenitor markers chromogranin A (CHGA) and neurogenin 3 (NGN3) was not detected in the NKX6.1+ 3D structures, while few structures were positive for NKX2.2, another endocrine progenitor marker, thereby shedding light on the origin of this novel population and its role in pancreatic endocrine development. Furthermore, SOX9 was highly expressed in the 3D structures, but cytokeratin 19, a main ductal marker, was not detected in these structures. CONCLUSIONS: These data support the existence of two independent NKX6.1+ MPC populations during human pancreatic development and the novel PDX1-/NKX6.1+ population may be involved in a unique trajectory to generate ß cells in humans.

Enhanced differentiation of human pluripotent stem cells into pancreatic progenitors co-expressing PDX1 and NKX6.1.

BACKGROUND: Pancreatic progenitors (PPs) co-expressing the two transcription factors (TFs) PDX1 and NKX6.1 are recognized as the indispensable precursors of functional pancreatic ß cells. Here, we aimed to establish an efficient protocol for maximizing generation of PDX1+/NKX6.1+ PPs from human pluripotent stem cells (hPSCs). METHODS: In order to enhance the PDX1+/NKX6.1+ population, we manipulated in vitro culture conditions during differentiation by dissociating densely formed endodermal cells and re-plating them at different densities. These dissociated cells were subjected to an augmented duration of retinoid and fibroblast growth factor (FGF)10 signaling to induce higher PDX1 and NKX6.1 expression. RESULTS: Our optimized protocol dramatically increased the expression of NKX6.1, leading to an increase in the proportion of PDX1+/NKX6.1+ progenitors (~90%) in monolayer, higher than the previously published protocols, as well as upregulated key TFs controlling pancreatic development. The improved efficiency of pancreatic differentiation was complemented by an inhibited hepatic specification and an increased proliferation of NKX6.1+ cells. Interestingly, we were able to enrich a novel PDX1-/NKX6.1+ population by manipulating the re-plating density; these oriented themselves in three-dimensional clusters. Further differentiation validated the ability of our PDX1+/NKX6.1+ progenitors to generate NGN3+ endocrine progenitors. CONCLUSIONS: We provide a novel technique that facilitates appropriate cellular rearrangement in monolayer culture to yield a high proportion of PDX1+/NKX6.1+ PPs with an elevated self-replicating capacity, thereby aiding scalable production of functional ß cells from hPSCs in vitro. Our innovative method also enriches a novel NKX6.1+/PDX1- population, with characteristics of proposed endocrine precursors, allowing further studies on deciphering routes to ß-cell development.

Pathways governing development of stem cell-derived pancreatic ß cells: lessons from embryogenesis.

The loss of functional ß cells leads to development of diabetes. Several studies have shown that ß 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 ß cells in vitro. Our current knowledge of the mechanisms involved in pancreatic ß 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 ß-cell development and survival. Furthermore, hPSCs provide a renewable source of functional pancreatic ß cells for cell replacement therapy as well as disease modelling. Herein, we discuss the signalling pathways involved in the development of pancreatic ß cells during embryogenesis. Additionally, we describe how these pathways are manipulated in vitro to differentiate hPSCs into functional ß cells. Finally, we highlight the progress that has been made for the applications of those cells in treating and modelling diabetes.