Vitronectin

hPSCs (WA09, passages 20–24; 58–80; DS1, passages 30–35; DS2U, passages 35–4018 (link)) were maintained under feeder free conditions by coating with vitronectin (Life technology) or matrigel (BD Biosciences). For 3-4 days of maintenance in E8 medium (Life technology), hPSCs were dissociated by using EDTA (Lonza, 1 mL /well) for 1-2 minutes in 37 degrees, and reseeded at the density of 1 × 10⁵ cells per well of a 6-well plate. For neural differentiation, hPSCs were detached by dispase (Life technology) or EDTA (Lonza) to form embryoid bodies (EBs), and then cultured in neural induction medium (NIM) as previously described11 (link): 500 ml of NIM contains 5 ml of N2 supplement, 5 ml of NEAA, and 490 ml of DMEM/F12. After floating for 7 days, EBs were attached on vitronectin coated surfaces. Rosette structures can be observed at d10–16. At d16, rosette colonies were detached by a 1-ml pipette manually. Non-neuroepithelial colonies can be removed at this stage. Neurospheres were continuous floated in NIM, and then dissociated by TrypLE (Life technology) and plated on vitronectin (Life technology) and poly-l-ornithine (Sigma) pre-coated coverslips for further neuronal differentiation.

The WTC-11 GCaMP6f hiPSC line was used in all experiments (Dr. Bruce Conklin, The Gladstone Institutes, San Francisco, CA, USA). hiPSCs were maintained on 10 cm² dishes coated with 5 μg/mL vitronectin (Thermo Fisher, Waltham, MA, USA) in a cell culture incubator (37 °C, 5% CO₂). hiPSCs were maintained in Essential 8 (E8) medium (Gibco, Waltham, MA, USA) and passaged every 4–5 days at 80% confluency with versene (0.5 M EDTA (Fisher, Waltham, MA, USA) and 1.1 mM D-glucose (MilliporeSigma, Cleveland, OH, USA) in DPBS without calcium and magnesium (Gibco)) onto new vitronectin-coated plates.

The TkDN4-M (4M) hiPSCs line was a kind gift from Dr. M Ohtsu at The Institute of Medical Science, The University of Tokyo. 4M cells were cultured as previously described with minor modifications [23 (link)]. They were maintained on mitomycin C (MMC; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)-treated SNL feeder cells in hiPSCs medium (DMEM/Ham`s F12 (FUJIFILM Wako) supplemented with 20% Knockout Serum Replacement (KSR; GIBCO BRL, Palo Alto, CA, USA), 1x MEM nonessential amino acids (FUJIFILM Wako), 0.5x penicillin streptomycin(PS; FUJIFILM Wako), 55μM 2-melcaptoethanol (2ME Gibco) and 7.5μg/ml basic fibroblast growth factor (FGF2; Peprotech, Rocky Hill, NJ, USA). For passage, 4M colonies were detached using CTK solution, chipped by pipetting, and seeded onto MMC-treated SNL feeder (ECACC, Salisbury, UK) in hiPSCs medium once a week. The 15M63 hiPSCs line was kindly provided from the Center for iPS Cell Research and Application of Kyoto University (Kyoto, Japan) and maintained on vitronectin (Invitrogen, CA, USA) coated dishes in StemFit. For passage, 15M63 was dissociated with accutase (Innovative Cell Technologies, San Diego, USA) by pipetting and seeded on vitronectin coated dishes in StemFit (Ajinomoto, Tokyo, Japan) supplemented with 10μM Y27632 (Cayman Chemical, Ann Arbor, MI, USA).

100 μg/μL of collagen IV (BD, UK), collagen I (BD, UK), fibronectin (Sigma, UK), vitronectin (Invitrogen, UK), or laminin (Sigma, UK) was preplated to Ibidi μ-Slide VI 0.4 Luer slides for 1 hour at 37°C. The Ibidi slide was then connected to the flow system as described above. The slide was washed for 2 min with 1% BSA in DPBS. A total of 4 × 10⁵ LO-EPC in 1.5 mL were perfused at a shear stress of 0.7 dyn/cm² for 4 min. An additional 2 min wash was applied to remove the nonadherent cells. Cells were subsequently fixed with 4% paraformaldehyde for 15 min at room temperature, washed twice with PBS, and stained with Hoechst dye (1 μg/mL) for 30 min. The number of adherent LO-EPC was imaged, counted, and expressed as adherent cells per square millimetre.