Y 27632 dihydrochloride

Cord blood mononuclear cells (CBMCs) were directly obtained from the Cord Blood Bank of Seoul St. Mary’s Hospital. The Institutional Review Board (IRB) of the Catholic University of Korea, Seoul St. Mary’s Hospital approved this study. The reprogramming of CBMCs into iPSCs was induced using the Cytotune-iPS Sendai Reprogramming Kit (Life Technologies, Carlsbad, CA, USA). Briefly, CBMCs were seeded in a 24-well plate (3 × 10⁵ cells/well) with StemSpan™ medium (STEMCELL Technologies, Seattle, WA, USA). After addition of the viral components, the plate was centrifuged at 1160×g at 25 °C for 30 min and then incubated at 37 °C in 5% CO₂. On the next day, the cells were transferred to a 12-well plate coated with vitronectin (Life Technologies). The plate was centrifuged at 1160×g at 25 °C for 10 min, and Essential 8™ medium (Thermo Fisher Scientific, Waltham, MA, USA) was added at a 1:1 ratio. The cells were maintained in E8 medium until iPSC colonies were generated. The colonies were maintained in E8 medium (Thermo Fisher Scientific) on vitronectin-coated culture dishes. iPSCs were passaged every 3–4 days using Accutase Cell Detachment Solution (Global Cell Solutions, North Garden, VA) with Y-27632 dihydrochloride (R&D Systems, Minneapolis, MN, 10 µM). The medium was changed every day.

For embryoid body (EBs) formation, iPSCs were incubated with 1 mg/mL of collagenase I (Sigma-Aldrich) in PBS with Ca⁺⁺ and Mg⁺⁺ and 20% FBS for 20 min at 37 °C. Subsequently, cells were dissociated to form small aggregates with 0.05% Trypsin-EDTA (Gibco) and mechanical scrapping. Aggregates were plated in ultra-low attachment plates (Corning) with the following basal culture medium: StemPro®-34 SFM (Gibco), 1% glutamine, 1% P/S, 150 μg/mL of transferrin (Roche Life Sciences), 0.039 μL/mL of monothioglycerol (Sigma-Aldrich), 50 μg/mL ascorbic acid (Sigma-Aldrich) and 1 μM of Y-27632 dihydrochloride (R&D Systems). For spontaneous differentiation into the three embryonic germ layers, EBs were cultured in suspension for 7 days in basal culture medium. Then, EBs were transferred back to adherent plates and cultured for another 7 days for immunofluorescence^{40 (link)}.