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  • Title: Culturing with modified EGM2 medium enhances porcine neonatal islet-like cell clusters resistance to apoptosis in islet xenotransplantation.
    Author: Ma X, Yang C, Zhang J, Wang J, Li W, Xu C, Rong P, Ye B, Wu M, Jiang J, Yi S, Wang W.
    Journal: Xenotransplantation; 2018 Jan; 25(1):. PubMed ID: 29131417.
    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.
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