Stemflex basal medium

Reprogramming of patient and parent-derived fibroblasts (NG1406-1 and NG1406-4) was performed via episomal vectors – pCXLEhOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL (Sgourdou et al., 2017 (link)). Human iPSCs were cultured as previously described (Sousa et al., 2017 (link)). Briefly, cells were seeded on Matrigel-coated culture plates (Cat: #356234, Corning, 1:60) and maintained in StemFlex Basal medium (Thermo Fisher Scientific; #A3349201) or Essential 8 Medium (Thermo Fisher Scientific, #A2858501). Cells were typically passaged with EDTA (0.5 mM) at room temperature (RT). After 3–5 min of incubation, the EDTA solution was removed and cells were gently detached from the dish with a small volume of medium, generating clumps of six to eight cells. In standard conditions (37°C, 5% CO₂), iPSC colonies typically grow within 4–5 days.

The iPS cells, generated as previously described through episomal reprogramming (Sousa et al., 2017 (link)), were maintained in StemFlex Basal medium (Thermo Fisher Scientific, #A3349201) or Essential 8™ medium (Thermo Fisher Scientific, #A2858501) on Matrigel-coated culture plates (1:60, Corning, #356234). To preserve pluripotency properties, iPS cells were passaged every 5–6 days at a 1:6–1:8 ratio. To induce colonies detachment, cells were incubated at room temperature (RT) with EDTA (0.5 mM) for 3–5 min. After EDTA removal, culture vessels were rinsed with cell media to collect small clumps of cells. Finally, iPS cells were plated according to the optimal split ratio in a new Matrigel-coated culture plate.

Human iPSCs (male WTC11 background, Coriell Catalog (Cat.) No. GM25256) were cultured in StemFlex Basal Medium (Gibco; Cat. No. A33493-01) on BioLite Cell Culture Treated Dishes (Thermo Fisher Scientific; assorted Cat. Nos.) coated with Growth Factor Reduced, Phenol Red-Free, LDEV-Free Matrigel Basement Membrane Matrix (Corning; Cat. No. 356231) diluted 1:100 in Knockout DMEM (GIBCO/Thermo Fisher Scientific; Cat. No. 10829-018). StemFlex was replaced every other day or every day once 50% confluent. When 70–80% confluent, cells were passaged by aspirating media, washing with DPBS (Gibco; Cat. No. 14190-144), incubating with StemPro Accutase Cell Dissociation Reagent (GIBCO/Thermo Fisher Scientific; Cat. No. A11105-01) at 37 °C for 7 min, diluting Accutase 1:5 in StemFlex, collecting cells in conicals, centrifuging at 220g for 5 min, aspirating supernatant, resuspending cell pellet in StemFlex supplemented with 10 nM Y-27632 dihydrochloride ROCK inhibitor (Tocris; Cat. No. 125410), counting and plating onto Matrigel-coated plates at the desired number. Human iPSC studies at the University of California, San Francisco were approved by the Human Gamete, Embryo and Stem Cell Research Committee.