Neonatal Islet-like Cell Clusters Research Articles

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

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.

In vitro exposure of pig neonatal isletlike cell clusters to human blood.

Pig islet grafts have been successful in treating diabetes in animal models. One remaining question is whether neonatal pig isletlike cell clusters (NICC) are resistant to the early loss of islets from the instant blood-mediated inflammatory reaction (IBMIR). Neonatal isletlike cell clusters were harvested from three groups of piglets-(i) wild-type (genetically unmodified), (ii) α1,3-galactosyltransferase gene-knockout (GTKO)/CD46, and (iii) GTKO/CD46/CD39. NICC samples were mixed with human blood in vitro, and the following measurements were made-antibody binding; complement activation; speed of islet-induced coagulation; C-peptide; glutamic acid decarboxylase (GAD65) release; viability. Time to coagulation and viability were both reduced in all groups compared to freshly drawn non-anticoagulated human blood and autologous combinations, respectively. Antibody binding to the NICC occurred in all groups. Neonatal isletlike cell clusters were subject to humoral injury with no difference associated to their genetic characteristics.

Long-term cultured neonatal islet cell clusters demonstrate better outcomes for reversal of diabetes: in vivo and molecular profiles.

Porcine neonatal islet-like cell clusters (NICC) are being considered as a source of β-cell replacement. However, the lag time to full function due to hormonal immaturity remains a problem. This study aimed to determine whether time in culture was important for NICC function in vivo. Neonatal islet-like cell clusters were isolated from piglets aged between 1 and 3 days, and cultured for up to 27 days post-isolation. Each week, NICC number, viability, and function were determined. Neonatal islet-like cell clusters cultured for 12, 19, and 27 days achieved normal blood glucose levels at 46 days (85% of animals), 32 days (100% of animals), and 35 days (81% of animals), respectively. By comparison, standard 6-day culture took a mean of 63 days to achieve normoglycemia in 35% of animals. Longer time in culture resulted in a significant loss of islet equivalent over time. However, insulin gene expression levels were significantly higher at days 12, 19, 27 compared to day 6. Glucagon gene expression was highest at day 12, and significantly higher than day 6 at all time points. Bcl-2 gene expression increased over time, and tissue factor (TF) gene expression was highest on day 6 and then decreased over the remaining time points. Culture of NICC for 12 days provides the best balance in vivo functional outcome for transplantation, shown by better reversal of diabetes, and higher levels of gene expression for insulin, glucagon and Bcl-2 and lower levels of TF expression with acceptable NICC number loss in terms of time and expense.

Functional and immunohistochemical evaluation of porcine neonatal islet-like cell clusters.

Porcine neonatal islet-like cell clusters (NICCs) may be an attractive source of insulin-producing tissue for xenotransplantation in type I diabetic patients. We examined the functional and immunohistochemical outcome of the islet grafts in vitro during long-term culture and in vivo after transplantation to athymic nude mice. On average we obtained 29,000 NICCs from each pancreas. In a perifusion system, NICCs responded poorly to a glucose challenge alone, but 10 mmol/L arginine elicited a fourfold increase in insulin secretion and 16.7 mmol/L glucose + 10 mmol/L arginine caused a sevenfold increase in insulin section indicating some sensitivity towards glucose. Hormone content as well as the number of hormone-containing cells increased for the first 14 days of culture. When NICCs were stained for hormones, proliferation (Ki67), and duct cells (CK7), some insulin- and glucagon-positive cells co-stained for proliferation. However no co-staining was observed between insulin- and glucagon-positive cells or between hormone-and CK-positive cells. Following transplantation of 2000 NICCs under the renal capsule of diabetic nude mice, BG levels were normalized within an average of 13 weeks. Oral and IP glucose tolerance tests revealed a normal or even faster clearance of a glucose load compared with normal controls. Immunohistochemical examination of the grafts revealed primarily insulin-positive cells. In summary, in vitro, NICCs responded to a challenge including glucose and arginine. There was a potential for expansion of the beta-cell mass of NICCs in vitro as well as in vivo where NICCs eventually may normalize blood glucose of diabetic mice.