Islet-like Cell Research Articles

Tankyrase inhibition promotes endocrine commitment of hPSC-derived pancreatic progenitors

Human pluripotent stem cells (hPSCs) have the potential to differentiate into various cell types, including pancreatic insulin-producing β cells, which are crucial for developing therapies for diabetes. However, current methods for directing hPSC differentiation towards pancreatic β-like cells are often inefficient and produce cells that do not fully resemble the native counterparts. Here, we report that highly selective tankyrase inhibitors, such as WIKI4, significantly enhances pancreatic differentiation from hPSCs. Our results show that WIKI4 promotes the formation of pancreatic progenitors that give rise to islet-like cells with improved β-like cell frequencies and glucose responsiveness compared to our standard cultures. These findings not only advance our understanding of pancreatic development, but also provide a promising new tool for generating pancreatic cells for research and potential therapeutic applications.

An INSULIN and IAPP dual reporter enables tracking of functional maturation of stem cell-derived insulin producing cells

ObjectiveHuman embryonic stem cell (hESC; SC)-derived pancreatic β cells can be used to study diabetes pathologies and develop cell replacement therapies. Although current differentiation protocols yield SCβ cells with varying degrees of maturation, these cells still differ from deceased donor human β cells in several respects. We sought to develop a reporter cell line that could be used to dynamically track SCβ cell functional maturation. MethodsTo monitor SCβ cell maturation in vitro, we created an IAPP-2A-mScar and INSULIN-2A-EGFP dual fluorescent reporter (INS2A-EGFP/+;IAPP2A-mScarlet/+) hESC line using CRISPR/Cas9. Pluripotent SC were then differentiated using a 7-stage protocol to islet-like cells. Immunohistochemistry, flow cytometry, qPCR, GSIS and electrophysiology were used to characterise resulting cell populations. ResultsWe observed robust expression of EGFP and mScarlet fluorescent proteins in insulin- and IAPP-expressing cells without any compromise to their differentiation. We show that the proportion of insulin-producing cells expressing IAPP increases over a 4-week maturation period, and that a subset of insulin-expressing cells remain IAPP-free. Compared to this IAPP-free population, we show these insulin- and IAPP-expressing cells are less polyhormonal, more glucose-sensitive, and exhibit decreased action potential firing in low (2.8 mM) glucose. ConclusionsThe INS2A-EGFP/+;IAPP2A-mScarlet/+ hESC line provides a useful tool for tracking populations of maturing hESC-derived β cells in vitro. This tool has already been shared with 3 groups and is freely available to all.

Scalable Bioreactor-based Suspension Approach to Generate Stem Cell-derived Islets From Healthy Donor-derived iPSCs.

Induced pluripotent stem cells (iPSCs) offer the potential to generate autologous iPSC-derived islets (iPSC islets), however, remain limited by scalability and product safety. Herein, we report stagewise characterization of cells generated following a bioreactor-based differentiation protocol. Cell characteristics were assessed using flow cytometry, quantitative reverse transcription polymerase chain reaction, patch clamping, functional assessment, and in vivo functional and immunohistochemistry evaluation. Protocol yield and costs are assessed to determine scalability. Differentiation was capable of generating 90.4% PDX1+/NKX6.1+ pancreatic progenitors and 100% C-peptide+/NKX6.1+ iPSC islet cells. However, 82.1%, 49.6%, and 0.9% of the cells expressed SOX9 (duct), SLC18A1 (enterochromaffin cells), and CDX2 (gut cells), respectively. Explanted grafts contained mature monohormonal islet-like cells, however, CK19+ ductal tissues persist. Using this protocol, semi-planar differentiation using 150 mm plates achieved 5.72 × 104 cells/cm2 (total 8.3 × 106 cells), whereas complete suspension differentiation within 100 mL Vertical-Wheel bioreactors significantly increased cell yield to 1.1 × 106 cells/mL (total 105.0 × 106 cells), reducing costs by 88.8%. This study offers a scalable suspension-based approach for iPSC islet differentiation within Vertical-Wheel bioreactors with thorough characterization of the ensuing product to enable future protocol comparison and evaluation of approaches for off-target cell elimination. Results suggest that bioreactor-based suspension differentiation protocols may facilitate scalability and clinical implementation of iPSC islet therapies.

Rat Ductal Cell-Derived Differentiation into Islet-Like Cells.

Regenerative medicine investigates the conversion of pancreatic ductal cells into functional islet cells, offering innovative treatments for conditions such as diabetes. Ductal cells, primarily supporting the pancreas' exocrine functions, can differentiate into various cell types, including islet cells, under specific conditions, opening new avenues in research and therapy. The outlined protocol elaborates on the conversion process, covering ductal cell differentiation induction, and insulin-producing capacity assessment. The primary objective is to address the shortage of insulin-secreting cells for transplantation, thereby advancing diabetes treatment methodologies.

Supragel-mediated efficient generation of pancreatic progenitor clusters and functional glucose-responsive islet-like clusters

Although several synthetic hydrogels with defined stiffness have been developed to facilitate the proliferation and maintenance of human pluripotent stem cells (hPSCs), the influence of biochemical cues in lineage-specific differentiation and functional cluster formation has been rarely reported. Here, we present the application of Supragel, a supramolecular hydrogel formed by synthesized biotinylated peptides, for islet-like cluster differentiation. We observed that Supragel, with a peptide concentration of 5 mg/mL promoted spontaneous hPSCs formation into uniform clusters, which is mainly attributable to a supporting stiffness of ∼1.5 kPa as provided by the Supragel matrix. Supragel was also found to interact with the hPSCs and facilitate endodermal and subsequent insulin-secreting cell differentiation, partially through its components: the sequences of RGD and YIGSR that interacts with cell membrane molecules of integrin receptor. Compared to Matrigel and suspension culturing conditions, more efficient differentiation of the hPSCs was also observed at the stages 3 and 4, as well as the final stage toward generation of insulin-secreting cells. This could be explained by 1) suitable average size of the hPSCs clusters cultured on Supragel; 2) appropriate level of cell adhesive sites provided by Supragel during differentiation. It is worth noting that the Supragel culture system was more tolerance in terms of the initial seeding densities and less demanding, since a standard static cell culture condition was sufficient for the entire differentiation process. Our observations demonstrate a positive role of Supragel for hPSCs differentiation into islet-like cells, with additional potential in facilitating germ layer differentiation.

Characterizing the effects of Dechlorane Plus on β-cells: a comparative study across models and species

ABSTRACT Epidemiological studies consistently link environmental toxicant exposure with increased Type 2 diabetes risk. Our study investigated the diabetogenic effects of a widely used flame retardant, Dechlorane Plus (DP), on pancreatic β-cells using rodent and human model systems. We first examined pancreas tissues from male mice exposed daily to oral gavage of either vehicle (corn oil) or DP (10, 100, or 1000 μg/kg per day) and fed chow or high fat diet for 28-days in vivo. DP exposure did not affect islet size or endocrine cell composition in either diet group. Next, we assessed the effect of 48-hour exposure to vehicle (DMSO) or DP (1, 10, or 100 nM) in vitro using immortalized rat β-cells (INS-1 832/3), primary mouse and human islets, and human stem-cell derived islet-like cells (SC-islets). In INS-1 832/3 cells, DP did not impact glucose-stimulated insulin secretion (GSIS) but significantly decreased intracellular insulin content. DP had no effect on GSIS in mouse islets or SC-islets but had variable effects on GSIS in human islets depending on the donor. DP alone did not affect insulin content in mouse islets, human islets, or SC-islets, but mouse islets co-exposed to DP and glucolipotoxic (GLT) stress conditions (28.7 mM glucose + 0.5 mM palmitate) had reduced insulin content compared to control conditions. Co-exposure of mouse islets to DP + GLT amplified the upregulation of Slc30a8 compared to GLT alone. Our study highlights the importance and challenges of using different in vitro models for studying chemical toxicity.

The generation of islet-like insulin-producing cells from Wharton's jelly-derived mesenchymal stem cells on the PES/fish gelatin scaffold

Diabetes Mellitus (DM) disrupts the body's capability to control blood glucose statuses. Type 1 diabetes mellitus (T1DM) arises from inadequate insulin production and is treated with insulin replacement therapy. Stem cell therapy is a hopeful treatment for T1DM that involves using adult stem cells to generate insulin-producing cells (IPCs). Mesenchymal stem cells (MSCs) are particularly advantageous for generating IPCs. The islet cells require interactions with the extracellular matrix for survival, which is lacking in conventional 2D culture systems. Natural or synthetic polymers create a supportive 3D microenvironment in tissue engineering. We aim to construct superior differentiation conditions employing polyethersulfone (PES)/Fish gelatin scaffolds to differentiate Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) to IPCs. In this study, the PES/fish gelatin scaffold (3D) was manufactured by electrospinning, and then its biocompatibility and non-toxicity were investigated by MTT assay. After that, scaffold-supportive effects on WJ-MSCs differentiation to IPCs were studied at the gene and protein levels. After exposure to the differentiation media, 2D and 3D (PES/Fish gelatin) cultured cells were slowly aggregated and developed spherical-shaped clusters. The viability of cells was found to be comparable in both 2D and 3D cultures. The gene expression analysis showed that efficiency of differentiation was more elevated in 3D culture. Additionally, ELISA results indicated that C-peptide and insulin release were more significant in 3D than in 2D culture. In conclusion, the PES/fish gelatin scaffold is highly promising for pancreatic tissue engineering because it supports the viability, growth, and differentiation of WJ-MSCs into IPCs.

Impact of Prevascularization on Immunological Environment and Early Engraftment in Subcutaneous Islet Transplantation.

The utilization of islet-like cells derived from pluripotent stem cells may resolve the scarcity of islet transplantation donors. The subcutaneous space is a promising transplantation site because of its capacity for graft observation and removal, thereby ensuring safety. To guarantee subcutaneous islet transplantation, physicians should ensure ample blood supply. Numerous methodologies, including prevascularization, have been investigated to augment blood flow, but the optimal approach remains undetermined. From C57BL/6 mice, 500 syngeneic islets were transplanted into the prevascularized subcutaneous site of recipient mice by implanting agarose rods with basic fibroblast growth factor at 1 and 2 wk. Before transplantation, the blood glucose levels, cell infiltration, and cytokine levels at the transplant site were evaluated. Furthermore, we examined the impact of the extracellular matrix capsule on graft function and the inflammatory response. Compared with the 1-wk group, the 2-wk group exhibited improved glycemic control, indicating that longer prevascularization enhanced transplant success. Flow cytometry analysis detected immune cells, such as neutrophils and macrophages, in the extracellular matrix capsules, whereas cytometric bead array analysis indicated the release of inflammatory and proinflammatory cytokines. Treatment with antitumor necrosis factor and anti-interleukin-6R antibodies in the 1-wk group improved graft survival, similar to the 2-wk group. In early prevascularization before subcutaneous transplantation, neutrophil and macrophage accumulation prevented early engraftment owing to inflammatory cytokine production.

Trends in the Application of Stem Cells for the Treatment of Diabetes

Diabetes mellitus is a global epidemic that showed a dramatic worldwide increase in its incidence and has been associated with life threatening complications if not detected and managed properly. Even though, insulin and oral anti-diabetic drugs were used for its management, lots of efforts are made to improve patient’s care by pancreas and islet transplantation along with stem cell therapies like embryonic stem cells, induced pluripotent stem cells and adult mesenchymal stem cells. Adult stem cells, which are also termed as somatic stem cells or resident stem cells, are a type of undifferentiated cells, resided in a differentiated tissue, organ or organism, in a specialized structural local microenvironment, called stem cell niche. The stem cell niche, is the in vivo microenvironment where stem cells are located and receive extrinsic signals interact and integrate to influence stem cell behavior that generally determine their fate. ESC showed unlimited differentiation into insulin producing β-cells with ethical concerns. MSCs possess the capacity to differentiate into islet-like insulin producing cells, to promote the regeneration of pancreatic islet beta cells and protect endogenous pancreatic islet beta cells from apoptosis through immunomodulatory mechanisms.

Transcriptomic profiling analysis of the effect of palmitic acid on 3D spheroids of β-like cells derived from induced pluripotent stem cells

Type 2 diabetes (T2D) is posing a serious public health concern with a considerable impact on human life and health expenditures worldwide. The disease develops when insulin plasma level is insufficient for coping insulin resistance, caused by the decline of pancreatic β-cell function and mass. In β-cells, the lipotoxicity exerted by saturated free fatty acids in particular palmitate (PA), which is chronically elevated in T2D, plays a major role in β-cell dysfunction and mass. However, there is a lack of human relevant in vitro model to identify the underlying mechanism through which palmitate induces β-cell failure. In this frame, we have previously developed a cutting-edge 3D spheroid model of β-like cells derived from human induced pluripotent stem cells. In the present work, we investigated the signaling pathways modified by palmitate in β-like cells derived spheroids. When compared to the 2D monolayer cultures, the transcriptome analysis (FDR set at 0.1) revealed that the 3D spheroids upregulated the pancreatic markers (such as GCG, IAPP genes), lipids metabolism and transporters (CD36, HMGSC2 genes), glucose transporter (SLC2A6). Then, the 3D spheroids are exposed to PA 0.5 mM for 72 h. The differential analysis demonstrated that 32 transcription factors and 135 target genes were mainly modulated (FDR set at 0.1) including the upregulation of lipid and carbohydrates metabolism (HMGSC2, LDHA, GLUT3), fibrin metabolism (FGG, FGB), apoptosis (CASP7). The pathway analysis using the 135 selected targets extracted the fibrin related biological process and wound healing in 3D PA treated conditions. An overall pathway gene set enrichment analysis, performed on the overall gene set (with pathway significance cutoff at 0.2), highlighted that PA perturbs the citrate cycle, FOXO signaling and Hippo signaling as observed in human islets studies. Additional RT-PCR confirmed induction of inflammatory (IGFBP1, IGFBP3) and cell growth (CCND1, Ki67) pathways by PA. All these changes were associated with unaffected glucose-stimulated insulin secretion (GSIS), suggesting that they precede the defect of insulin secretion and death induced by PA. Overall, we believe that our data demonstrate the potential of our spheroid 3D islet-like cells to investigate the pancreatic-like response to diabetogenic environment.

Dual-targeted nano-encapsulation of neonatal porcine islet-like cell clusters with triiodothyronine-loaded bifunctional polymersomes

There is growing evidence that neonatal porcine islet-like cell clusters (NPCCs) isolated from piglets can be used to treat type 1 diabetes in humans. However, graft rejection is a common complication in humans owing to the prevalence of xenoantigens in porcine. Therefore, researchers have investigated various islet encapsulation techniques that could protect against these antigens. To this end, this study presents a robust nano-encapsulation method based on bifunctional polymersomes (PSomes), in which N-hydroxysuccinimide (NHS) and maleimide (Mal) groups conjugated to the PSomes terminal interact with the amine and thiol groups on the surface of NPCCs to induce dual targeting via two covalent bonds. The findings indicate that the ratio of NHS to Mal on PSomes is optimal for dual targeting. Moreover, triiodothyronine (T3) is known to promotes pancreatic islet maturation and differentiation of endocrine cells into beta cells. T3 encapsulated in PSomes is shown to increase the glucose sensitivity of NPCCs and enhance insulin secretion from NPCCs. Furthermore, improvements in the nano-encapsulation efficiency and insulin-secreting capability of NPCCs through dual targeting via dual-Psomes are demonstrated. In conclusion, the proposed nano-encapsulation technique could pave the way for significant advances in islet nano-encapsulation and the imprevement of NPCC immaturity via T3 release.

Reduction of Activin A gives rise to comparable expression of key definitive endoderm and mature beta cell markers.

Aim: Cell therapies for diabetes rely on differentiation of stem cells into insulin-producing cells, which is complex and expensive. Our goal was to evaluate production costs and test ways to reduce it. Methods: Cost of Goods (COGs) analysis for differentiation was completed and the effects of replacement or reduction of the most expensive item was tested using qRT-PCR, immunohistochemistry, flow cytometry along with glucose-stimulated insulin release. Results: Activin A (AA) was responsible for significant cost. Replacement with small molecules failed to form definitive endoderm (DE). Reducing AA by 50% did not negatively affect expression of beta cell markers. Conclusion: Reduction of AA concentration is feasible without adversely affecting DE and islet-like cell differentiation, leading to significant cost savings in manufacturing.

Islet Like Cells Induced from Umbilical Cord Mesenchymal Stem Cells with Neonatal Bovine Pancreatic Mesenchymal Exosomes for Treatment of Diabetes Mellitus.

To investigate the safety and efficacy of the islet-like cell (cell) induced from human umbilical cord mesenchymal stem cell (UCMSC) with different methods for the treatment of diabetic animal model. UCMSCs were induced to βcells with cytokines (CY) and neonatal bovine pancreatic mesenchymal cell exosomes (Ex) combined with CY (EX+CY). The insulin secretion of UCMSC and βcell was measured with ELISA when the cells were growing in different concentrations of glucose media for different times. UCMSCs (4×105) and the same number of cells prepared with two methods were transplanted to type I diabetic rat models. UCMSCs could be induced into islet βcells by CY or EX+CY in vitro. The insulin secretion of the prepared β cells growing in 25.0 mM glucose medium was over 5-fold of that in 6.0 mM glucose. The transplantation of the βcells to type I diabetic rat models could reduce the blood glucose and prolong the survival time. The β cells induced by EX+CY had much more significant effects on decreasing blood glucose and increasing survival time (p<0.01). The cells did not affect blood sugar level and had no serious side-effects in human health. UCMSC could be induced to islet βcells with either CY or EX+CY. The transplantation of the induced islet βcells could reduce blood glucose and prolong the survival time of diabetic animal models. Although the cells induced with EX+CY had more significant effects on diabetic rats, they did not affect blood glucose level and had no serious side-effects in human health.

410.5: Prolactin modulates the differentiation of pancreatic ductal cell to islet-like cell

410.5: Prolactin modulates the differentiation of pancreatic ductal cell to islet-like cell

PAX4 loss of function increases diabetes risk by altering human pancreatic endocrine cell development

The coding variant (p.Arg192His) in the transcription factor PAX4 is associated with an altered risk for type 2 diabetes (T2D) in East Asian populations. In mice, Pax4 is essential for beta cell formation but its role on human beta cell development and/or function is unknown. Participants carrying the PAX4 p.His192 allele exhibited decreased pancreatic beta cell function compared to homozygotes for the p.192Arg allele in a cross-sectional study in which we carried out an intravenous glucose tolerance test and an oral glucose tolerance test. In a pedigree of a patient with young onset diabetes, several members carry a newly identified p.Tyr186X allele. In the human beta cell model, EndoC-βH1, PAX4 knockdown led to impaired insulin secretion, reduced total insulin content, and altered hormone gene expression. Deletion of PAX4 in human induced pluripotent stem cell (hiPSC)-derived islet-like cells resulted in derepression of alpha cell gene expression. In vitro differentiation of hiPSCs carrying PAX4 p.His192 and p.X186 risk alleles exhibited increased polyhormonal endocrine cell formation and reduced insulin content that can be reversed with gene correction. Together, we demonstrate the role of PAX4 in human endocrine cell development, beta cell function, and its contribution to T2D-risk.

231-LB: Prolactin Modulates the Differentiation of Pancreatic Ductal Cell to Islet-Like Cell

Objective: Pancreatic ductal cells will be differentiated into islet-like cell with the activation of PRL-PRLR signaling pathway in vitro. Methods: Primary mouse and human pancreatic ductal cells were obtained by CK-19 labelling and MACS sorting of digested pancreatic tissue, and selected cells were cultured with recombinant mouse prolactin and recombinant human prolactin. Subsequently, the cell clusters formed by the pancreatic ductal cells were stained by anti-insulin, anti-GCK, anti-GLUT2, anti-PC1/3 Abs. In vitro, glucose stimulated insulin secretion (GSIS) was quantified. Prolactin related signaling pathway, glucose transportation and insulin secretion related proteins were assessed by immunoblotting. Lastly, the in vitro formed islet-like cells were transplanted under the kidney capsule of diabetic mice. To assess the role for blood glucose regulation of transplanted islet-like cells, blood glucose, c-peptide and intravenous glucose tolerance tests were performed and immunofluorescence staining for insulin and glucagon was performed on the grafts. Results: At day 7, the pancreatic ductal cells, also called progenitor cells, expressed CK-19; at day 14, the ductal cells were aggregated and formed sphere-shaped cell clusters. At day 21, the cell clusters stained positive for insulin and etc. By in vitro GSIS test, we measured a 2.58±0.32-fold increase of insulin secretion in response to a 16.7 mM glucose stimulation. Immunoblotting demonstrated that PDX-1, MafA and other genes were expressed. At day 21, the cell clusters expressed PDX-1, insulin and glucagon. In vivo studies showed that diabetic mice transplanted with islet-like cells, maintained blood glucose levels below 300 mg/dL with c-peptide detected in the serum. Further tissue staining indicated that the transplanted islet-like cells contained both β-cells and α-cells. Conclusion: Out study shows that in vitro stimulation by prolactin might differentiate pancreatic ductal cells into islet-like cells. Disclosure Y. Wang: None. Funding National Natural Science Foundation of China (81802504); Sichuan Science and Technology Bureau (2022YFH0005, 2023YFH0010)

Promoting β-cells function by the recapitulation of in vivo microenvironmental differentiation signals.

The study aims to transdifferentiate rat bone marrow-derived mesenchymal stem cells (BM-MSCs) more efficiently into islet-like cells and encapsulate and transplant them with vital properties like stability, proliferation, and metabolic activity enhanced for the treatment of T1DM. Trans-differentiation of BM-MCs into islet-like cells induced by high glucose concentration combined with Nicotinamide, ꞵ-Mercaptoethanol, ꞵ-Cellulin, and IGF-1. Glucose challenge assays and gene expression profiles were used to determine functionality. Microencapsulation was performed using the vibrating nozzle encapsulator droplet method with a 1% alginate concentration. Encapsulated ꞵ-cells were cultured in a fluidized-bed bioreactor with 1850 μL/min fluid flow rates and a superficial velocity of 1.15cm/min. The procedure was followed by transplanting transdifferentiated cells into the omentum of streptozotocin (STZ)-induced diabetic Wistar rats. Changes in weight, glucose, insulin, and C-peptide levels were monitored for 2months after transplantation. PDX1, INS, GCG, NKx2.2, NKx6.1, and GLUT2 expression levels revealed the specificity of generated β-cells with higher viability (about 20%) and glucose sensitivity about twofold more. The encapsulated β-cells decreased the glucose levels in STZ-induced rats significantly (P < 0.05) 1week after transplantation. Also, the weight and levels of insulin and C-peptide reached the control group. In contrast to the treated, the sham group displayed a consistent decline in weight and died when loss reached > 20% at day ~ 55. The coated cells secrete significantly higher amounts of insulin in response to glucose concentration changes. Enhanced viability and functionality of β-cells can be achieved through differentiation and culturing, a promising approach toward insulin therapy alternatives.

Human induced pluripotent stem cells (hiPSC), enveloped in elastin-like recombinamers for cell therapy of type 1 diabetes mellitus (T1D): preliminary data.

Introduction: Therapeutic application and study of type 1 diabetes disease could benefit from the use of functional β islet-like cells derived from human induced pluripotent stem cells (hiPSCs). Considerable efforts have been made to develop increasingly effective hiPSC differentiation protocols, although critical issues related to cost, the percentage of differentiated cells that are obtained, and reproducibility remain open. In addition, transplantation of hiPSC would require immunoprotection within encapsulation devices, to make the construct invisible to the host's immune system and consequently avoid the recipient's general pharmacologic immunosuppression. Methods: For this work, a microencapsulation system based on the use of "human elastin-like recombinamers" (ELRs) was tested to envelop hiPSC. Special attention was devoted to in vitro and in vivo characterization of the hiPSCs upon coating with ERLs. Results and Discussion: We observed that ELRs coating did not interfere with viability and function and other biological properties of differentiated hiPSCs, while in vivo, ELRs seemed to afford immunoprotection to the cell grafts in preliminary in vivo study. The construct ability to correct hyperglycemia in vivo is in actual progress.

Inhibition of the type 1 diabetes candidate gene PTPN2 aggravates TNF-α-induced human beta cell dysfunction and death.

TNF-α plays a role in pancreatic beta cell loss in type 1 diabetes mellitus. In clinical interventions, TNF-α inhibition preserves C-peptide levels in early type 1 diabetes. In this study we evaluated the crosstalk of TNF-α, as compared with type I IFNs, with the type 1 diabetes candidate gene PTPN2 (encoding protein tyrosine phosphatase non-receptor type 2 [PTPN2]) in human beta cells. EndoC-βH1 cells, dispersed human pancreatic islets or induced pluripotent stem cell (iPSC)-derived islet-like cells were transfected with siRNAs targeting various genes (siCTRL, siPTPN2, siJNK1, siJNK3 or siBIM). Cells were treated for 48 h with IFN-α (2000 U/ml) or TNF-α (1000 U/ml). Cell death was evaluated using Hoechst 33342 and propidium iodide staining. mRNA levels were assessed by quantitative reverse transcription PCR (qRT-PCR) and protein expression by immunoblot. PTPN2 silencing sensitised beta cells to cytotoxicity induced by IFN-α and/or TNF-α by 20-50%, depending on the human cell model utilised; there was no potentiation between the cytokines. We silenced c-Jun N-terminal kinase (JNK)1 or Bcl-2-like protein 2 (BIM), and this abolished the proapoptotic effects of IFN-α, TNF-α or the combination of both after PTPN2 inhibition. We further observed that PTPN2 silencing increased TNF-α-induced JNK1 and BIM phosphorylation and that JNK3 is necessary for beta cell resistance to IFN-α cytotoxicity. We show that the type 1 diabetes candidate gene PTPN2 is a key regulator of the deleterious effects of TNF-α in human beta cells. It is conceivable that people with type 1 diabetes carrying risk-associated PTPN2 polymorphisms may particularly benefit from therapies inhibiting TNF-α.

CDK8/19 inhibition plays an important role in pancreatic β-cell induction from human iPSCs

BackgroundTransplantation of differentiated cells from human-induced pluripotent stem cells (hiPSCs) holds great promise for clinical treatments. Eliminating the risk factor of malignant cell transformation is essential for ensuring the safety of such cells. This study was aimed at assessing and mitigating mutagenicity that may arise during the cell culture process in the protocol of pancreatic islet cell (iPIC) differentiation from hiPSCs.MethodsWe evaluated the mutagenicity of differentiation factors used for hiPSC-derived pancreatic islet-like cells (iPICs). We employed Ames mutagenicity assay, flow cytometry analysis, immunostaining, time-resolved fluorescence resonance energy transfer-based (TR-FRET) cell-free dose–response assays, single-cell RNA-sequencing and in vivo efficacy study.ResultsWe observed a mutagenic effect of activin receptor-like kinase 5 inhibitor II (ALK5iII). ALK5iII is a widely used β-cell inducer but no other tested ALK5 inhibitors induced β-cells. We obtained kinase inhibition profiles and found that only ALK5iII inhibited cyclin-dependent kinases 8 and 19 (CDK8/19) among all ALK5 inhibitors tested. Consistently, CDK8/19 inhibitors efficiently induced β-cells in the absence of ALK5iII. A combination treatment with non-mutagenic ALK5 inhibitor SB431542 and CDK8/19 inhibitor senexin B afforded generation of iPICs with in vitro cellular composition and in vivo efficacy comparable to those observed with ALK5iII.ConclusionOur findings suggest a new risk mitigation approach for cell therapy and advance our understanding of the β-cell differentiation mechanism.