Modelling human diabetes ex vivo: a glance at maturity onset diabetes of the young.

Diabetes is a complex metabolic disease which most commonly has a polygenic origin; however, in rare cases, diabetes may be monogenic. This is indeed the case in both Maturity Onset Diabetes of the Young (MODY) and neonatal diabetes. These disease subtypes are believed to be simpler than Type 1 (T1D) and Type 2 Diabetes (T2D), which allows for more precise modelling. During the three last decades, many studies have focused on rodent models. These investigations provided a wealth of knowledge on both pancreas development and beta cell function. In particular, they allowed the establishment of a hierarchy of the transcription factors and highlighted the role of microenvironmental factors in the control of progenitor cell proliferation and differentiation. Transgenic mice also offered the possibility to decipher the mechanisms that define the functional identity of the pancreatic beta cells. Despite such interest in transgenic mice, recent data have also indicated that important differences exist between mice and human. To overcome these limitations, new human models are necessary. In the present review, we describe these ex vivo models, which are created using stem cells and organoids, and represent an important step toward islet cell therapy and drug discovery.

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 ß cells, which are crucial for developing therapies for diabetes. However, current methods for directing hPSC differentiation towards pancreatic ß-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 ß-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.

Acquisition of durable insulin-producing cells from human adipose tissue-derived mesenchymal stem cells as a foundation for cell- based therapy of diabetes mellitus.

This study aimed to identify the suitable induction protocol to produce highly qualified insulin producing cells (IPCs) from human adipose tissue derived stem cells (ADSCs) and evaluate the efficacy of the most functionally IPCs in management of diabetes mellitus (DM) in rats. The ADSCs were isolated and characterized according to the standard guidelines. ADSCs were further induced to be IPCs in vitro using three different protocols. The success of trans-differentiation was assessed in vitro through analysis of pancreatic endocrine genes expression, and insulin release in response to glucose stimulation. Then, the functionalization of the generated IPCs was evaluated in vivo. The in vitro findings revealed that the laminin-coated plates in combination with insulin-transferrin-selenium, B27, N2, and nicotinamide could efficiently up-regulate the expression of pancreatic endocrine genes. The in vivo study indicated effectual homing of the PKH-26-labelled IPCs in the pancreas of treated animals. Moreover, IPCs infusion in diabetic rats induced significant improvement in the metabolic parameters and prompted considerable up-regulation in the expression of the pancreatic related genes. The regenerative effect of infused IPCs was determined through histological examination of pancreatic tissue. Conclusively, the utilization of laminin-coated plates in concomitant with extrinsic factors promoting proliferation and differentiation of ADSCs could efficiently generate functional IPCs.

Enhancing differentiation and functionality of insulin-producing cells derived from iPSCs using esterified collagen hydrogel for cell therapy in diabetes mellitus.

BACKGROUND: Islet transplantation is a recommended treatment for type 1 diabetes but is limited by donor organ shortage. This study introduces an innovative approach for improving the differentiation and functionality of insulin-producing cells (IPCs) from iPSCs using 3D spheroid formation and hydrogel matrix as an alternative pancreatic islet source. The extracellular matrix (ECM) is crucial for pancreatic islet functionality, but finding the ideal matrix for ß-cell differentiation has been challenging. We aimed to advance IPC differentiation and maturation through an esterified collagen hydrogel, comparing its effectiveness with conventional basement membrane extract (BME) hydrogels. METHODS: iPSCs were differentiated into IPCs using a small molecule-based sequential protocol, followed by spheroid formation in concave microwells. Rheological analysis, scanning electron microscopy, and proteomic profiling were used to characterize the chemical and physical properties of each matrix. IPCs, both in single-cell form and as spheroids, were embedded in either ionized collagen or BME hydrogels, which was followed by assessments of morphological changes, pancreatic islet-related gene expression, insulin secretion, and pathway activation using comprehensive analytical techniques. RESULTS: Esterified collagen hydrogels markedly improved the structural integrity, insulin expression, and cell-cell interactions in IPC spheroids, forming densely packed insulin-expressing clusters, in contrast to the dispersed cells observed in BME cultures. Collagen hydrogel significantly enhanced the mRNA expression of crucial endocrine markers and maturation factors, with IPC spheroids showing accelerated differentiation from day 5, suggesting a faster differentiation compared to single cells in hydrogel encapsulation. Insulin secretion in response to glucose in collagen environments, with a GSIS index of 2.46 ± 0.05, exceeded those in 2D and BME, demonstrating superior pancreatic islet functionality. Pathway analysis highlighted enhanced insulin secretion capabilities, evidenced by the upregulation of genes like Secretogranin III and Chromogranin A in collagen cultures. In vivo transplantation results showed that collagen hydrogel enhanced cluster integrity, tissue integration, and insulin secretion compared to non-embedded IPCs and BME groups. CONCLUSION: Esterified collagen hydrogels demonstrated superior efficacy over 2D and BME in promoting IPC differentiation and maturation, possibly through upregulation of the expression of key secretion pathway genes. Our findings suggest that using collagen hydrogels presents a promising approach to enhance insulin secretion efficiency in differentiating pancreatic ß-cells, advancing cell therapy in diabetes cell therapy.

Safeguarding genomic integrity in beta-cells: implications for beta-cell differentiation, growth, and dysfunction.

The maintenance of optimal glucose levels in the body requires a healthy reserve of the insulin producing pancreatic beta-cells. Depletion of this reserve due to beta-cell dysfunction and death results in development of diabetes. Recent findings highlight unresolved DNA damage as a key contributor to beta-cell defects in diabetes. Beta-cells face various stressors and metabolic challenges throughout life, rendering them susceptible to DNA breaks. The post-mitotic, long-lived phenotype of mature beta-cells further warrants robust maintenance of genomic integrity. Failure to resolve DNA damage during beta-cell development, therefore, can result in an unhealthy reserve of beta-cells and predispose to diabetes. Yet, the molecular mechanisms safeguarding beta-cell genomic integrity remain poorly understood. Here, we focus on the significance of DNA damage in beta-cell homeostasis and postulate how cellular expansion, epigenetic programming, and metabolic shifts during development may impact beta-cell genomic integrity and health. We discuss recent findings demonstrating a physiological role for DNA breaks in modulating transcriptional control in neurons, which share many developmental programs with beta-cells. Finally, we highlight key gaps in our understanding of beta-cell genomic integrity and discuss emerging areas of interest.

Human mesenchymal stem/stromal cell based-therapy in diabetes mellitus: experimental and clinical perspectives.

Diabetes mellitus (DM), a chronic metabolic disease, poses a significant global health challenge, with current treatments often fail to prevent the long-term disease complications. Mesenchymal stem/stromal cells (MSCs) are, adult progenitors, able to repair injured tissues, exhibiting regenerative effects and immunoregulatory and anti-inflammatory responses, so they have been emerged as a promising therapeutic approach in many immune-related and inflammatory diseases. This review summarizes the therapeutic mechanisms and outcomes of MSCs, derived from different human tissue sources (hMSCs), in the context of DM type 1 and type 2. Animal model studies and clinical trials indicate that hMSCs can facilitate pleiotropic actions in the diabetic milieu for improved metabolic indices. In addition to modulating abnormally active immune system, hMSCs can ameliorate peripheral insulin resistance, halt beta-cell destruction, preserve residual beta-cell mass, promote beta-cell regeneration and insulin production, support islet grafts, and correct lipid metabolism. Moreover, hMSC-free derivatives, importantly extracellular vesicles, have shown potent experimental anti-diabetic efficacy. Moreover, the review discusses the diverse priming strategies that are introduced to enhance the preclinical anti-diabetic actions of hMSCs. Such strategies are recommended to restore the characteristics and functions of MSCs isolated from patients with DM for autologous implications. Finally, limitations and merits for the wide spread clinical applications of MSCs in DM such as the challenge of autologous versus allogeneic MSCs, the optimal MSC tissue source and administration route, the necessity of larger clinical trials for longer evaluation duration to assess safety concerns, are briefly presented.

Islet cell spheroids produced by a thermally sensitive scaffold: a new diabetes treatment.

The primary issues in treating type 1 diabetes mellitus (T1DM) through the transplantation of healthy islets or islet ß-cells are graft rejection and a lack of available donors. Currently, the majority of approaches use cell encapsulation technology and transplant replacement cells that can release insulin to address transplant rejection and donor shortages. However, existing encapsulation materials merely serve as carriers for islet cell growth. A new treatment approach for T1DM could be developed by creating a smart responsive material that encourages the formation of islet cell spheroids to replicate their 3D connections in vivo and controls the release of insulin aggregates. In this study, we used microfluidics to create thermally sensitive porous scaffolds made of poly(N-isopropyl acrylamide)/graphene oxide (PNIPAM/GO). The material was carefully shrunk under near-infrared light, enriched with mouse insulinoma pancreatic ß cells (ß-TC-6 cells), encapsulated, and cultivated to form 3D cell spheroids. The controlled contraction of the thermally responsive porous scaffold regulated insulin release from the spheroids, demonstrated using the glucose-stimulated insulin release assay (GSIS), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay. Eventually, implantation of the spheroids into C57BL/6 N diabetic mice enhanced the therapeutic effect, potentially offering a novel approach to the management of T1DM.

Alpha- to Beta-Cell Transdifferentiation in Neonatal Compared with Adult Mouse Pancreas in Response to a Modest Reduction in Beta-Cells Using Streptozotocin.

Following the near-total depletion of pancreatic beta-cells with streptozotocin (STZ), a partial recovery of beta-cell mass (BCM) can occur, in part due to the alpha- to beta-cell transdifferentiation with an intermediary insulin/glucagon bi-hormonal cell phenotype. However, human type 2 diabetes typically involves only a partial reduction in BCM and it is not known if recovery after therapeutic intervention involves islet cell transdifferentiation, or how this varies with age. Here, we used transgenic mouse models to examine if islet cell transdifferentiation contributes to BCM recovery following only a partial depletion of BCM. Cell lineage tracing was employed using Glucagon-Cre/yellow fluorescent protein (YFP) transgenic mice treated with STZ (25 mg/kg-neonates; 70 mg/kg-adults) or vehicle alone on 3 consecutive days. Mice were euthanized 2-30 days later with a prior glucose tolerance test on day 30, and immunofluorescence histology performed on the pancreata. Beta-cell abundance was reduced by 30-40% two days post STZ in both neonates and adults, and subsequently partially recovered in adult but not neonatal mice. Glucose tolerance recovered in adult females, but not in males or neonates. Bi-hormonal cell abundance increased 2-3-fold in STZ-treated mice vs. controls in both neonates and adults, as did transdifferentiated cells expressing insulin and the YFP lineage tag, but not glucagon. Transdifferentiated cell presence was an order of magnitude lower than that of bi-hormonal cells. We conclude that alpha- to beta-cell transdifferentiation occurs in mice following only a moderate depletion in BCM, and that this was accompanied by a partial recovery of BCM in adults.

Microgravity Effect on Pancreatic Islets.

We previously demonstrated that boundary cap neural crest stem cells (BCs) induce the proliferation of beta-cells in vitro, increase survival of pancreatic islets (PIs) in vivo after transplantation, and themselves strongly increase their proliferation capacity after exposure to space conditions. Therefore, we asked if space conditions can induce the proliferation of beta-cells when PIs are alone or together with BCs in free-floating or 3D-printed form. During the MASER 15 sounding rocket experiment, half of the cells were exposed to 6 min of microgravity (µg), whereas another group of cells were kept in 1 g conditions in a centrifuge onboard. The proliferation marker EdU was added to the cells just before the rocket reached µg conditions. The morphological assessment revealed that PIs successfully survived and strongly proliferated, particularly in the free-floating condition, though the fusion of PIs hampered statistical analysis. Proliferation of beta-cells was displayed in 3D-printed islets two weeks after µg exposure, suggesting that the effects of µg may be delayed. Thus, PIs in 3D-printed scaffolds did not fuse, and this preparation is more suitable than free-floating specimens for morphological analysis in µg studies. PIs maintained their increased proliferation capacity for weeks after µg exposure, an effect that may not appear directly, but can emerge after a delay.

Generation of a pancreas derived hydrogel for the culture of hiPSC derived pancreatic endocrine cells.

Stem cell-derived ß-cells (SC-BCs) represent a potential source for curing diabetes. To date, in vitro generated SC-BCs display an immature phenotype and lack important features in comparison to their bona-fide counterparts. Transplantation into a living animal promotes SC-BCs maturation, indicating that components of the in vivo microenvironment trigger final SC-BCs development. Here, we investigated whether cues of the pancreas specific extracellular matrix (ECM) can improve the differentiation of human induced pluripotent stem cells (hiPSCs) towards ß-cells in vitro. To this aim, a pancreas specific ECM (PanMa) hydrogel was generated from decellularized porcine pancreas and its effect on the differentiation of hiPSC-derived pancreatic hormone expressing cells (HECs) was tested. The hydrogel solidified upon neutralization at 37 °C with gelation kinetics similar to Matrigel. Cytocompatibility of the PanMa hydrogel was demonstrated for a culture duration of 21 days. Encapsulation and culture of HECs in the PanMa hydrogel over 7 days resulted in a stable gene and protein expression of most ß-cell markers, but did not improve ß-cell identity. In conclusion, the study describes the production of a PanMa hydrogel, which provides the basis for the development of ECM hydrogels that are more adapted to the demands of SC-BCs.

ß-Cell Regeneration Is Driven by Pancreatic Plasticity.

The pancreas has been considered a non-regenerative organ. ß cells lost in diabetes are not replaced due to the inability of the pancreas to regenerate. However, ample evidence generated in the last few decades using murine models has demonstrated that the pancreas has a remarkable plasticity wherein differentiated cells can change cell fate toward a ß-like cell phenotype. Although this process is observed after using rather artificial stimuli and the conversion efficiency is very limited, these findings have shed some light on novel pathways for ß-cell regeneration. In this chapter, we will summarize the different cellular interconversion processes described to date, the experimental details and molecular regulation of such interconversions, and the genomic technologies that have allowed the identification of potential new ways to generate ß cells.

A capsule-based scaffold incorporating decellularized extracellular matrix and curcumin for islet beta cell therapy in type 1 diabetes mellitus.

The transplantation of islet beta cells offers an alternative to heterotopic islet transplantation for treating type 1 diabetes mellitus (T1DM). However, the use of systemic immunosuppressive drugs in islet transplantation poses significant risks to the body. To address this issue, we constructed an encapsulated hybrid scaffold loaded with islet beta cells. This article focuses on the preparation of the encapsulated structure using 3D printing, which incorporates porcine pancreas decellularized extracellular matrix (dECM) to the core scaffold. The improved decellularization method successfully preserved a substantial proportion of protein (such as Collagen I and Laminins) architecture and glycosaminoglycans in the dECM hydrogel, while effectively removing most of the DNA. The inclusion of dECM enhanced the physical and chemical properties of the scaffold, resulting in a porosity of 83.62% ± 1.09% and a tensile stress of 1.85 ± 0.16 MPa. In teams of biological activity, dECM demonstrated enhanced proliferation, differentiation, and expression of transcription factors such as Ki67, PDX1, and NKX6.1, leading to improved insulin secretion function in MIN-6 pancreatic beta cells. In the glucose-stimulated insulin secretion experiment on day 21, the maximum insulin secretion from the encapsulated structure reached 1.96 ± 0.08 mIU ml-1, representing a 44% increase compared to the control group. Furthermore, conventional capsule scaffolds leaverage the compatibility of natural biomaterials with macrophages to mitigate immune rejection. Here, incorporating curcumin into the capsule scaffold significantly reduced the secretion of pro-inflammatory cytokine (IL-1ß, IL-6, TNF-α, IFN-γ) secretion by RAW264.7 macrophages and T cells in T1DM mice. This approach protected pancreatic islet cells against immune cell infiltration mediated by inflammatory factors and prevented insulitis. Overall, the encapsulated scaffold developed in this study shows promise as a natural platform for clinical treatment of T1DM.

Protocol for CRISPR-Cas12a genome editing of protein tyrosine phosphatases in human pluripotent stem cells and functional ß-like cell generation.

Gene editing of human pluripotent stem cells is a promising approach for developing targeted gene therapies for metabolic diseases. Here, we present a protocol for generating a CRISPR-Cas12a gene knockout of protein tyrosine phosphatases in human embryonic stem cells. We describe steps for differentiating the edited clones into pancreatic islet-like spheroids rich in ß-like cells. We then detail procedures for implanting these spheroids under the murine kidney capsule for in vivo maturation.

From iPSCs to Pancreatic ß Cells: Unveiling Molecular Pathways and Enhancements with Vitamin C and Retinoic Acid in Diabetes Research.

Diabetes mellitus, a chronic and non-transmissible disease, triggers a wide range of micro- and macrovascular complications. The differentiation of pancreatic ß-like cells (PßLCs) from induced pluripotent stem cells (iPSCs) offers a promising avenue for regenerative medicine aimed at treating diabetes. Current differentiation protocols strive to emulate pancreatic embryonic development by utilizing cytokines and small molecules at specific doses to activate and inhibit distinct molecular signaling pathways, directing the differentiation of iPSCs into pancreatic ß cells. Despite significant progress and improved protocols, the full spectrum of molecular signaling pathways governing pancreatic development and the physiological characteristics of the differentiated cells are not yet fully understood. Here, we report a specific combination of cofactors and small molecules that successfully differentiate iPSCs into PßLCs. Our protocol has shown to be effective, with the resulting cells exhibiting key functional properties of pancreatic ß cells, including the expression of crucial molecular markers (pdx1, nkx6.1, ngn3) and the capability to secrete insulin in response to glucose. Furthermore, the addition of vitamin C and retinoic acid in the final stages of differentiation led to the overexpression of specific ß cell genes.

Peroxyredoxin 6 Protects RIN-M5F Pancreatic Beta Cells Against Streptozotocin-Induced Senescence.

BACKGROUND/AIMS: There are evidences that a decrease in the functional activity of pancreatic ß-cells under type 2 diabetes conditions may be associated with their senescence, therefore, senotherapy may be a prospective strategy for the diabetes treatment. METHODS: The senotherapeutic potential of peroxiredoxin 6 (PRDX6) was studied in RIN-m5F pancreatic ß-cells with streptozotocin-induced senescence by measuring markers, associated with senescence. RESULTS: Exposure to streptozotocin (STZ) resulted in the senescence of the ß-cells. The addition of PRDX6 to the culture medium of RIN-m5F ß-cells before treatment with STZ decreased the levels of the following senescence markers: the percentage of SA-ß-Gal-positive cells, the phosphorylation of histone H2AX and p21 proteins, and the secretion of the proinflammatory cytokine IL-6 but not the anti-inflammatory cytokine IL-10. These effects were accompanied by a decrease in the production of reactive oxygen species (ROS) and the restoration of impaired NF-κB activation. In addition, PRDX6 altered the production of the heat shock protein HSP90: the production of the constitutive form of HSP90-beta decreased, while the level of inducible HSP90-alpha increased. CONCLUSION: PRDX6 prevented the senescence of RIN-m5F cells in response to the DNA damage-inducing agent streptozotocin, indicating a potential protective role of PRDX6 in type 2 diabetes mellitus.

An investigation of functionalized chitosan and alginate multilayer conformal nanocoating on mouse beta cell spheroids as a model for pancreatic islet transplantation.

Multilayer conformal coatings have been shown to provide a nanoscale barrier between cells and their environment with adequate stability, while regulating the diffusion of nutrition and waste across the cell membrane. The coating method aims to minimize capsule thickness and implant volume while reducing the need for immunosuppressive drugs, making it a promising approach for islet cell encapsulation in clinical islet transplantation for the treatment of Type 1 diabetes. This study introduces an immunoprotective nanocoating obtained through electrostatic interaction between quaternized phosphocholine-chitosan (PC-QCH) and tetrahydropyran triazole phenyl-alginate (TZ-AL) onto mouse ß-cell spheroids. First, successful synthesis of the proposed polyelectrolytes was confirmed with physico-chemical characterization. A coating with an average thickness of 540 nm was obtained with self-assembly of 4-bilayers of PC-QCH/TZ-AL onto MIN6 ß-cell spheroids. Surface coating of spheroids did not affect cell viability, metabolic activity, or insulin secretion, when compared to non-coated spheroids. The exposure of the polyelectrolytes to THP-1 monocyte-derived macrophages lead to a reduced level of TNF-α secretion and exposure of coated spheroids to RAW264.7 macrophages showed a decreasing trend in the secretion of TNF-α and IL-6. In addition, coated spheroids were able to establish normoglycemia when implanted into diabetic NOD-SCID mice, demonstrating in vivo biocompatibility and cellular function. These results demonstrate the ability of the PC-QCH/TZ-AL conformal coating to mitigate pro-inflammatory responses from macrophages, and thus can be a promising candidate towards nanoencapsulation for cell-based therapy, particularly in type 1 diabetes, where the insulin secreting ß-cells are subjected to inflammation and immune cell attack.

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

OBJECTIVE: Human embryonic stem cell (hESC; SC)-derived pancreatic ß cells can be used to study diabetes pathologies and develop cell replacement therapies. Although current differentiation protocols yield SCß cells with varying degrees of maturation, these cells still differ from deceased donor human ß cells in several respects. We sought to develop a reporter cell line that could be used to dynamically track SCß cell functional maturation. METHODS: To monitor SCß 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. RESULTS: We 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. CONCLUSIONS: The INS2A-EGFP/+;IAPP2A-mScarlet/+ hESC line provides a useful tool for tracking populations of maturing hESC-derived ß cells in vitro. This tool has already been shared with 3 groups and is freely available to all.

Single cell glucose-stimulated insulin secretion assay using nanowell-in-microwell plates.

Pancreatic ß cells secrete insulin in response to elevated levels of glucose. Stem cell derived ß (SCß) cells aim to replicate this glucose-stimulated insulin secretion (GSIS) function, but current preparations cannot provide the same level of insulin as natural ß cells. Here, we develop an assay to measure GSIS at the single cell level to investigate the functional heterogeneity of SCß cells and donor-derived islet cells. Our assay involves randomly depositing single cells and insulin capture microbeads in open-top nanowells (40 × 40 × 55 µm3) fabricated on glass-bottom imaging microwell plates. Insulin secreted from single cells is captured on microbeads and then stained using a detection antibody. The nanowell microstructure limits diffusion of secreted insulin. The glass substrate provides an optically flat surface for quantitative microscopy to measure the concentration of secreted insulin. We used this approach to measure GSIS from SCß cells and donor-derived islet cells after 15 minutes exposure to 3.3 mM and 16.7 mM glucose. Both cell types exhibited significant GSIS heterogeneity, where elite cells (<20%) produced the majority of the secreted insulin (55-78%). This assay provides an immediate readout of single cell glucose-stimulated insulin secretion in a flexible well plate-based format.

Vascular and immune interactions in islets transplantation and 3D islet models.

The aim of regenerative medicine is to restore specific functions to damaged cells or tissues. A crucial aspect of success lies in effectively reintegrating these cells or tissues within the recipient organism. This is particularly pertinent for diabetes, where islet function relies on the close connection of beta cells to the bloodstream for glucose sensing and insulin release. Central to this approach is the need to establish a fast connection with the host's vascular system. In this review, we explore the intricate relationships between endocrine, vascular, and immune cell interactions in transplantation outcomes. We also delve into recent strategies aimed at enhancing engraftment, along with the utilization of in vitro platforms to model cellular interactions.

Generation and application of novel hES cell reporter lines for the differentiation and maturation of hPS cell-derived islet-like clusters.

The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells, including functional ß-cells, have been based on a detailed understanding of the underlying developmental mechanisms. However, the final differentiation steps, leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells, remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells), which include a lack of ß-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS+ and GCG+ cells and their resolution into mono-hormonal cells (INSeGFP, INSeGFP/GCGmCHERRY) as well as ß-cell maturation (INSeGFP/MAFAmCHERRY) and function (INSGCaMP6). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive, insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions, we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and, using this approach, we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal ß-cells without affecting the number of α-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets.