Ultra low adherent 6 well plates

The in vitro differentiation potential of iPSCs was assessed by their spontaneous differentiation into embryoid bodies (EBs). iPSCs were cultured in ultra-low adherent 6-well plates (Corning) for EB formation in Essential 6™ Medium. The medium was changed every 2–3 days, until day 20, when total RNA was extracted from EBs. Scorecard assay was performed by Thermo-Fisher Scientific on day 20-EBs compared to the corresponding iPSC clones. Scorecard values correspond to algorithm scores for the samples, showing upregulation or downregulation of the pluripotent (self-renewal), ectoderm, mesoderm and endoderm markers relative to a reference set of nine undifferentiated pluripotent stem cell lines.

Twenty four hours after transfection with indicated constructs, cells were subjected to analysis. For soft agar colony formation assay, 1 mL of 1% noble agar (Sigma‐Aldrich, St.Louis, MO, USA) in 40 °C culture medium was added to each well of a 6‐well plate and allowed to solidify in a cell culture hood for 30 min. Cells were suspended in 0.35% agar in a growth medium and 0.5 mL of the agar‐cell mixture was plated on top of a solid layer of 1% agar (5000 cells per well). Colonies were photographed and counted 12 days later. For sphere formation assay, cells were grown in FBS‐free DMEM‐F12 (Invitrogen, Carlsbad, CA, USA), supplemented with 20 ng mL⁻¹ epidermal growth factor (EGF) and bFGF and 2% B27 (Invitrogen), and plated at 1000 cells/well in ultra‐low adherent 6‐well plates (Corning, NY, USA). EGF and bFGF were added every 3 days. Two weeks later, spheres were photographed and counted. For transwell assay, 3 × 10⁴ cells in serum‐free growth medium were seeded in the upper wells of 24‐well invasion or migration chambers (Corning). The lower side of the chamber contained the growth medium with 10% FBS. After 24 h of seeding, the lower side cells of the chamber were fixed, stained, and counted.