In vitro transduction of HD and WAS patients’ derived BM or MPB CD34+ cells
MPB, CD7-APCR700, CD15-BV510
BM CD34+ cells isolated from HD were purchased from Lonza (2M-101C), while MPB G+P CD34+ cells isolated from healthy subjects were purchased from StemExpress (MLEGP34005C). Frozen BM CD34+ cells before GT from Pt2, Pt3, Pt4 and Pt9 and frozen MPB CD34+ cells before GT from Pt8, Pt9, Pt12 and Pt14 were thawed to perform in vitro transduction experiments. An aliquot of cells from both HD and MPB, before transduction, was labeled with:Mouse anti-human Lineage cocktail (anti-CD3/CD14/CD16/CD19/CD20/CD56)-BV510 (Clones: OKT3, M5E2, 3G8, HIB19, 2H7, HCD56; Biolegend, 348807), Verified Reactivity: Human; Application: Flow cytometric analysis of antibody surface-stained cells. Dilution: 1:10Mouse anti-human CD15-BV510 (Clone: W6D3; Biolegend, 323028), Verified Reactivity: Human; Application: Flow cytometric analysis of antibody surface-stained cells. Dilution: 1:50Mouse anti-human CD34-PB (Clone: 581; Biolegend, 343512), Verified Reactivity: Human; Reported Reactivity: Cynomolgus; Application: Flow cytometric analysis of antibody surface-stained cells. Dilution: 1:20Mouse anti-human CD38-PC5 (Clone: HIT2; Biolegend, 303508), Verified Reactivity: Human; Reported Reactivity: Chimpanzee, Horse, Cow; Application: Flow cytometric analysis of antibody surface-stained cells. Dilution: 1:50Mouse anti-human CD10-PC7 (Clone: HI10a; Biolegend, 312214), Verified Reactivity: Human, Cynomolgus, Rhesus; Reported Reactivity: African Green, Baboon, Capuchin monkey, Chimpanzee; Application: Flow cytometric analysis of antibody surface-stained cells. Dilution: 1:33Mouse anti-human CD7-APCR700 (Clone: M-T701; BD Biosciences, 659124), Verified Reactivity: Human; Application: Flow cytometry. Dilution: 1:20Mouse anti-human CD135-PE (Clone: BV10A4H2; Biolegend, 313306), Verified Reactivity: Human; Application: Flow cytometric analysis of antibody surface-stained cells. Dilution: 1:33Mouse anti-human CD45RA-APCH7 (Clone: HI100; Biolegend, 304128), Verified Reactivity: Human; Reported Reactivity: Chimpanzee; Application: Flow cytometric analysis of antibody surface-stained cells. Dilution: 1:20All the antibodies were purchased from Biolegend and BD Biosciences and they are well characterized and validated by providers.Here below the alternative gating strategies.Briefly, after gating on CD34+LIN- cells, we identify CD34All stained samples were acquired through BD Symphony A5 (BD Biosciences) cytofluorimeters after Rainbow beads (Spherotech) calibration Raw data were collected through DIVA software Version 8.0.2 and analyzed with FlowJo software Version 10.5.3 (BD Biosciences). The graphical output was generated through Prism v9.1.0 (GraphPad software). Technically validated results were always included in the analyses, and we did not apply any exclusion criteria for outliers.