Chir99021

The hiCFs were induced according to a previously published protocol^{68 (link)}. A culture of hiPSCs grown to 80% confluence in PSCeasyII medium was first exposed to RPMI + B27 medium supplemented with 4 μM CHIR99021 (Selleck, S2924) for 48 hr. After the initial treatment, CHIR99021 was removed, and the cells were cultured in RPMI + B27 medium for another 24 hr. On day 3, 5 μM Wnt pathway inhibitor IWR-1 (Selleck, S7086) was added to the medium. On day 5, the cells were cultured in the RPMI + B27 medium. On day 6, the cells were dissociated using Accutase (Thermo Fisher Scientific, A1110501) and centrifuged at 200 × g for 3 min. The cells were then resuspended in advanced DMEM/F-12 medium (Thermo Fisher Scientific, 12634010) supplemented with 1% GlutaMax (Thermo Fisher Scientific, 35050061), 5 μM CHIR99021, 2 μM retinoid acid (Sigma, R2625), 5 μM Y-27632 (ROCK inhibitor), and 1% FBS (Gibco). Subsequently, the cells were plated onto Matrigel-coated cell culture dishes at a density of 2 × 10⁴ per cm². On day 7, the medium was replaced with advanced DMEM/F-12 medium supplemented with 1% GlutaMax, 5 μM CHIR99021, and 2 μM retinoid acid. On day 9, the medium was replaced with advanced DMEM/F-12 medium supplemented with 1% GlutaMax. On day 11, the cells were dissociated using Accutase, centrifuged at 200 × g for 3 min, resuspended in the medium with advanced DMEM/F-12 medium supplemented with 1% GlutaMax and 2 μM SB431542 (Selleck, S1067), and seeded onto Matrigel-coated cell culture dishes at a split ratio ranging from 1:3 to 1:6, with the medium changed every other day. Upon 90–100% confluence, the cells were dissociated using Accutase, centrifuged, resuspended in PromoCell Fibroblast Growth Medium (PromoCell, C-23025) supplemented with 20 ng/mL of bFGF (STEMCELL Technologies, 78003) and 10 μM SB431542, and then seeded onto a Matrigel-coated cell culture dish at a density of 1 × 10⁴ cells per cm² for culturing for 4 days, with the medium changed every other day. Subsequently, the resultant hiCFs were passaged and seeded again onto a Matrigel-coated cell culture dish at a density of 1 × 10⁴ cells per cm² and cultured in PromoCell Fibroblast Growth Medium (PromoCell, C-23025) containing 20 ng/mL of bFGF and 10 μM SB431542, for two days. The resultant hiCFs were kept in PromoCell Fibroblast Growth Medium (PromoCell, C-23025) containing 10 μM SB431542, with the medium changed every other day until used for the experiment.

hPSCs maintained on a Matrigel-coated surface in mTeSR1 were dissociated into single cells with Accutase (Life Technologies) at 37°C for 5 min and then seeded onto a Matrigel-coated cell culture dish at 50,000 cell/cm² in mTeSR1 supplemented with 5 μM ROCK inhibitor Y-27632 (Selleckchem) (day −3) for 24 hr. Cells were then cultured in mTeSR1, changed daily. At day 0, cells were treated with 6–10 μM CHIR99021 (Selleckchem) for 2 days in LaSR basal medium, which consists of Advanced DMEM/F12, 2.5 mM GlutaMAX, and 60 μg/ml ascorbic acid (Sigma, A8960). After 2 days, CHIR99021-containing medium was aspirated and cells were maintained in LaSR basal medium without CHIR99021 for 3–4 additional days. PD0325901 was purchased from Tocris Bioscience.

Human iPSCs [iPS(IMR90)-4 (72 (link)), iPS-DF 19-9-11T (73 (link)), and hESCs (H9) (29 (link))] were maintained on Matrigel (Corning)–coated surfaces in mTeSR1 (STEMCELL Technologies), as previously described (74 (link)). Before differentiation, hPSCs were singularized with Accutase (Innovative Cell Technologies) and plated onto Matrigel-coated plates at a density between 25 × 10³ and 50 × 10³ cells/cm² in mTeSR1 supplemented with 10 μM Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Selleckchem). hPSCs were expanded in mTeSR1 for 3 days. For differentiation on defined substrates, singularized hPSCs were plated onto the vitronectin-coated (Thermo Fisher Scientific) or Synthemax-coated (Corning) surface at a density between 25 × 10³ and 50 × 10³ cells/cm² in mTeSR1 supplemented with 10 μM ROCK inhibitor Y-27632 (Selleckchem). To initiate differentiation at day 0, cells were treated with 6 μM CHIR99021 (Selleckchem) in DeSR1: DMEM/Ham’s F12 (Thermo Fisher Scientific), 1× MEM-NEAA (Thermo Fisher Scientific), 0.5× GlutaMAX (Thermo Fisher Scientific), and 0.1 mM β-mercaptoethanol (Sigma). After 24 hours, the medium was changed to DeSR2: DeSR1 plus 1× B27 (Thermo Fisher Scientific) every day for another 5 days. At day 6, the medium was switched to hECSR1: hESFM (Thermo Fisher Scientific) supplemented with bFGF (20 ng/ml), 10 μM RA, and 1× B27. After 2 days of culture in hECSR1 medium, day 8 cells were dissociated with Accutase and plated at 1 × 10⁶ cells/cm² in hECSR1 onto 48-well tissue culture plates or 1.12-cm² Transwell-Clear permeable inserts (0.4 μm pore size) coated with Matrigel (100 μg/ml). For cells differentiated on vitronectin or Synthemax substrates, cells were dissociated with Accutase and plated at 1 × 10⁶ cells/cm² in hECSR1 onto 48-well tissue culture plates or 1.12-cm² Transwell-Clear permeable inserts (0.4 μm pore size) coated with human placenta–derived collagen IV (400 μg/ml)/human plasma–derived fibronectin (100 μg/ml). At day 9, the medium was changed to hECSR2 (hECSR1 without RA or bFGF) for longer-term maintenance. Y-27632 was added to increase the attachment (75 (link)) of cells differentiated in the absence of RA and was permitted to ensure confluent monolayer formation and confirm that the reduction in TEER was not only a result of incomplete monolayer formation.