(F1136) IN VITRO FUNCTIONAL CHARECTERIZATION OF CLINICAL-GRADE HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS AND THEIR DIFFERENTIATED INSULIN PRODUCING BETA CELLS
Research Scholar K.S. Hegde Medical Academy, Karnataka, India
Abstract: Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are the most preferred source of multipotent stem cells due to no ethical issues and immune rejection risk, and high proliferation and differentiation ability. Type 1 diabetes mellitus (T1DM) is an autoimmune disease with treatment options of insulin therapy and islet transplantation. But both methods pose many challenges. Alternatively, UC-MSCs can be successfully differentiated into insulin-producing beta cells (IBPCs) both in vitro and in vivo, helping to restore beta cell mass and its functions. Most studies focused on fetal bovine serum (FBS) supplemented media for cells expansion, but it has proven to be unsuitable for therapeutic use. Hence, serum-free medium (SFM) and optimized differentiation protocol are essential for ensuring the safety, efficacy, and functionality of UC-MSCs derived IPBCs. With this, the present study aimed to characterize clinical-grade UC-MSCs and their differentiated IPBCs on their functional properties. The functional features of UC-MSC derived beta cells were assessed by dithizone staining, a glucose-stimulated insulin secretion assay, and the confirmation of insulin secretion by ELISA. The expression of selected pancreatic and mature beta cell markers was analysed using immunofluorescence and qPCR. Established UC-MSCs exhibited a small, spindle-like appearance with high proliferation rate and a low population doubling time. Flow cytometry analysis showed higher levels of surface markers CD44, CD73, and CD90, and low expression of CD34, CD45, and HLA-DR. The results also revealed that UC-MSCs had a greater colony-forming ability with very low senescence activity. UC-MSCs secreted different types of growth factors and pro- and anti-inflammatory cytokines, and successfully differentiated into mesodermal lineages. Further, UC-MSCs derived IPBCs were positive for dithizone stain and secreted insulin under low glucose stimulation. Later, the cells exhibited a differential expression of markers, such as PDX1, Nestin, Insulin, Glut-2, PAX6, NKX6.1, MafA, and MafB. Improving the differentiation protocol to enhance the purity and maturity of beta cells would further facilitate in establishing functional clinical-grade IPBCs from UC-MSCs for treating T1DM.
