Rhlaminin 521

The H1 hESC line was purchased from WiCell Institute. The H1-iCas9 ESC line is a gift from Danwei Huangfu’s laboratory. The wild-type iPSC line was reprogrammed and well characterized in previous studies [44 (link), 56 (link), 57 (link)]. The study was approved by the KAUST Institutional Biosafety and Bioethics Committee (IBEC). All hPSCs were cultured in Essential 8 medium (ThermoFisher, Cat# A1517001) in rhLaminin-521 (ThermoFisher, Cat# A29249) coated wells with medium change daily. The peripheral blood mononuclear cells were isolated from the whole blood of a healthy donor via a standard Ficoll-Paque-based protocol and further cultured in StemSpan™-ACF Erythroid Expansion medium (STEMCELL Technology, Cat# 09860) for 13 days with medium change every 3 days to expand the erythroid progenitors. The erythroid progenitors were analyzed by FACS before CRISPR-Cas9 editing.

We used a previously well-characterized wild-type iPSC line^{26 ,33} (46XX) to perform homozygous knockout of the WAS gene using CRISPR-Cas9. To knock out the entire WAS gene, sgRNAs targeting upstream and downstream of the WAS gene with the following protospacer and protospacer-adjacent motif (PAM) sequences–5’-CCAAGCTCAGCCTAACGAGG-AGG-3’ (upstream) and 5’-GGATTTCACCCCCTAAGGGC-AGG-3’ (downstream)–were synthesized by in vitro transcription using the T7 MEGAshortscript kit (Thermo Fisher Scientific). Neon transfection system (Thermo Fisher Scientific; 1600 v/10 ms/3 pulses) was used for electroporating 200 k single cells with 50 pmol Cas9 protein and 25 pmol each sgRNA ribonucleoprotein complexes (RNPs) in buffer R (provided by Neon kit). After electroporation, iPSCs were cultured in Essential 8 medium (Thermo Fisher Scientific) with ROCKi (Abcam) on rhLaminin521 (Thermo Fisher Scientific) coated 24-well plates. The cells were dissociated by TrypleE after 48 h and subsequently 1000 single cells were seeded on laminin521 coated six-well plates. After 7 days half of the colony was picked for genotyping by PCR using the following primers: (F1, 5’-GTAGTAACCCTTCCGGACTA-3’ and R1, 5’-CGTAAAGGCGGATGAAGTAG-3’; F2, 5’-TCACACTCACCCAACAATCC-3’ and R2, 5’-TAACTGGGCCGACATTTCTC-3’, see Fig. 1d). The genotyping results were confirmed by Sanger sequencing.

Progenitor‐enriched cell populations were isolated from muscle samples as described previously (Vaughan & Lamia, 2019 ), and cultured on Matrigel (BD Biosciences, Franklin Lake, NJ, USA) at 39°C in Dulbecco's modified Eagle's medium (DMEM) high glucose with 1% P/S, supplemented with 20% fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA, USA) and 5 ng mL^–1 basic FGF (PeproTech, London, UK). Cells were trypsinized and passaged every 2−3 days. MPCs were differentiated using a protocol adapted from Hausman & Poulos (2005 (link)). In short, MPCs were plated on rh‐Laminin 521 (Life Technologies)‐coated wells (1000 cells mm^–2) and, when they reached 70% confluency, media was changed to DMEM high glucose with antibiotics supplemented with 10% FBS and 80 nm dexamethasone (Sigma‐Aldrich). Forty‐eight hours later or when cells reached full confluence (day 0), media was changed to DMEM high glucose with antibiotics supplemented with 2% FBS, 1% of insulin‐transferrin‐selenium (Life Technologies) and, in some experiments, human FGF21 (1−100 ng mL^–1; #100‐42; PeproTech), and maintained for up to 7 days. All experiments with cells were performed using triplicate wells.

Cells were differentiated as per a previously published protocol^{63 (link)} with minor changes. In short, MDPCs (5 × 10⁴) were seeded in triplicate on rh-Laminin 521 (0.5 µg/cm², Life Technologies)-coated 24-well or 96-well plates plates with growth media (DMEM supplemented with 10% FBS, 5 ng/ml bFGF and 1% PS). Once cells reached 70% confluency, media was changed to muscle proliferation media containing 80 nM dexamethasone (Sigma Aldrich), 10% FBS and 1% PS, until they reached full confluence, at which point (Day 0) media was replaced with serum free medium supplemented with 1% Insulin-Transferrin-Selenium (100×, Life Technologies) and 1% PS. Samples were taken on Days 0, 3 and 5 in TRIzol reagent and stored at – 80 °C, or cells on Day 5 were fixed in 4% PFA.