Hematopoietic progenitor cells from healthy donors and FA‐A patients

Healthy donor CD34⁺ cells were provided by the Centro de Transfusiones de la Comunidad de Madrid from umbilical cord blood samples (CB) scheduled for discard, after written informed consent from the mothers. Mononuclear cells from pooled CBs were obtained by fractionation in Ficoll–Hypaque according to manufacturer's instructions (GE Healthcare). Purified CD34⁺ cells were obtained using the MACS CD34 Micro‐Bead kit (Miltenyi Biotec). Cells were grown in StemSpam (StemCell Technologies) supplemented with 1% GlutaMAX™ (Gibco), 1% penicillin/streptomycin solution (Gibco), 100 ng/ml SCF and Flt3‐ligand, and 20 ng/ml TPO and IL6 (all from EuroBiosciences).

A small number of mobilized peripheral blood (mPB) CD34⁺ cells from FA‐A patients that remained in cell collection bags and tubes from the CliniMACS^® System (Miltenyi Biotec), used for the collection (FANCOSTEM trial; {"type":"clinical-trial","attrs":{"text":"NCT02931071","term_id":"NCT02931071"}}NCT02931071) and the subsequent transduction of CD34⁺ cells with lentiviral vectors (LVs) (FANCOLEN Trial: {"type":"clinical-trial","attrs":{"text":"NCT03157804","term_id":"NCT03157804"}}NCT03157804), was used after the informed consent was signed and after the approval of the corresponding ethics committees. Cells were grown in StemSpam (StemCell Technologies) supplemented with 1% GlutaMAX™ (Gibco), 1% penicillin/streptomycin solution (Gibco), 100 ng/ml SCF and Flt3‐ ligand, 20 ng/ml TPO and IL6 (all from EuroBiosciences), 10 μg/ml anti‐TNF‐α (Enbrel‐Etanercept), and 1 mM N‐acetylcysteine (Pharmazam) under hypoxic conditions (5% of O₂).