Abstract Inhibiting pro-inflammatory cytokine activity can reverse inflammation mediated dysfunction of islet grafts. Human amniotic epithelial cells (hAECs) possess regenerative, immunomodulatory and anti-inflammatory properties. We hypothesized that hAECs could protect islets from cellular damage induced by pro-inflammatory cytokines. To verify our hypothesis hAECs monocultures, rat islets (RI), or RI-hAEC co-cultures where exposed to a pro-inflammatory cytokine cocktail (Interferon γ: IFN-γ, Tumor necrosis factor α: TNF-α and Interleukin-1β: IL-1β). The secretion of anti-inflammatory cytokines and gene expression changes in hAECs and viability and function of RI were evaluated. The expression of non-classical Major Histocompatibility Complex (MHC) class I molecules by hAECs cultured with various IFN-γ concentrations were assessed. Exposure to the pro-inflammatory cocktail significantly increased the secretion of the anti-inflammatory cytokines IL6, IL10 and G-CSF by hAECs, which was confirmed by upregulation of IL6, and IL10 gene expression. HLA-G, HLA-E and PDL-1 gene expression was also increased. This correlated with an upregulation of STAT1, STAT3 and NF-κB1gene expression levels. RI co-cultured with hAECs maintained normal function after cytokine exposure compared to RI cultured alone, and showed significantly lower apoptosis rate. Our results show that exposure to pro-inflammatory cytokines stimulates secretion of anti-inflammatory and immunomodulatory factors by hAECs through the JAK1/2 – STAT1/3 and the NF-κB1 pathways, which in turn protects islets against inflammation-induced damages. Integrating hAECs in islet transplants appears as a valuable strategy to achieve to inhibit inflammation mediated islet damage, prolong islet survival, improve their engraftment and achieve local immune protection allowing to reduce systemic immunosuppressive regimens.
Abstract Inhibiting pro-inflammatory cytokine activity can reverse inflammation mediated dysfunction of islet grafts. Human amniotic epithelial cells (hAECs) possess regenerative, immunomodulatory and anti-inflammatory properties. We hypothesized that hAECs could protect islets from cellular damage induced by pro-inflammatory cytokines. To verify our hypothesis hAECs monocultures, rat islets (RI), or RI-hAEC co-cultures where exposed to a pro-inflammatory cytokine cocktail (Interferon γ: IFN-γ, Tumor necrosis factor α: TNF-α and Interleukin-1β: IL-1β). The secretion of anti-inflammatory cytokines and gene expression changes in hAECs and viability and function of RI were evaluated. The expression of non-classical Major Histocompatibility Complex (MHC) class I molecules by hAECs cultured with various IFN-γ concentrations were assessed. Exposure to the pro-inflammatory cocktail significantly increased the secretion of the anti-inflammatory cytokines IL6, IL10 and G-CSF by hAECs, which was confirmed by upregulation of IL6, and IL10 gene expression. HLA-G, HLA-E and PDL-1 gene expression was also increased. This correlated with an upregulation of STAT1, STAT3 and NF-κB1gene expression levels. RI co-cultured with hAECs maintained normal function after cytokine exposure compared to RI cultured alone, and showed significantly lower apoptosis rate. Our results show that exposure to pro-inflammatory cytokines stimulates secretion of anti-inflammatory and immunomodulatory factors by hAECs through the JAK1/2 – STAT1/3 and the NF-κB1 pathways, which in turn protects islets against inflammation-induced damages. Integrating hAECs in islet transplants appears as a valuable strategy to achieve to inhibit inflammation mediated islet damage, prolong islet survival, improve their engraftment and achieve local immune protection allowing to reduce systemic immunosuppressive regimens.
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.
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 β 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 β cell replacement therapies in more favourable transplantation sites than the liver.
Background: Inflammation is a primary contributor to early graft loss and poor islet engraftment. Human amniotic epithelial cells (hAEC) possess regenerative, immunomodulatory and anti-inflammatory properties. In particular, these cells express HLA-G and HLA-E, involved in immunomodulation and immune tolerance. Here, we hypothesized that hAECs could protect islets from cellular damage induced by proinflammatory cytokines and we assessed the cytokine-induced expression of HLA-G and HLA-E in hAECs. Methods: Rat islets were cultured with or without hAECs for 24 hours, followed by 48- hour exposure to IFN-γ, TNF-α and IL-1β. Controls included mono or cocultures without cytokines. For all conditions, glucose stimulated insulin secretion (GSIS), apoptosis by detection of histone-associated DNA fragments, and Th1/Th2 cytokines secreted in the culture media were evaluated by ELISA. Gene expression modifications were assessed by qPCR. hAEC surface marker expression (CD105, CD90, CD326, HLA-E, HLA-G, SSEA-4) was assessed by flow cytometry after culture in control culture medium or in medium containing various concentrations of human recombinant IFN-γ for 24–48H. Results: Exposure to a pro-inflammatory cocktail significantly increased the secretion of the anti-inflammatory cytokines IL6, IL10 and G-CSF by hAECs at both 24H and 48H. IL6, IL8 and IL10 gene expression was significantly upregulated, as well as HLA-G and HLAE. This correlated with an upregulation of STAT1, STAT3 and NF-κB1gene expression levels. RI co-cultured with hAECs maintained a normal insulin secretion after cytokine exposure compared to RI cultured alone, and a significantly lower apoptosis rate. Conclusions: In conclusion, hAECs increase their anti-inflammatory and immunomodulatory potentials when exposed to inflammation in vitro, and protect pancreatic islets against pro-inflammatory cytokines in a coculture set-up.
Background: Replacing damaged organs with biological substitutes capable of protecting the islets and facilitating vascularization is a great objective in the field of islet transplantation. A decellularized placenta includes a large number of cotyledons with a conserved vessel structure of the native organ. Our goal is to obtain a perfect decellularization protocol to generate pre-vascularized organoids by recellularizing this ECM with HUVECs and pancreatic islets. Methods: After blood removal, cotyledons were dissected from the placentas and decellularized using a bioreactor. Cell removal was assessed by histology and quantification of residual DNA. Presence of structural proteins and ECM structure were analyzed using SEM, CT scan and mass spectrometry. Recellularization protocols were conducted, with HUVECs or BOECs as endothelial cell sources, and with Ins-1E cells or rat islets as insulin secreting cell sources. Function of recellularized cotyledons was assessed in vitro with glucose stimulated insulin secretion tests (GSIS). To assess in vivo biocompatibility and function of the scaffolds, we transplanted in diabetic NSG mice. Glycaemia was measured every day to monitor normalization of blood glucose levels. Results: Our protocol led to successful decellularization, as evidenced by the absence of cells and the preserved ECM structure. Moreover, DNA quantification did not reveal any residual DNA. Quantification of GAG and hydroxyproline, and mass spectrometry analysis show that structural proteins are conserved. SEM and CT scan images revealed that the ECM structure was preserved after the decellularization protocol. Cells after recellularization showed a good vascularization after 7 days. The GSIS test shows a perfect organ response in the production of insulin. Conclusions: The decellularized cotyledon is the perfect scaffold to reproduce a prevascularized insulin-producing organ, which allows transplanted cells to survive during the peri-transplantation period.
Abstract Inhibiting pro-inflammatory cytokine activity can reverse inflammation mediated dysfunction of islet grafts. Human amniotic epithelial cells (hAECs) possess regenerative, immunomodulatory and anti-inflammatory properties. We hypothesized that hAECs could protect islets from cellular damage induced by pro-inflammatory cytokines. To verify our hypothesis, hAEC monocultures, rat islets (RI), or RI-hAEC co-cultures where exposed to a pro-inflammatory cytokine cocktail (Interferon γ: IFN-γ, Tumor necrosis factor α: TNF-α and Interleukin-1β: IL-1β). The secretion of anti-inflammatory cytokines and gene expression changes in hAECs and viability and function of RI were evaluated. The expression of non-classical Major Histocompatibility Complex (MHC) class I molecules by hAECs cultured with various IFN-γ concentrations were assessed. Exposure to the pro-inflammatory cocktail significantly increased the secretion of the anti-inflammatory cytokines IL6, IL10 and G-CSF by hAECs, which was confirmed by upregulation of IL6, and IL10 gene expression. HLA-G, HLA-E and PDL-1 gene expression was also increased. This correlated with an upregulation of STAT1, STAT3 and NF-κB1gene expression levels. RI co-cultured with hAECs maintained normal function after cytokine exposure compared to RI cultured alone, and showed significantly lower apoptosis rate. Our results show that exposure to pro-inflammatory cytokines stimulates secretion of anti-inflammatory and immunomodulatory factors by hAECs through the JAK1/2 – STAT1/3 and the NF-κB1 pathways, which in turn protects islets against inflammation-induced damages. Integrating hAECs in islet transplants appears as a valuable strategy to achieve to inhibit inflammation mediated islet damage, prolong islet survival, improve their engraftment and achieve local immune protection allowing reducing systemic immunosuppressive regimens. Graphical Abstract This study focuses on the cytoprotective effect of isolated hAECs on islets exposed to pro-inflammatory cytokines in vitro. Exposure to pro-inflammatory cytokines stimulated secretion of anti-inflammatory and immunomodulatory factors by hAECs putatively through the JAK1/2 – STAT1/3 and the NF-κB1 pathways. This had protective effect on islets against inflammation-induced damages. Taken together our results indicate that incorporating hAECs in islet transplants could be a valuable strategy to inhibit inflammation mediated islet damage, prolong islet survival, improve their engraftment and achieve local immune protection allowing to reduce systemic immunosuppressive regimens.
Background Recent advancements in tissue engineering shows that generating multicellular spheroids by combining different cell types can enhance their regenerative capacity. Human amniotic epithelial cells (hAECs) gained great interest in regenerative medicine due to their availability, safety, regenerative, immunomodulatory and anti-inflammatory properties. The aim of this study was to determine whether combination of hAECs with islet cells in the same spheroid would further improve islet cell survival and function in vitro under normoxic and hypoxic conditions. 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. Engineered islet homo- and heterospheroids were cultured under normoxic and hypoxic conditions for 16 h. For all conditions, cell viability, GSIS, total islet cellular insulin content were detected. Hypoxia-induced changes in gene expression were assessed by real time PCR analysis. Next we considered possible molecular mechanisms behind the beneficial effect of hAECs on islet cell function. For this purpose, the mRNA expression levels of Hif-1α, Casp3, Casp8, Casp9 and Bcl2 were determined. Results Confocal laser scanning microscopy showed uniform distribution of islet cells and hAECs throughout of hybrid spheroids, without evidence of cell loss. Quantifications of the insulin positive area ratios to nuclei, showed that heterospheroids expressed more insulin compared to homospheroids. Moreover, islet heterospheroids expressed significantly more E-cadherin, key cell-to-cell adhesion molecule then homospheroids. The stimulation index of the heterospheroids was significantly higher than those of homospheroids. Exposure to hypoxia rapidly caused fragmentation of homospheroids and augmented cell membrane permeability. In contrary considerably less dead signals were observed within heterospheroids. As anticipated, glucose responsiveness of homospheroids was seriously impaired; the average glucose stimulation index of the heterospheroids was significantly higher than that of the homospheroids (1.7±0.4 vs 0.9±0.5, p < 0.05). This was correlated with downregulation of apoptotic genes Casp3, Casp8 and Casp9 and 2 fold upregulation of antiapoptotic gene Bcl2. Conclusions These data indicate that incorporation of hAECs into the islet heterospheroids improves the secretory function and viability of islet cells both in conventional culture and in hypoxic conditions.
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