Search

Abstract: Background: Hypoxia‐induced damage is one of the key factors associated with islet graft dysfunction. Mesenchymal stem cells (MSCs) could be used to enhance the therapeutic effect of islet transplantation due to their paracrine potential such as exosomes. In this study, we investigated whether exosomes from human umbilical cord‐derived MSC‐conditioned medium (hu‐MSC‐CM) could increase the survival and function of neonatal porcine islet cell clusters (NICCs) exposed to hypoxia. Methods: Neonatal porcine islet cell clusters were cultured with hu‐MSC‐CM, with or without exosomes, and native medium RPMI‐1640 (Control) under hypoxic conditions (1% O2). The effects of exosomes on NICCs viability and function in vitro were examined by FACS, the Loops system, and the Extracellular Flux assay, respectively. Results: Compared with NICCs cultured in RPMI‐1640 medium and hu‐MSC‐CM without exosomes, the survival ratio, viability, and function increased in NICCs cultured in hu‐MSC‐CM with exosomes. Conclusions: This study found that hu‐MSC‐CM could protect NICCs from hypoxia‐induced dysfunction, and exosomes played an important role in hypoxic resistance, suggesting a potential strategy to improve islet transplantation outcomes. [ABSTRACT FROM AUTHOR]

Abstract: Background: Neonatal pig islet‐like cell clusters (NICC) are an attractive source of insulin‐producing tissue for potential transplantation treatment of type 1 diabetic patients. However, a considerable loss of NICC after their transplantation due to apoptosis resulted from islet isolation and instant blood‐mediated inflammatory reaction remains to be overcome. Methods: EGM2 medium depleted with hydrocortisone and supplemented with 50 mmol/L isobutylmethylxanthine, 10 mmol/L nicotinamide, and 10 mmol/L glucose was used to culture NICC at day 1, the day after isolation and changed every other day. NICC cultured with EGM2 or control Ham's F‐10 medium were collected at day 7 of culture for the following assays. The viability of NICC was evaluated by AO/EB staining and FACS. Static assay and oxygen consumption rate analysis were performed to assess the function of NICC. Insulin and glucagon gene expression were measured by real‐time PCR. Tubing loops model and TUNEL assay were performed to confirm the apoptosis‐resistant ability of NICC cultured with modified EGM2 medium. Serum starvation and hypoxia treatment were used to test the tolerant capability of NICC in the microenvironment of hypoxia/nutrient deficiency in vitro. The molecules involved in apoptosis pathways in NICC were analyzed by Western blotting. Results: Compared with Ham's F‐10 medium, culturing NICC with EGM2 medium led to increased number and viability of NICC with higher stimulation index, upregulated gene expression of both insulin and glucagon, and enhanced mitochondria function. Furthermore, fewer modified EGM2 medium cultured NICC were found under apoptosis when evaluated in an in vitro tubing loop model of IBMIR. Moreover, EGM2 medium cultured NICC demonstrated much less apoptotic cells under either serum starvation or hypoxia condition than their Ham's F‐10 medium cultured counterparts. The enhanced capability of EGM2 cultured NICC to resist apoptosis was associated with their elevated protein levels of anti‐apoptotic Bcl‐2 family member Mcl‐1. Conclusion: Culturing NICC with EGM2 provides a simple and effective approach not only to increase NICC yield, viability, and maturation but also to enhance their resistance to apoptosis to preserve the initial graft mass for successful islet xenotransplantation. [ABSTRACT FROM AUTHOR]

Background A high immunosuppressive burden is required for long-term islet xenograft survival in non-human primates even using genetically modified donor pigs. Aims We aimed to investigate the capacity of baboon regulatory T cells (Treg) to suppress islet xenograft rejection, thereby developing a potential immunoregulatory or tolerance therapy that could be evaluated in NHP models of xenotransplantation. Materials & Methods Baboon Treg expanded with stimulation by porcine peripheral blood mononuclear cells ( PBMC) were characterized by cell phenotyping and suppressive activity assays in vitro. Their function in vivo was evaluated in neonatal porcine islet cell clusters ( NICC) transplanted NOD- SCID IL-2rγ−/− ( NSG) mice receiving baboon PBMC alone or with expanded autologous Treg. Results The majority of expanded Treg coexpressed Foxp3 and CD39 and were highly suppressive of the baboon anti-pig xenogeneic T cell response in vitro. Reconstitution of mice with baboon PBMC alone resulted in NICC xenograft rejection within 35 days. Cotransfer with baboon PBMC and Treg prolonged islet xenograft survival beyond 100 days, correlating with Treg engraftment, intragraft CD39 and Foxp3 gene expression, and reduced graft infiltrating effector T cells and reduced interferon-γ production. Discussion & Conclusion Our data supports the capacity of ex vivo expanded CD39+ baboon Treg to suppress islet xenograft rejection in primatized mice, suggesting it has potential as an adjunctive immunotherapy in preclinical NHP models of xenotransplantation. [ABSTRACT FROM AUTHOR]

The article discusses the third World Health Organization (WHO) Global conference on regulatory requirements for Xenotransplantation clinical trials that was held in Changsha, China on 12–14 December, 2018. It mentions recent advances in the development of technologies for solid organ xenotransplantation; and also mentions Dr. Megan Sykes reviewed the current technologies and studies in the development of tolerance and use of regulatory T cells.

In order to investigate the mechanism by which CD4+ T cells and macrophages interact in the xenogeneic immune response, murine CD4+ T cells and macrophages were used as responder cells in culture with irradiated fetal pig spleen cells (FPSC) as pig xenogeneic stimulators. In this in vitro model, murine CD4+ T cells and macrophages were cultured individually, or together with FPSC. In addition, mouse CD4+ T cells were also cultured with autologous macrophages which were previously stimulated by FPSC. The cultured murine cells were analyzed for expression of CD4+ T cell and macrophage activation markers (cell surface markers and cytokines) as well as cytokine production. CD4+ T cells and macrophages cultured alone or together without FPSC showed unchanged low levels of expression of activation markers. Coculture of macrophages with FPSC and in the absence of CD4+ T cells induced increased expression levels of all the activation markers examined except B7.2 and ICAM-1. Addition of CD4+ T cells to the coculture further enhanced this up-regulation. Coculture of CD4+ T cells with FPSC-stimulated macrophages, but not naive macrophages, or FPSC alone, resulted in significantly increased numbers of CD4+ T cells coexpressing their activation markers, especially IFN-gamma and CD40L, and this expression was enhanced further by including FPSC in the coculture. The activation of both CD4+ T cells and macrophages in their coculture with FPSC was suppressed by neutralizing IFN-gamma but not IL-4. Our results demonstrated that interaction of CD4+ T cells and autologous macrophages was required for their optimal activation in response to pig xenogeneic stimulation. The mechanisms involved included cell-cell and/or cytokine interactions, and in particular IFN-gamma mediated communication was involved. Macrophages activated by pig cells in the absence of CD4+ T cells were able to activate naive CD4+ T cells, thus providing an important communication pathway between innate immune activation and a T cell mediated response in xenograft rejection.