Porcine Neonatal Islet-like Cell Clusters Cultured with EGM2 Medium Enhance their Resistance to Apoptosis in Islet Xenotransplantation

Abstract

Introduction Pig neonatal 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. Materials and Methods EGM2 medium was depleted with hydrocortisone and supplemented with 50 mM isobutylmethlxanthine, 10 mM nicotinamide,10 mM glucose. Modified EGM2 medium was used to culture NICC at day 1, the day after isolation and changed every other day. When NICC cultured with modified EGM2 or control medium Ham’s F-10 at day 7, they were collected to do the following assays. AO/EB staining were used to evaluate the viability of NICC. Static assay and oxygen consumption rate analysis were performed to assess the function of NICC. Insulin and glucagon expression was measured by real-time PCR. Serum starvation and Cocl2 treatment was used to simulate the microenvironment post islet transplantation. The viability of NICC was evaluated by FACS and the proposed mechanism was detected by western blotting. Results Compared with Ham’s F-10 medium, culturing NICC with modified EGM2 medium led to increased number and viability of NICC with higher stimulation index, upregulated gene expression of both insulin and glucagon and also higher mitochondria function. Moreover, modified EGM2 medium cultured NICC demonstrated much less apoptotic cells under either serum starvation or CoCl2-induced 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. Discussion NICC cultured with EGM2 medium not only improved their viability and mitochondria function but also enhanced their resistance to apoptosis under the hypoxia environment. Conclusion Culturing NICC with modified EGM2 provides a simple and effective approach to enhance their resistance to apoptosis to preserve the initial graft mass for successful islet xenotransplantation. This project was supported by NSFC 81201171 and Hunan Provincial Natural Science Foundation of China 2017JJ3423.