Rock inhibitor y 27632

NPC43 cells were cultured in Roswell Park Memorial Institute 1 × 1640 medium (Gibco, Franklin Lakes, NJ, USA) with 10% fetal bovine serum (FBS, Gibco, USA), 0.2% 2 mM rock inhibitor Y-27632 (ENZO, Broomfield, CO, USA), and 1% antibiotic antimycotic (Gibco, USA). NPC43 cells were maintained in an incubator at 37 °C with 5% CO₂. The medium was changed every two days, and the cells were passaged when they reached 70% confluency.

Human astrocyte differentiation was performed as described by^{79 (link)} with minor adaptations. hNPCs were passaged with Accutase at 80% confluency and counted using a Neubauer improved cell counting chamber (Carl Roth, PK361). Cells were resuspended in astrocyte differentiation medium (50 % DMEM/F12-GlutaMAX, 50% Neurobasal supplemented with 1% B27 (17504044, Life Technologies), 0.5% N2, 0.5x GlutaMaxx (Thermo Fisher Scientific, 35050061), 0.5x non-essential amino acids, 0.02% beta-mercaptoethanol, 2.5 µg/ml human Insulin (Sigma, I3536-100MG), 5 ng/ml CNTF (Miltenyi Biotec, 130-096-337), 10 ng/ml BMP4 (Miltenyi Biotec, 130-111-165), 10 ng/ml EGF (Miltenyi Biotec, 130-093-825), 8 ng/ml FGF2 (Miltenyi Biotec, 130-093-839), 10 ng/ml Heregulin1/Neuregulin1b (Biomol, 97642.1) containing 10 µM ROCK inhibitor Y27632 (Enzo Life Sciences, ALX-270333-M005) and were seeded on 15 µg/mL poly-L-ornithine and 10 µg/mL laminin coated 6-well plates at a density of 500.000 cells/well. Astrocytes were differentiated in astrocyte differentiation medium at 37 °C, 7% CO₂, 21% O₂ for at least 35 days with daily medium changes. At 80 % confluency, astrocytes were passaged with Accutase and seeded on 15 µg/mL poly-L-ornithine and 10 µg/mL laminin coated plates.

The hUiPSCs line was developed from urine-derived cells as previously described [28 (link)]. Cells were routinely grown on Matrigel Matrix (Invitrogen) in mTeSR_1 medium (STEMCELL Technologies) at 37°C degree in an incubator with 5% CO₂ and 3% O₂. Cells were subcultured using STEMPRO ACCUTASE (Life Technologies) and ROCK-Inhibitor Y-27632 (Enzo Life Sciences).

iPSCs were dissociated into single cells with Accutase Enzyme Cell Detachment Medium (Innovative Cell Technologies). Cell suspensions were then passed through a pre-separation filter (Miltenyi Biotec) to remove cell aggregates and cell counts were obtained. Up to 2x10⁶ cells were resuspended in 100 μL of hESC medium supplemented with 5 μM ROCK inhibitor Y-27632 (Enzo Life Sciences) and incubated for 10 min at 4°C with 10 μL of anti-TRA-1-60-PE or anti-SSEA4-PE antibody (Miltenyi Biotec). Typically 2–3 wells of a 6-well plate containing the original reprogrammed colonies were used for the first round of sorting. After antibody labeling, the cells were washed with 2 mL of hESC medium and incubated for 15 min at 4°C with 20 μL of anti-PE magnetic microbeads (Miltenyi Biotec) in 80 μL of fresh hESC medium. After washing, cells were suspended in 0.5 mL fresh hESC medium supplemented with 5 μM Y-27632 and loaded onto LS separation columns (Miltenyi Biotec) and antibody labeled cells were retained under a magnetic field. After extensive washing of the column with hESC medium, antibody labeled cells were eluted in fresh hESC medium and directly plated for culture.

Footprint-free iPSCs were generated as previously described in our recent study^{26 (link)} by seeding of 5 × 10⁴ renal epithelial cells obtained from 100–200 ml urine of healthy donors per well of a 12-well plate coated with 0.1% gelatin. The reprogramming was performed by transfection with 0.5 µg self-replicating RNA (VEE-OKSiM-GFP RNA). The generated iPSC colonies were detached and seeded onto 0.5 µg/cm² vitronectin-coated (Thermo Fisher Scientific, Waltham, USA) tissue culture flasks. The cells were cultivated in Essential 8 (E8) medium (Thermo Fisher Scientific) at 37 °C and 5% CO₂ with daily medium changes and passaged every 4–6 days. After reaching confluence, iPSCs were washed with Dulbecco’s phosphate-buffered saline (DPBS, Thermo Fisher Scientific) and detached by 5 min incubation with DPBS containing 0.5 mM ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich, St. Louis, USA). After detachment, the EDTA solution was aspirated and the cells were rinsed with E8 medium. 2 × 10⁵ cells were seeded per well of vitronectin-coated 6-well plates in E8 medium containing 10 µg/ml ROCK inhibitor Y-27632 (Enzo Life Sciences, Lausen, Switzerland).

Tissue and biopsy collection were approved by the UK Research and Ethics Council (REC references 06/Q0505 and 11/LO/1522). Primary airway cells were isolated from routine airway endoscopy procedures and lung resections. All samples were transported on ice in a medium containing streptomycin (50 μg/mL), penicillin (50 IU/mL), and amphotericin B (1 μg/mL). Epithelial cells were isolated by explant expansion or by first digesting tissue overnight in 0.15% (w/v) pronase in DMEM at 4°C on a rotator. DMEM containing 10% fetal bovine serum (FBS) was then used to neutralize the pronase solution at a ratio of 2:1. Samples were then centrifuged at 300 g for 5 min to form a cell pellet before resuspension in epithelial growth medium containing 5 μM ROCK inhibitor Y-27632 (Enzo Life Sciences, Exeter, United Kingdom) and seeding into flasks containing a mitomycin C-treated 3T3-J2 feeder layer as previously described.^{20 (link),23 (link)}Primary human lung fibroblasts (a kind gift from Prof. Robin McAnulty; University College London, United Kingdom) were maintained in DMEM (Gibco, Hemel Hempstead, United Kingdom) containing 10% FBS and were used no later than passage 10.^{24 (link)}