Vanguard Consortium. Background: This study aimed to engineer a functional, vascularized endocrine pancreas by utilizing decellularized human placental cotyledons (hPLCs), human blood outgrowth endothelial cells (BOECs), and pancreatic islets. BOECs, which can be obtained from recipients, were used for revascularizing the construct. Methods: hPLCs were decellularized using 1% SDS and 0.1% Triton. Acellular hPLCs were repopulated with BOECs and then seeded with 1500 human islet equivalents (IEQ). Recellularization was confirmed through histological and immunohistochemical methods, while endocrine function was assessed by glucose-stimulated insulin secretion tests. Vascularized endocrine constructs were transplanted into the subcutaneous (SC) space of STZ diabetic NSG mice (PLCs+Islets+BOECs). Control mice were transplanted with non-endothelialized scaffolds (PLCs+Islets) containing the same number of islets and free islets in prevascularized subcutaneous spaces (SC) and under the kidney capsule (KC). Results: Engineered vascularized endocrine constructs exhibited a continuous CD31+ endothelial cell network within the hPLC, with islets embedded in the vascular bed. These constructs also demonstrated physiological insulin release in response to glucose stimulation. In the first week following transplantation, 80% of mice in the PLCs+Islets+BOECs group achieved normalized blood glucose levels, compared to 60% in the PLCs+Islets group. Mice transplanted with islets in the prevascularized subcutaneous space did not return to a normoglycemic state. Graft-bearing construct removal led to hyperglycemia recurrence in all mice within 24 hours. Immunohistochemical staining revealed a larger β-cell mass, as assessed by insulin-positive area per field, in the PLCs+Islets+BOECs group compared to the PLCs+Islets group 90 days post-transplant. CD31-stained explanted grafts indicated significantly higher vessel density in the PLCs+Islets+BOECs samples compared to the PLCs+Islet samples. Conclusions: The engineered vascularized endocrine pancreas provides a fully biocompatible construct that closely mimics the native islets’ matrix environment and offers mechanical protection, enabling transplanted islets to engraft and function long-term. This work was supported by grants from the European Commission (Horizon 2020 Framework Program; VANGUARD grant 874700), the Juvenile Diabetes Research Foundation (JDRF; grant 3-SRA-2020-926-S-B), the Shota Rustaveli National Science Foundation (grant FR-19-19760) and the Swiss National Science Foundation (grant 310030_173138 and grant CRSII5_209417).
Abstract NRLP3 inflammasome is a protein complex involved in the maturation of IL1β. In the onset of type 1 diabetes as well as in islet transplantation, IL-1β is one of the cytokines involved in the recruitment of immune cells in islets and eventually in islet destruction. Whether IL-1β is produced by islet cells is still under debate and NLRP3 inflammasome-dependent IL-1β production has not been yet determined in human islets. The aim of this study was to determine the expression and the regulation of the NRLP3 inflammasome in human islets. Human islets were stimulated with LPS and successively with ATP (LPS + ATP) in the presence or absence of the inflammasome inhibitor glyburide. Islets were also incubated in hypoxic or normoxic conditions for 24 h in the presence or absence of glyburide. Then, IL1B and NLRP3 expression was studied by real time PCR, protein expression by western blot, protein localization by immunofluorescence and protein secretion by ELISA. LPS + ATP increased gene expression of NRLP3 and IL1B . Glyburide partially prevented this effect. IL-1β protein was localized in β and non-β cells. Moreover, LPS + ATP increased IL-1β protein expression and production, which were prevented by glyburide. Hypoxia increased gene expression of NRLP3 and IL1B and induced IL-1β and caspase-1 production. Finally, hypoxia-induced cell death which was not prevented by inhibition of NLRP3 inflammasome. NRLP3 inflammasome is expressed and plays a role in IL-1β production by human islets. By contrast, NRLP3 inflammasome activation is not involved in islet cell death induced by hypoxia.
In this study, we identified new lipid species associated with the loss of pancreatic β-cells triggering diabetes. We performed lipidomics measurements on serum from prediabetic mice lacking β-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 β-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 β-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 β-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 β-cells and that support the secretory function of the remaining β-cells.Circulating lysophosphatidylinositols (lysoPIs) are increased in situations associated with β-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 β-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 β-cells and supporting the function of the remaining β-cells.
Background Human amniotic epithelial cells (hAECc) derived from placental tissue are widely available and possess immunomodulatory, anti-inflammatory and regenerative properties. In this study we have generated islet heterospheroids composed of hAECs and dispersed islet cells (ICs) aiming to improve viability, engraftment and vascularization of the transplanted spheroids. Methods Functional Islet spheroids were generated on 3D agarose-patterned microwells. To form homospheroids dispersed rat islet cells (ICs) and hAECs (128,000 cells/mold and 500cell/spheroid) were seeded alone. Heterospheroids were formed by mixing ICs and hAECs at ratio of 1:1 (128,000 cells/mold and 500cell/spheroid). Marginal mass (150 IEQ) of islet heterospheroids (islet + AEC group), islet homospheroids (islet-only group) or hAEC spheroids (hAEC alone group) was transplanted under the kidney capsule of diabetic SCID mice. Blood glucose levels were monitored daily and IPGTTs were carried out. Grafts and serum were harvested at 1, 2, 6 and 12 weeks after transplantation to assess outcome. Results Mice transplanted with islet heterospheroids exhibited enhanced glycemic control as measured by glucose tolerance, serum insulin/c-peptide level and diabetes reversal rate, compared with mice in islet alone group. The cumulative percentage of animals reaching normoglycemia was 74% in the islet+hAEC group versus 26% in the islets-alone group. The median time to reverse hyperglycaemia for islet+hAEC grafts was 5 ± 0.9 days and 30 ± 7 days for islet-alone recipients (p < 0.0001, n = 26). Between groups, the morphology of islet grafts showed significant differences in size and composition of grafted endocrine tissues. A two-fold increase in graft revascularization was seen in islet + hAEC grafts, which was mainly attributed to stimulating vascular endothelial growth factor-A (VEGF-A) production. The rapid revascularization led to improved graft perfusion and recovery from hypoxia. Conclusion These data indicate that hAECs may have a significant potential to protect islet cells and may be employed to improve islet cell survival and function prior to transplantation. Hence, hAEC-enriched pseudoislets may represent a novel approach to increase the success rate of islet transplantation.
Authors have elaborated new method of surgical correction of hepaticocholedoch strictures. The experiments were carried out on 25 mongrel dogs of both sexes with body weight 15-25 kg. The surgical procedures were done under endotracheal ether narcosis. The hepaticocholedoch stricture was induced preliminarily in all animals. After the modelling, stricture was cut out, the anatomical integrity of the common bile duct was restored by autovein (n=10) and GORE-TEX vascular prosthesis (n=10). The performed investigations have shown that autovenous transplant was subjected to incrustation by bile salts, later on it became necrotic. Therefore the autovenous transplant could not be used in biliary reconstruction. The results of biliary reconstruction by GORE-TEX vascular prosthesis were promising. The GORE-TEX vascular prosthesis is well adapting with bile ducts tissue and do not incrust by bile salts.
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.
Type 1 diabetes is characterized by the destruction of pancreatic β 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 β-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 β cell mass from α cells.
