Abstract
BACKGROUND/AIM: Brain microvascular endothelial cells (BMECs), which constitute the blood-brain barrier (BBB), are essential for maintaining central nervous system homeostasis. Like BMECs, multipotent mesenchymal stem cells (MSCs) originate from the mesodermal lineage. Thus, MSCs may serve as a direct and efficient cellular source for BMEC-like differentiation. Notably, differentiation of human induced pluripotent stem cells (hiPSCs) into BMECs typically involves a 2-step protocol: inducing mesodermal commitment followed by endothelial specification. In contrast, direct differentiation from MSCs could bypass the initial mesodermal induction step, offering a streamlined alternative. This study tested a novel strategy for differentiating MSCs into brain-like endothelial cells (BLECs), circumventing the conventional mesodermal induction step. MATERIALS AND METHODS: Our differentiation protocol integrates developmental cues through the application of hypoxia, retinoic acid (RA), cobalt chloride (CoCl(2)), and-for the first time in this context-sodium sulfite (Na(2)SO(3)) to promote endothelial specification. Various basal media, including IMDM, EGM-2, and Endopan, were tested in combination with B27 supplement or fetal bovine serum (FBS) to optimize differentiation conditions. MSC viability under CoCl(2) and Na(2)SO(3) treatment was evaluated using the MTT assay to determine appropriate concentrations. The endothelial functionality of the resulting BLECs was assessed via tube formation assays. RESULTS: Immunocytochemical analysis confirmed the expression of key BMEC markers, including ZO-1, CD31, and occludin, showing both phenotypic and functional characteristics of brain microvascular endothelium. CONCLUSION: This MSC-based differentiation approach provides a robust and physiologically relevant in vitro BBB model with potential applications in studying neurological disease mechanisms and screening therapeutic agents.