Fibroblast Reprogramming to iPSC

The CHM3 fibroblasts were seeded on day (D) −1 at a density of 2 × 10⁵ cells per 9.4 cm² in high glucose DMEM containing GlutaMAX and sodium pyruvate (Gibco) and supplemented with 10% FCS, 1% non-essential amino acids (Gibco), 1 mM L-ascorbic acid (Sigma-Aldrich, Saint Quentin Fallavier, France) and 10 ng/ml bFGF (Peprotech, Neuilly Sur Seine, France). On D0, cells were reprogrammed using the CytoTune-iPS 1.0 Reprogramming kit (Life Technologies, ThermoFisher Scientific) containing four Sendai virus-based reprogramming vectors expressing OCT4, SOX2, KLF4 and c-MYC at an MOI of 3. On D1, the media was refreshed, and, on D5, the transduced fibroblasts were passaged onto feeder cells (prepared as described^{28 (link)}) at a density of 10⁵ cells per 9.4 cm². On day 6, the media was changed to ES media: Knockout DMEM (Gibco) supplemented with 20% KO serum replacement (Gibco), 200 mM GlutaMAX (Gibco), 1% non-essential amino acids, 0.1% β-mercaptoethanol (Gibco), 1% penicillin-streptomycin (Gibco) and 10 ng/ml bFGF, and (until D11) 500 µM Valproic acid (Sigma-Aldrich). Resulting iPSC were mechanically passaged and subsequently adapted to feeder-free conditions on a 1/100 dilution Corning Matrigel HESC-qualified matrix (Analytic Lab, St Mathieu de Treviers, France) and in Essential (E) 8 media (Gibco). Passages were subsequently performed using Versene solution (Gibco).