Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

TNFα and MMP1 in brain microvascular endothelial cells regulate blood-brain barrier dysfunction in psychotic disorders

“…The impervious nature of the human BBB is attributed primarily to the structure and function of BMECs, comprising physical, enzymatic, transport, and immunological processes that are vital for proper BBB functioning 3 . We had previously adapted and optimized a protocol for differentiating human iPSCs to generate BMEC-like cells with morphological and molecular marker expression (TJP1, OCLN, CLDN5, CD31 and SLC2A1), physical barrier properties with high transendothelial electrical resistance (TEER) ≥ 2000  x cm 2 , enzymatic transport activity for ABCB1 and ABCC1, and angiogenic potential in the sprouting assay that are characteristic features of BMECs [19][20][21] . Human iPSCs were grown in Essential 6 (E6) medium for four days to prime the cells for differentiation and then switched to human endothelial serum-free culture medium (hESFM) supplemented with 20ng/mL basic fibroblast growth factor (bFGF), 10 μM retinoic acid (RA), and B27 supplement to induce BMEC differentiation 19 .…”

“…We had previously adapted and optimized a protocol for differentiating human iPSCs to generate BMEC-like cells with morphological and molecular marker expression (TJP1, OCLN, CLDN5, CD31 and SLC2A1), physical barrier properties with high transendothelial electrical resistance (TEER) ≥ 2000  x cm 2 , enzymatic transport activity for ABCB1 and ABCC1, and angiogenic potential in the sprouting assay that are characteristic features of BMECs [19][20][21] . Human iPSCs were grown in Essential 6 (E6) medium for four days to prime the cells for differentiation and then switched to human endothelial serum-free culture medium (hESFM) supplemented with 20ng/mL basic fibroblast growth factor (bFGF), 10 μM retinoic acid (RA), and B27 supplement to induce BMEC differentiation 19 . On the sixth day, cells were sub-cultured onto a collagen IV and fibronectin 1 matrix hESFM plus B27, bFGF, and RA resulting in a purified BMEC population by day 8 21 (Fig.…”

“…Differentiation of iPSCs to BMECs. iPSCs were differentiated into BMEC-like cells following a protocol we have described earlier 19,20 . In brief, iPSCs were washed with DPBS and dissociated using Accutase for 5 min at 37°C.…”

“…While, there is no consensus on which models are best for interrogating BBB function, stem cell-based BBB models are promising avenues since they are able to recapitulate the intrinsic contribution of disease-and patient-specific genetic backgrounds 3,18 . We recently adapted and validated a method to model BBB function in vitro using human induced pluripotent stem cell (iPSC)-derived BMECs 19 , in order to gain new insights into the biology underlying SZ and BD.…”

Modeling Human Brain Tumors and the Microenvironment Using Induced Pluripotent Stem Cells

“…The key steps of endothelial induction and BMEC specification and purification were adopted later in the iBMEC protocols reviewed by Workman and Svendsen [79]. The successful protocols resulted in optimized iPSC seeding density [80], faster differentiation [81], accurate developmental course [82,83], simplified defined media [84], increased iBMEC purity [85], and capacity for cryopreservation [86,87]. However, limitations of the iBMEC protocols still exist and mainly include the generation of a homogeneous epithelial cell population.…”