Differentiation of hPSC-Derived Brain Microvascular Endothelial Cells

IMR90-4 and DF19-9-11T iPSCs and H9 hESCs were maintained between passages 26–42 on Matrigel (BD Biosciences) in mTeSR1 medium (STEMCELL Technologies) or on irradiated mouse embryonic fibroblasts (MEFs) in standard unconditioned medium (UM) as previously described18 (link). For differentiation, cells were passaged onto Matrigel in mTeSR1 medium for 2–3 days of expansion and then switched to unconditioned medium (UM) lacking bFGF for 6 days. Human endothelial serum-free medium (hESFM; Life Technologies) supplemented with 20 ng/mL bFGF (R&D Systems) and 1% platelet-poor plasma derived bovine serum (Biomedical Technologies, Inc.) was then added for an additional 2–4 days. All-trans RA (Sigma) was reconstituted in DMSO and included at concentrations of 1–10 μM depending on the experiment. Cells were then dissociated with Versene (Life Technologies) and plated onto 12-well tissue culture polystyrene plates or 1.12 cm² Transwell-Clear® permeable inserts (0.4 μm pore size) coated with a mixture of collagen IV (400 μg/mL; Sigma) and fibronectin (100 μg/mL; Sigma) in H₂O. Culture plates were incubated with the coating for at least 30 min at 37°C, while the inserts were incubated for a minimum of 4 h at 37°C. Resultant, purified hPSC-derived BMECs were then grown in EC medium for 24 h (with or without RA); in some experiments, primary pericytes or fibroblasts were co-cultured with BMECs during these 24 h (see description below). After this 24 h period, BMECs were continued as monoculture or co-cultured as described below. In our previous publication, we had utilized dispase for subculturing the BMECs18 (link), but non-enzymatic treatment of the BMECs with Versene results in less debris attached to the BMEC monolayer. We had also used hPSCs exclusively maintained on MEFs prior to differentiation18 (link), but in this study no noticeable differences in BBB properties were observed between hPSCs maintained on MEFs and hPSCs maintained under feeder-independent conditions and we now exclusively use hPSCs maintained in mTeSR1 on Matrigel.