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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 For xenotransplantation to truly succeed, we must develop immunomodulatory strategies to suppress the xenoimmune response but by minimizing immunosuppression over the long term. Regulatory macrophages (Mreg) have been shown to suppress polyclonal T-cell proliferation in vitro and prolong allograft survival in vivo. However, the question of whether they are capable of suppressing xenoimmune responses remains unknown. This study assessed the potential of human Mreg to be used as an effective immunomodulatory method in xenotransplantation. Methods CD14+ monocytes selected from human peripheral blood mononuclear cells ( PBMC) were cultured with macrophage colony-stimulating factor (M- CSF) for 7 days with IFN-γ added at day 6 for Mreg induction. Mreg phenotyping was performed by flow cytometric analysis, and the in vitro suppressive function was assessed by mixed lymphocyte reaction ( MLR) using irradiated pig PBMC as the xenogeneic stimulator cells, human PBMC as responder cells, and autologous Mreg as suppressor cells. To assess mRNA expression of Mreg functional molecules indoleamine-2,3-dioxygenase ( IDO), IL-10, inducible nitric oxide synthase ( iNOS) and TGF-β were measured by real-time PCR. Supernatants were collected from the MLR cultures for IDO activity assay by high-performance liquid chromatography ( HPLC). The effects of the IDO inhibitor 1-D/L-methyl-tryptophan (1- MT), iNOS inhibitor NG-monomethyl-l-arginine (L- NMMA), and anti- IFN-γ or anti- TGF-β monoclonal antibody ( mAb) treatment on Mreg suppressive capacity were tested from the supernatants of the MLR assays. Results We demonstrated that induced Mreg with a phenotype of CD14low CD16−/low CD80low CD83−/low CD86+/hi HLA- DR+/hi were capable of suppressing proliferating human PBMC, CD4+, and CD8+ T cells, even at a higher responder:Mreg ratio of 32:1 in a pig-human xenogeneic MLR. The strong suppressive potency of Mreg was further correlated with their upregulated IDO expression and activity. The IDO upregulation of Mreg was associated with an increased production of IFN-γ, an IDO stimulator, by xenoreactive responder cells in the xenogeneic MLR. While no effect on Mreg suppressive potency was detected by addition of the iNOS inhibitor L- NMMA or anti- TGF-β mAb into the MLR assays, inhibition of IDO activity by neutralizing IFN-γ or by IDO inhibitor 1- MT substantially impaired the capacity of Mreg to suppress the xenogeneic response, indicating the importance of upregulated IDO activity in Mreg-mediated suppression of the xenogeneic response in vitro. Conclusion This study demonstrates that human Mreg are capable of suppressing the xenoimmune response in vitro via IDO-involved mechanism(s), suggesting their potential role as an effective immunomodulatory tool in 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.