Human Bone Marrow-Mesenchymal Stem Cells Differentiation Into Brain-Like Endothelial Cells|

Human pluripotent stem cells (hPSCs) have been frequently utilized to produce robust monolayers of brain microvascular endothelial cells (BMECs). During the differentiation process, hPSCs are to first differentiate into mesenchymal lineage cells. Accordingly, Mesenchymal stem cells obtained from human bone marrow (BM-MSCs) may offer a method for producing fully functioning human BMECs that may be utilized to construct the blood-brain barrier (BBB) for study aims. Looking through the literature, it was shown that BM-MSCs may differentiate into a range of cell types, endothelial cells (ECs) included. However, there is no strategy for the conversion of BM-MSCs into endothelial cells with brain characteristics, which precludes their wide applications. Therefore, we developed a new protocol for brain-like endothelial cells (BLECs) differentiation from BM-MSCs, inspired by embryologically developmental procedures and previously published iPSCs-BMECs protocols. To develop the differentiation protocol, we optimized the seeding densities of BM-MSCs and the components of the differentiation medium by using three different differentiation media: Endopan, EGM-2, and IMDM. Then, in order to enhance the development of BLECs, we looked into adding retinoic acid (RA) to the differentiation media at various concentrations. Aside from that, we examined the control of the hypoxic environment during endothelial cell differentiation by chemical HIF-1α regulators, cobalt chloride (CoCl2), and sodium sulfite (Na2SO3) to mimic the embryological developmental environment and observed significantly increased expression of brain endothelial cell markers. Different combinations of the basal media used to formulate the expansion and differentiation media were shown to impact how differentiated cells behaved, with IMDM found to favor BLEC differentiation over LG-DMEM. The effect of using animal-derived serum (fetal bovine serum-FBS) and synthetic serum, B27, during differentiation, was also tested, and FBS proved to be more effective than B27, in particular when the differentiation media was supplemented with CoCl2 and Na2SO3. The use of the IMDM medium in conjunction with the addition of 3 μM RA shortened the differentiation time of BM-MSCs into BLECs from 14 to 9 days. The addition of 200 μM CoCl2 for two days of differentiation followed by standard differentiation medium or the addition of 4 mM Na2SO3 throughout the differentiation period enhanced occludin, CD-31, ZO-1, and claudin-5 expressions in BLECs. In addition, we could prove the functionality of the BLECs by tube structure formation when cultured on Matrigel. In conclusion, we have provided a protocol for the differentiation of BM-MSCs into BLECs that can be used to construct fully functional, physiologically relevant, human cell-based BBB models for the study of brain-related diseases and the testing of various novel drugs for the treatment of neurological disorders