Artemisinins Target GABAA Receptor Signaling and Impair α Cell Identity.

Type 1 diabetes is characterized by the destruction of pancreatic ß cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional ß-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic ß cell mass from α cells.

Identification of newly synthetized proteins by mass spectrometry to understand palmitate-induced early cellular changes in pancreatic islets.

Obesity and lipid metabolism dysregulation are often associated with insulin resistance, and can lead to type 2 diabetes. However, mechanisms linking insulin resistance, high levels of plasma free fatty acids (FFA), and ß cell failure remain unclear. The aim of this work was to search for proteins whose synthesis was modified by a short exposure to FFA. This could help in the future to identify molecular mechanisms underlying islet dysfunction in the presence of FFA. Therefore, we assessed by mass spectrometry de novo protein synthesis of freshly isolated rat islets after palmitate short exposure. Quantitative proteome and secretome analyses were performed by combining metabolic incorporation of azidohomoalanine (AHA) and pulse labeling with stable isotope labeling by amino acids in cell culture (SILAC). We showed that pancreatic islets, in response to 4-h exposure to palmitate, increased the synthesis of ribosomal proteins and proteins of the cytoskeleton, and increased their secretion of proteins involved in insulin synthesis and insulin secretion, as well as insulin itself. First, these results show that de novo protein quantification analysis by LC-MS/MS is a useful method to investigate cellular modifications induced by FFA on pancreatic islets. Also, these results show that short exposure to palmitate increases the expression of ribosomal proteins and proteins involved in insulin secretion, and it remains to be determined if these effects are responsible or linked to the harmful effect of palmitate on ß cells.NEW & NOTEWORTHY These results show that pancreatic rat islets cultured with palmitate mainly increase synthesis of ribosomal proteins and some proteins of the cytoskeleton. They also show a significant increase of secreted proteins involved in insulin synthesis and insulin secretion, as well as insulin itself. These data provide information to understand the mechanisms of ß cell failure induced by lipotoxicity via the identification of all newly synthesized proteins in islets in response to short-term exposure to palmitate.

The Relevance of Advanced Therapy Medicinal Products in the Field of Transplantation and the Need for Academic Research Access: Overcoming Bottlenecks and Claiming a New Time.

The field of transplantation has witnessed the emergence of Advanced Therapy Medicinal Products (ATMPs) as highly promising solutions to address the challenges associated with organ and tissue transplantation. ATMPs encompass gene therapy, cell therapy, and tissue-engineered products, hold immense potential for breakthroughs in overcoming the obstacles of rejection and the limited availability of donor organs. However, the development and academic research access to ATMPs face significant bottlenecks that hinder progress. This opinion paper emphasizes the importance of addressing bottlenecks in the development and academic research access to ATMPs by implementing several key strategies. These include the establishment of streamlined regulatory processes, securing increased funding for ATMP research, fostering collaborations and partnerships, setting up centralized ATMP facilities, and actively engaging with patient groups. Advocacy at the policy level is essential to provide support for the development and accessibility of ATMPs, thereby driving advancements in transplantation and enhancing patient outcomes. By adopting these strategies, the field of transplantation can pave the way for the introduction of innovative and efficacious ATMP therapies, while simultaneously fostering a nurturing environment for academic research.

Impact of ischemia time on islet isolation success and posttransplantation outcomes: A retrospective study of 452 pancreas isolations.

Many variables impact islet isolation, including pancreas ischemia time. The ischemia time upper limit that should be respected to avoid a negative impact on the isolation outcome is not well defined. We have performed a retrospective analysis of all islet isolations in our center between 2008 and 2018. Total ischemia time, cold ischemia time, and organ removal time were analyzed. Isolation success was defined as an islet yield ≥200 000 IEQ. Of the 452 pancreases included, 288 (64%) were successfully isolated. Probability of isolation success showed a significant decrease after 8 hours of total ischemia time, 7 hours of cold ischemia time, and 80 minutes of organ removal time. Although we observed an impact of ischemia time on islet yield, a probability of isolation success of 50% was still present even when total ischemia time exceeds 12 hours. Posttransplantation clinical outcomes were assessed in 32 recipients and no significant difference was found regardless of ischemia time. These data indicate that although shorter ischemia times are associated with better islet isolation outcomes, total ischemia time >12 hours can provide excellent results in appropriately selected donors.

Mini-organs forum: how to advance organoid technology to organ transplant community.

The generation of human mini-organs, the so-called organoids, is one of the biggest scientific advances in regenerative medicine. This technology exploits traditional three-dimensional culture techniques that support cell-autonomous self-organization responses of stem cells to derive micrometer to millimeter size versions of human organs. The convergence of the organoid technology with organ transplantation is still in its infancy but this alliance is expected to open new venues to change the way we conduct both transplant and organoid research. In this Forum we provide a summary on early achievements facilitating organoid derivation and culture. We further discuss on early advances of organoid transplantation also offering a comprehensive overview of current limitations and challenges to instruct organoid maturation. We expect that this Forum sets the ground for initial discussions between stem cell biologists, bioengineers, and the transplant community to better direct organoid basic research to advance the organ transplantation field.

Bio-Engineering of Pre-Vascularized Islet Organoids for the Treatment of Type 1 Diabetes.

Lack of rapid revascularization and inflammatory attacks at the site of transplantation contribute to impaired islet engraftment and suboptimal metabolic control after clinical islet transplantation. In order to overcome these limitations and enhance engraftment and revascularization, we have generated and transplanted pre-vascularized insulin-secreting organoids composed of rat islet cells, human amniotic epithelial cells (hAECs), and human umbilical vein endothelial cells (HUVECs). Our study demonstrates that pre-vascularized islet organoids exhibit enhanced in vitro function compared to native islets, and, most importantly, better engraftment and improved vascularization in vivo in a murine model. This is mainly due to cross-talk between hAECs, HUVECs and islet cells, mediated by the upregulation of genes promoting angiogenesis (vegf-a) and ß cell function (glp-1r, pdx1). The possibility of adding a selected source of endothelial cells for the neo-vascularization of insulin-scereting grafts may also allow implementation of ß cell replacement therapies in more favourable transplantation sites than the liver.

[Total pancreatectomy and islet auto-transplantation: current state and new perspectives]. / Pancréatectomie totale et autotransplantation d'îlots†: état actuel et nouvelles perspectives.

Total pancreatectomy is a procedure primarily performed for chronic pancreatitis refractory to conservative therapy. It may nevertheless be indicated in the event of a malignant tumor, either as a treatment for a surgical complication or as a prevention of anastomotic leakage. If possible, islet auto-transplantation should be combined with total pancreatectomy for benign disease, in order to prevent a severe diabetes. Until recently, malignant disease was considered an absolute contraindication to islet auto-transplantation. A recent series from Milan showed promising oncological results in auto-transplantation for malignant disease, opening up new perspectives for total pancreatectomy for cancer.

La pancréatectomie totale est une procédure principalement effectuée pour une pancréatite chronique réfractaire au traitement conservateur. Elle peut néanmoins être indiquée en cas de tumeur maligne, soit comme traitement d'une complication chirurgicale, soit en prévention de fuite anastomotique. Dans la mesure du possible, une autogreffe d'îlots de Langerhans devrait être associée à une pancréatectomie totale pour maladie bénigne, dans le but de prévenir un diabète pancréatoprive. Jusqu'à récemment, une pathologie maligne était considérée comme une contre-indication absolue à une autogreffe d'îlots. Une série récente de Milan a montré des résultats oncologiques prometteurs en cas d'autogreffe pour pathologies malignes, ouvrant de nouvelles perspectives à la pancréatectomie totale pour cancer.

Shielding islets with human amniotic epithelial cells enhances islet engraftment and revascularization in a murine diabetes model.

Hypoxia is a major cause of considerable islet loss during the early posttransplant period. Here, we investigate whether shielding islets with human amniotic epithelial cells (hAECs), which possess anti-inflammatory and regenerative properties, improves islet engraftment and survival. Shielded islets were generated on agarose microwells by mixing rat islets (RIs) or human islets (HI) and hAECs (100 hAECs/IEQ). Islet secretory function and viability were assessed after culture in hypoxia (1% O2 ) or normoxia (21% O2 ) in vitro. In vivo function was evaluated after transplant under the kidney capsule of diabetic immunodeficient mice. Graft morphology and vascularization were evaluated by immunohistochemistry. Both shielded RIs and HIs show higher viability and increased glucose-stimulated insulin secretion after exposure to hypoxia in vitro compared with control islets. Transplant of shielded islets results in considerably earlier normoglycemia and vascularization, an enhanced glucose tolerance, and a higher ß cell mass. Our results show that hAECs have a clear cytoprotective effect against hypoxic damages in vitro. This strategy improves ß cell mass engraftment and islet revascularization, leading to an improved capacity of islets to reverse hyperglycemia, and could be rapidly applicable in the clinical situation seeing that the modification to HIs are minor.

Immunomodulatory Properties of Amniotic Membrane Derivatives and Their Potential in Regenerative Medicine.

PURPOSE OF REVIEW: During the last decades, the field of regenerative medicine has been rapidly evolving. Major progress has been made in the development of biological substitutes applying the principles of cell transplantation, material science, and bioengineering. RECENT FINDINGS: Among other sources, amniotic-derived products have been used for decades in various fields of medicine as a biomaterial for the wound care and tissue replacement. Moreover, human amniotic epithelial and mesenchymal cells have been intensively studied for their immunomodulatory capacities. Amniotic cells possess two major characteristics that have already been widely exploited. The first is their ability to modulate and suppress the innate and adaptive immunities, making them a true asset for chronic inflammatory disorders and for the induction of tolerance in transplantation models. The second is their multilineage differentiation capacity, offering a source of cells for tissue engineering. The latter combined with the use of amniotic membrane as a scaffold offers all components necessary to create an optimal environment for cell and tissue regeneration. This review summarizes beneficial properties of hAM and its derivatives and discusses their potential in regenerative medicine.

Generation of insulin-secreting organoids: a step toward engineering and transplanting the bioartificial pancreas.

Diabetes is a major health issue of increasing prevalence. ß-cell replacement, by pancreas or islet transplantation, is the only long-term curative option for patients with insulin-dependent diabetes. Despite good functional results, pancreas transplantation remains a major surgery with potentially severe complications. Islet transplantation is a minimally invasive alternative that can widen the indications in view of its lower morbidity. However, the islet isolation procedure disrupts their vasculature and connection to the surrounding extracellular matrix, exposing them to ischemia and anoikis. Implanted islets are also the target of innate and adaptive immune attacks, thus preventing robust engraftment and prolonged full function. Generation of organoids, defined as functional 3D structures assembled with cell types from different sources, is a strategy increasingly used in regenerative medicine for tissue replacement or repair, in a variety of inflammatory or degenerative disorders. Applied to ß-cell replacement, it offers the possibility to control the size and composition of islet-like structures (pseudo-islets), and to include cells with anti-inflammatory or immunomodulatory properties. In this review, we will present approaches to generate islet cell organoids and discuss how these strategies can be applied to the generation of a bioartificial pancreas for the treatment of type 1 diabetes.

Decellularized human placenta supports hepatic tissue and allows rescue in acute liver failure.

Tissue engineering with scaffolds to form transplantable organs is of wide interest. Decellularized tissues have been tested for this purpose, although supplies of healthy donor tissues, vascular recellularization for perfusion, and tissue homeostasis in engineered organs pose challenges. We hypothesized that decellularized human placenta will be suitable for tissue engineering. The universal availability and unique structures of placenta for accommodating tissue, including presence of embedded vessels, were major attractions. We found decellularized placental vessels were reendothelialized by adjacent native cells and bridged vessel defects in rats. In addition, implantation of liver fragments containing all cell types successfully hepatized placenta with maintenance of albumin and urea synthesis, as well as hepatobiliary transport of 99m Tc-mebrofenin, up to 3 days in vitro. After hepatized placenta containing autologous liver was transplanted into sheep, tissue units were well-perfused and self-assembled. Histological examination indicated transplanted tissue retained hepatic cord structures with characteristic hepatic organelles, such as gap junctions, and hepatic sinusoids lined by endothelial cells, Kupffer cells, and other cell types. Hepatocytes in this neo-organ expressed albumin and contained glycogen. Moreover, transplantation of hepatized placenta containing autologous tissue rescued sheep in extended partial hepatectomy-induced acute liver failure. This rescue concerned amelioration of injury and induction of regeneration in native liver. The grafted hepatized placenta was intact with healthy tissue that neither proliferated nor was otherwise altered. CONCLUSION: The unique anatomic structure and matrix of human placenta were effective for hepatic tissue engineering. This will advance applications ranging from biological studies, drug development, and toxicology to patient therapies. (Hepatology 2018;67:1956-1969).

Single-cell transcriptomes reveal characteristic features of human pancreatic islet cell types.

Pancreatic islets of Langerhans contain several specialized endocrine cell types, which are commonly identified by the expression of single marker genes. However, the established marker genes cannot capture the complete spectrum of cellular heterogeneity in human pancreatic islets, and existing bulk transcriptome datasets provide averages across several cell populations. To dissect the cellular composition of the human pancreatic islet and to establish transcriptomes for all major cell types, we performed single-cell RNA sequencing on 70 cells sorted from human primary tissue. We used this dataset to validate previously described marker genes at the single-cell level and to identify specifically expressed transcription factors for all islet cell subtypes. All data are available for browsing and download, thus establishing a useful resource of single-cell expression profiles for endocrine cells in human pancreatic islets.

Ischemic preconditioning affects long-term cell fate through DNA damage-related molecular signaling and altered proliferation.

Despite the potential of ischemic preconditioning for organ protection, long-term effects in terms of molecular processes and cell fates are ill defined. We determined consequences of hepatic ischemic preconditioning in rats, including cell transplantation assays. Ischemic preconditioning induced persistent alterations; for example, after 5 days liver histology was normal, but γ-glutamyl transpeptidase expression was observed, with altered antioxidant enzyme content, lipid peroxidation, and oxidative DNA adducts. Nonetheless, ischemic preconditioning partially protected from toxic liver injury. Similarly, primary hepatocytes from donor livers preconditioned with ischemia exhibited undesirably altered antioxidant enzyme content and lipid peroxidation, but better withstood insults. However, donor hepatocytes from livers preconditioned with ischemia did not engraft better than hepatocytes from control livers. Moreover, proliferation of hepatocytes from donor livers preconditioned with ischemia decreased under liver repopulation conditions. Hepatocytes from donor livers preconditioned with ischemia showed oxidative DNA damage with expression of genes involved in MAPK signaling that impose G1/S and G2/M checkpoint restrictions, including p38 MAPK-regulated or ERK-1/2-regulated cell-cycle genes such as FOS, MAPK8, MYC, various cyclins, CDKN2A, CDKN2B, TP53, and RB1. Thus, although ischemic preconditioning allowed hepatocytes to better withstand secondary insults, accompanying DNA damage and molecular events simultaneously impaired their proliferation capacity over the long term. Mitigation of ischemic preconditioning-induced DNA damage and deleterious molecular perturbations holds promise for advancing clinical applications.

Effect of islet alone or islets after kidney transplantation on quality of life in type 1 diabetes: A systematic review.

BACKGROUND: Pancreatic islet transplantation for type 1 diabetes mellitus (T1DM) is efficacious in supressing severe hypoglycaemic episodes (SHE) and restoring glycaemic regulation, which are both pivotal in increasing health-related quality of life (HRQoL). Therefore, a systematic assessment of reports detailing HRQoL outcomes is warranted to better understand the benefits of islet transplantation. To this end, we performed a systematic review of the literature to assess the impact of islet transplantation on HRQoL in individuals with T1DM, whether as a standalone procedure (ITA) or following renal transplantation (IAK). METHOD: All studies providing a quantitative assessment of HRQoL following ITA or IAK were included. Selected studies had to meet the following criteria: they had to (i) involve adult recipients of islet grafts for T1DM, (ii) use either generic or disease-specific QoL assessment tools, (iii) provide a comparative analysis of QoL metrics between the pre- and post-transplantation state or between the post-transplantation state and other pre-transplant patients or the general population. RESULTS: Seven studies that met the inclusion criteria provided data on 205 subjects. In the included studies, HRQoL was measured using both generic instruments, such as the 36-item Short Form Health Survey (SF-36) and the Health Status Questionnaire (HSQ) 2.0, and disease-specific instruments, such as the Diabetes Distress Scale (DDS), the Diabetes Quality of Life Questionnaire, and the Hypoglycaemia Fear Survey (HFS). These instruments cover physical, mental, social, or functional health dimensions. We found that pancreatic islet transplantation was associated with improvements in all HRQoL dimensions compared with the pre-transplant baseline. CONCLUSIONS: Our systematic review demonstrates that islet transplantation significantly enhances quality of life in individuals with T1DM who are experiencing SHE. To our knowledge, this is the most extensive systematic review conducted to date, evaluating the impact of islet transplantation on HRQoL.

Lysophosphatidylinositols Are Upregulated After Human ß-Cell Loss and Potentiate Insulin Release.

In this study, we identified new lipid species associated with the loss of pancreatic ß-cells triggering diabetes. We performed lipidomics measurements on serum from prediabetic mice lacking ß-cell prohibitin-2 (a model of monogenic diabetes) patients without previous history of diabetes but scheduled for pancreaticoduodenectomy resulting in the acute reduction of their ß-cell mass (∼50%), and patients with type 2 diabetes (T2D). We found lysophosphatidylinositols (lysoPIs) were the main circulating lipid species altered in prediabetic mice. The changes were confirmed in the patients with acute reduction of their ß-cell mass and in those with T2D. Increased lysoPIs significantly correlated with HbA1c (reflecting glycemic control), fasting glycemia, and disposition index, and did not correlate with insulin resistance or obesity in human patients with T2D. INS-1E ß-cells as well as pancreatic islets isolated from nondiabetic mice and human donors exposed to exogenous lysoPIs showed potentiated glucose-stimulated and basal insulin secretion. Finally, addition of exogenous lysoPIs partially rescued impaired glucose-stimulated insulin secretion in islets from mice and humans in the diabetic state. Overall, lysoPIs appear to be lipid species upregulated in the prediabetic stage associated with the loss of ß-cells and that support the secretory function of the remaining ß-cells. ARTICLE HIGHLIGHTS: Circulating lysophosphatidylinositols (lysoPIs) are increased in situations associated with ß-cell loss in mice and humans such as (pre-)diabetes, and hemipancreatectomy. Pancreatic islets isolated from nondiabetic mice and human donors, as well as INS-1E ß-cells, exposed to exogenous lysoPIs exhibited potentiated glucose-stimulated and basal insulin secretion. Addition of exogenous lysoPIs partially rescued impaired glucose-stimulated insulin secretion in islets from mice and humans in the diabetic state. LysoPIs appear as lipid species being upregulated already in the prediabetic stage associated with the loss of ß-cells and supporting the function of the remaining ß-cells.

Generation of Insulin-Producing Multicellular Organoids.

Clinical islet transplantation (CIT) is an established noninvasive treatment for type I diabetes (T1D) and has demonstrated improved glycemic control, preventing the occurrence of severe hypoglycemia. However, CIT has several limitations, such as the need for multiple donors, lifelong immunosuppression, and suboptimal long-term graft function. Most of the transplanted islets are lost due to inflammation, ischemic damage, and delayed revascularization.Generation of organoids have gained increasing interest in regenerative medicine in recent years. In the context of beta-cell replacement, it offers a possibility to address limitations of CIT by allowing to produce uniform organoids from single or multiple cell types facilitating revascularization and anti-inflammatory and/or immunomodulatory protection. We have previously generated multicellular insulin-secreting organoids composed of islet cells and the human amniotic epithelial cells (hAECs). These 3D insulin-secreting structures demonstrated improved viability and function both in vitro and in vivo. Here we detail a stepwise methodology to generate insulin-secreting organoids using two different methods. In addition, quality assessment in vitro tests are also described.

Intercellular contacts affect secretion and biosynthesis of pancreatic islet cells.

Cell protein biosynthesis is regulated by different factors, but implication of intercellular contacts on alpha and beta cell protein biosyntheses activity has not been yet investigated. Islet cell biosynthetic activity is essential in regulating not only the hormonal reserve within cells but also in renewing all the proteins involved in the control of secretion. Here we aimed to assess whether intercellular interactions affected similarly secretion and protein biosynthesis of rat alpha and beta cells. Insulin and glucagon secretion were analyzed by ELISA or reverse hemolytic plaque assay, and protein biosynthesis evaluated at single cell level using bioorthogonal noncanonical amino acid tagging. Regarding beta cells, we showed a positive correlation between insulin secretion and protein biosynthesis. We also observed that homologous contacts increased both activities at low or moderate glucose concentrations. By contrast, at high glucose concentration, homologous contacts increased insulin secretion and not protein biosynthesis. In addition, heterogeneous contacts between beta and alpha cells had no impact on insulin secretion and protein biosynthesis. Regarding alpha cells, we showed that when they were in contact with beta cells, they increased their glucagon secretion in response to a drop of glucose concentration, but, on the other hand, they decreased their protein biosynthesis under any glucose concentrations. Altogether, these results emphasize the role of intercellular contacts on the function of islet cells, showing that intercellular contacts increased protein biosynthesis in beta cells, except at high glucose, and decreased protein biosynthesis in alpha cells even when glucagon secretion is stimulated.

Guidelines to Analyze Preclinical Studies Using Perinatal Derivatives.

The last 18 years have brought an increasing interest in the therapeutic use of perinatal derivatives (PnD). Preclinical studies used to assess the potential of PnD therapy include a broad range of study designs. The COST SPRINT Action (CA17116) aims to provide systematic and comprehensive reviews of preclinical studies for the understanding of the therapeutic potential and mechanisms of PnD in diseases and injuries that benefit from PnD therapy. Here we describe the publication search and data mining, extraction, and synthesis strategies employed to collect and prepare the published data selected for meta-analyses and reviews of the efficacy of PnD therapies for different diseases and injuries. A coordinated effort was made to prepare the data suitable to make statements for the treatment efficacy of the different types of PnD, routes, time points, and frequencies of administration, and the dosage based on clinically relevant effects resulting in clear increase, recovery or amelioration of the specific tissue or organ function. According to recently proposed guidelines, the harmonization of the nomenclature of PnD types will allow for the assessment of the most efficient treatments in various disease models. Experts within the COST SPRINT Action (CA17116), together with external collaborators, are doing the meta-analyses and reviews using the data prepared with the strategies presented here in the relevant disease or research fields. Our final aim is to provide standards to assess the safety and clinical benefit of PnD and to minimize redundancy in the use of animal models following the 3R principles for animal experimentation.

Perturbations in ataxia telangiectasia mutant signaling pathways after drug-induced acute liver failure and their reversal during rescue of animals by cell therapy.

Superior insights into molecular mechanisms of liver failure, which are not fully understood, will help strategies for inducing liver regeneration. We examined hepatotoxic mechanisms in mice homozygous for the severe combined immune deficiency mutation in the protein kinase, DNA-activated, catalytic polypeptide. Mice were treated with rifampicin, phenytoin, and monocrotaline. The ensuing acute liver failure was characterized by serological, histological, and mRNA studies. Subsequently, we studied whether transplantation of hepatocytes could rescue animals with liver failure. We found extensive liver damage in these animals, with mortality over several days. The expression of multiple hepatic genes was rapidly altered, including those representing pathways in oxidative/metabolic stress, inflammation, DNA damage-repair, and ataxia telangiectasia mutant (Atm) signaling pathways. This led to liver cell growth arrest involving cyclin-dependent kinase inhibitor 1A. Transplantation of hepatocytes with microcarriers in the peritoneal cavity efficiently rescued animals with liver failure. Molecular abnormalities rapidly reversed, including in hepatic Atm and downstream signaling pathways; and residual hepatocytes overcame cyclin-dependent kinase inhibitor 1A-induced cell growth arrest. Reseeding of the liver with transplanted hepatocytes was not required for rescue because native hepatocytes overcame cell growth-arrest to regenerate the liver. This likely resulted from paracrine signaling from hepatocytes in the peritoneal cavity. We concluded that Atm signaling played critical roles in the pathological features of liver failure. These studies should help redirect examination of pathophysiologic and therapeutic mechanisms in liver failure.