Glutamax and sodium pyruvate

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).

Neonatal FBs and KCs from two newborns were isolated in our laboratory from foreskins donated to the Presbyterian Hospital (Columbia University Institutional Review Board protocol AAAB2666). Adult FBs (Promocell) were from the abdominal skin of a 75-year-old female. After isolation, cells were subcultured for three passages before being used in the experiments. FBs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAX and sodium pyruvate (Gibco, #10569010) supplemented with 10% fetal bovine serum (Gibco, #16000069) and antibiotic-antimycotic (Gibco, #15240062). KCs were cultured in collagen I peptide–coated dishes (Corning, #354450) with Epilife medium (Gibco, #MEPI500CA) supplemented with S7 (Gibco, #S0175). The normal adult HDBECs were purchased from Promocell (#C-12225, used for prevascularization of hindlimb grafts), while the green fluorescent protein (GFP)–tagged HDBECs were purchased from Angioproteomie (#cAP-0005GFP, used in vitro to assess the development of the blood vasculature in our model). The HDBECs were grown on dishes coated with Quick Coating Solution (Angioproteomie, #cAP-01) using endothelial growth medium MV (Promocell, #C-22120) and expanded for three passages before being used in the experiments. FBs and HDBECs were dissociated using trypsin-EDTA 0.05% (Gibco, #25300054), while Accutase (Gibco, #A1110501) was used for KCs.

Hek293 cells (ATCC) were maintained in DMEM, high glucose with Glutamax and sodium pyruvate (ThermoFisher), 10% Fetal Bovine Serum, and supplemented with 1% penicillin/streptomycin. Cells were transfected using the Mirus TransIT 293 kit, according to manufacturer’s instructions. Briefly, 120,000 cells were seeded in a well of a 24-well plate 24 h pre-transfection. Cells were transfected with 750 ng plasmid pAF003 expressing SaCas9 driven by CMV promoter and 250 ng of linear DNA fragment expressing gRNAs driven by U6 promoter (125 ng each guide). Following expansion, cells were trypsinized, diluted and re-plated in 96-well plates at a dilution of approximately 1 cell per every 3 wells. Cells were visually monitored to ensure single cell colonies and expanded into 24-well plates. To determine editing, genomic DNA was isolated from clones using the Agencourt DNAdvance kit (Beckman Coulter) according to manufacturer’s instructions. Clones were screened by ddPCR and verified by Sanger sequencing.

U-2 OS cells (ATCC) were maintained in DMEM, high glucose with Glutamax and sodium pyruvate (ThermoFisher), 10% Fetal Bovine Serum, and supplemented with 1% penicillin/streptomycin. Cells were transfected by Lonza nucleofection using the 4D nucleofector system. Briefly, 250,000 cells were transfected with 1.5μg plasmid pAF003 expressing SaCas9 driven by CMV promoter and 500 ng of linear DNA fragment expressing gRNAs driven by U6 promoter (250 ng each guide). Cells were nucleofected using the SE kit and pulse code DN-100 and plated in 6-well plates. Cells were cultured for 3 days post-nucleofection and 3 transfection technical replicates were pooled together. Genomic DNA was isolated using the Agencourt DNAdvance kit (Beckman Coulter) according to manufacturer’s instructions.