Mesenchymal stem cell conditioned medium improves hypoxic injury to protect islet graft function

Objectives: Islet transplantation is one of the most promising curative methods for type 1 diabetes mellitus (T1DM), but early hypoxic death of the graft post-transplantation impedes successful treatment. To improve the efficacy of islet transplantation and enhance islet cell resistance to hypoxia, reducing hypoxic injury before revascularization is crucial. Mesenchymal stem cells (MSCs) are known to regulate immune responses and protect against hypoxic damage through paracrine mechanisms. This study aims to verify the protective effects of MSC-conditioned medium (CM) in enhancing islet cells' tolerance to hypoxic conditions and preserving islet graft function.

Methods: MIN6 cells were cultured under hypoxic conditions (1% oxygen), and their viability was assessed at different time points using AO/PI staining, observed through fluorescence microscopy. MIN6 cells were treated with varying concentrations of MSC-CM under normal and hypoxic conditions. At different time points, cell viability was measured by Annexin/PI flow cytometry, and insulin secretion capacity was assessed through glucose-stimulated insulin secretion tests. A NCG T1DM mouse model was established, and islet cells from BALB/c mice were co-incubated with MSC-CM for 24 hours. The islet cells were then transplanted under the renal capsule of NCG T1DM mice. Mice body weight and blood glucose levels were monitored, and glucose tolerance tests were conducted to evaluate graft function. Graft survival was further assessed by HE staining and insulin immunohistochemistry.

Results: Under hypoxic conditions, MIN6 cell death increased with prolonged hypoxia. Flow cytometry showed that after 48 hours of hypoxia, the survival rate of MIN6 cells was significantly lower than that of the normoxic group [(68.07±7.90)% vs (94.57±2.12)%, P<0.01)]. MSC-CM treatment restored the insulin secretion function of MIN6 cells under hypoxia, with the stimulation index (SI) increasing from 1.43±0.06 to 1.77±0.02 (P<0.001). Both 10% and 20% MSC-CM effectively mitigated hypoxic damage, whereas 30% MSC-CM had weaker effects. Glucose-stimulated insulin secretion results showed trends consistent with cell survival. Primary mouse islet cells pretreated with 10% MSC-CM and transplanted under the renal capsule of T1DM mice showed a sustained decrease in blood glucose levels 5 days post-surgery. HE staining and insulin immunohistochemistry indicated that the islet cells in the MSC-CM group maintained more intact morphology and higher insulin secretion. Glucose tolerance tests demonstrated better graft function in the MSC-CM group.

Conclusions: Hypoxia significantly reduces the survival of MIN6 cells and suppresses their insulin secretion function. However, MSC-CM can significantly improve hypoxia-induced cell death and functional decline, and protect islet graft function in a T1DM mouse transplantation model.