N2b27 medium

H9 human ESCs purchased from WiCell Research institute were cultured on mitomycin C-inactivated mouse embryonic fibroblasts (MEFs) or matrigel (BD Biosciences) (Liu et al., 2012 (link)). Human VECs were cultured in EGM-2 (Lonza) medium supplemented with 50 ng/mL VEGF (HumanZyme), 10 μmol/L SB431542 (Selleck) and 20 ng/mL FGF2 (Joint Protein Central, JPC). Human VSMCs were cultured in N2B27 medium (1:1 (v/v) of DMEM/F12 medium (Gibco) and Neurobasal medium (Gibco) supplemented with 2% B27 (Gibco), 1% N2 (Gibco), 55 μmol/L β-mercaptoethanol (Gibco) and 1% penicillin/streptomycin (Gibco)) supplemented with 10 ng/mL PDGF-BB (Peprotech). Human MSCs were cultured in 90% α-MEM medium (Gibco) supplemented with 10% fetal bovine serum (Ausbian, VS500T), 1% penicillin/streptomycin (Gibco), and 1 ng/mL FGF2 (JPC).

Unless specified, all cell culture products were from ThermoFisher Scientific (Gibco). Murine E14 Tg2a ESCs (BayGenomics) cells were passaged using 0.05% Trypsin-0.01% EDTA solution under standard feeder-free conditions on gelatinized tissue culture dishes in mouse ESC media: knockout DMEM supplemented with 15% ES cell-qualified fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 1 × non-essential amino acids, 50 μM β-mercaptoethanol, and 500 U/ml of bacterially expressed in-house hLIF. For reprogramming experiments standard N2B27 medium (Gibco) was used, supplemented with 500 U/ml hLIF, 3 μM GSK3 inhibitor CHIR99021 (Axon), and 1 μM MEK inhibitor PD0325901 (Axon), and 3 μg/ml doxycycline. Neural differentiation was performed as described previously with minor changes [21 (link), 22 (link)].

In vitro mesodermal lineage-derived vascular SMC differentiation from hESCs was performed using a previously reported protocol with minor modification^{33 (link)}. Briefly, dissociated hESCs were seeded onto Matrigel-coated 6-well plates at 400,000 cells per well in mTeSR1 supplemented with 5 μM Y-27632 for 24 h (day 0). At day 1, the medium was replaced with N2/B27 medium (1:1 of DMEM/F12 and Neurobasal medium [Gibco] with B27 and N2 supplements [Thermo Fisher Scientific] and 0.1% β-mercaptoethanol) supplemented with 8 μM CHIR99021 and 25 ng/ml human BMP4 (R&D), which was maintained for 3 days. On days 4 and 5, the medium was replaced with fresh N2/B27 medium supplemented with 10 ng/ml PDGF-BB (Peprotech) and 2 ng/ml Activin-A (Peprotech). Typically, mesodermal vascular SMCs (platelet-derived growth factor receptor-β [PDGFRB]⁺SM22⁺) show up from day 6 onward.

mESCs at a density of 500 cells/μL were loaded into the device with 2% AF-PEG hydrogel. The PEG hydrogel was supplemented with 100 μg/mL laminin (Invitrogen) and optionally with 1 nM of ZZ-domain for protein immobilization conditions. For co-culture condition, SNLP cells were cultured in the side compartment at a density of 0, 600 or 1200 cells/μL in 1.5% W-PEG supplemented with 100 μM RGD. Reservoir was filled with the N2B27 medium supplemented with 1% Knockout Serum Replacement (KSR) (Gibco). For the soluble LIF gradient condition, cells were exposed to 0, 10 or 20 ng/mL Fc-LIF from the source reservoir. For the tethered LIF gradient, hydrogel was locally treated from the source reservoir with 120 ng/mL Fc- for 0, 4 or 8 hours and then extensively washed with culture medium. Cells were cultured for 4 days and media reservoirs were renewed at day 2. At the end of the culture, cells were fixed with 4% PFA (Gibco) and stained with DAPI (Sigma-Aldrich) and Alexa635-phalloidin (Invitrogen).

Embryonic stem cell lines were isolated from the inner cell mass (ICM) of E3.5 blastula‐stage embryos obtained after interbreeding of Rbm14^+/− male and female mice. The embryos were flushed from the uteruses of the pregnant female mice and plated on the dishes coated with fibronectin (Millipore) at 37°C, 5% CO₂ in N2B27 medium (Gibco) supplemented with GlutaMAX‐I (2 mmol/L; Gibco), beta‐mercaptoethanol (beta‐ME, 0.01 mmol/L; Gibco), leucocyte inhibitory factor (LIF, 20 ng/mL; R&D systems), CHIR99021 (3 μmol/L; R&D systems) and PD0325901 (1 μmol/L; R&D systems). The outgrowths of the embryos formed bowl‐like ESC colonies in less than a week. The ESC colonies were then picked and propagated by passaging every 2 days.

iPSC differentiation into motor neurons was performed as previously described (55,56). Briefly, iPSCs were dissociated with Accutase and plated at a density of 100,000 cells/cm² in mTESR1 media substituted with a 10 µM ROCK inhibitor (DNSK International, Y-27632). The next day, media was replaced with N2B27 medium (50% DMEM:F12, 50% Neurobasal, supplemented with NEAA, Glutamax, N2 and B27; Gibco) supplemented with SB431542 (10 µM, DNSK International), LDN-193189 (100 nM, DNSK International), Retinoic Acid (1 µM RA, Sigma) and Smoothened-Agonist (1 µM SAG, DNSK International) and fed daily with the same media for 6 days. On day 7, media was switched to N2B27 supplemented with 1 µM RA, 1 µM SAG, 5 µM DAPT (DNSK International) and 4 µM SU5402 (DNSK International) and fed daily with the same media for 7 days. Cell were then dissociated using TrypLE Express (Gibco) supplemented with DNase I (Worthington), plated onto pre-coated Matrigel-coated surfaces (BD Biosciences) and cultured in Neurobasal medium supplemented with NEAA, Glutamax, N2, B27, Ascorbic acid (0.2 µg/mL) and BDNF, CNTF and GDNF (10 ng/mL, R&D systems). For imaging analysis, motor neurons (MNs) were seeded on pre-plated mouse glia cells.