Pakistan Journal of Pharmaceutical Sciences

Abstract: Background: Current diabetes therapies often fail to address the loss of insulin-producing ?-cells. Bone marrow mesenchymal stem cells (BMSCs) can differentiate into insulin-producing cells (IPCs), but functional maturity remains limited. Toll-like receptor 9 (TLR9) agonists may enhance BMSC maturation. Objectives: To investigate the ability of TLR9 agonists to promote the maturation of BMSCs in diabetic rats. Methods: BMSCs were isolated from rat bone marrow and identified by trilineage differentiation tests and flow cytometry. BMSCs were then differentiated into IPCs and treated with different concentrations of TLR9 agonists. PCR detected transcriptional levels of pancreatic endocrine-related genes. Diabetic model in rats was established using streptozotocin. BMSCs were encapsulated in TheraCyte capsules and subcutaneously transplanted into diabetic rats. Intraperitoneal injection of TLR9 agonists was performed, with serum insulin, C-peptide and fasting blood glucose (FBG) levels monitored in the transplanted rats. The recovered BMSCs were further characterized by immunofluorescence staining after the rats were killed. Results: Differentiated BMSCs displayed characteristics of IPCs, such as the ability to secrete insulin and C-peptide in vitro. Further, when exposed to gradient concentrations of TLR9 agonists, differentiated MSCs exhibited enhanced expression of pancreatic-associated endocrine genes and transcription factor levels. In vivo, differentiated BMSC transplantation combined with TLR9 agonist administration notably improved diabetic conditions, as evidenced by suppressed FBG levels and elevated serum insulin and C-peptide levels, comparable to those in the normal group. However, withdrawal of TLR9 agonists attenuated the therapeutic effects. Finally, immunofluorescence staining confirmed that a TLR9 agonist promoted IPC maturation, as evidenced by co-expression of insulin and C-peptide in vivo. Conclusion: TLR9 agonists promote the differentiation and functional maturation of BMSCs into IPCs, thereby improving metabolic parameters in diabetic rats. This "stem cell-immunomodulator" strategy provides a foundation for developing functional ?-cell replacement therapies.