Anti cd33

All samples were processed within 12 h from collection. Whole blood was lysed prior to staining using the RBC Lysis solution (Qiagen, Germany) according to manufacturer's specification. Immune populations were identified by staining with fluorophore-conjugated anti-CD11b, anti-CD33, anti-CD3, anti-HLA-DR, anti-CD45, anti-CD68, anti-CD206, anti-CD163, (BioLegend), anti-CD14, anti-CD15 (eBioscience) antibodies on ice for 30 min. Fluorescence data was acquired using BD Accuri C6 (BD Biosciences), Cyan and/or CytoFLEX (Beckman Coulter) cytometers. Normalised population statistics –including the median fluorescence intensities (MFI) were determined using FlowJo (BD Biosciences, formerly developed by FlowJo LLC).
Where indicated cell death was assessed by propidium iodide staining of cells after 72 h incubation with Gemtuzumab ozogamicin (Gift from Pfizer).

Briefly, cells were harvested and washed with PBS before staining and then incubated with antibodies for 30 min in the dark at 4 °C. For intracellular staining, surface-stained cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences, USA) for 20 min at 4 °C and then were stained with antibodies for 30 min at 4 °C. The following antibodies were used: anti-CD33 (BioLegend, #366622), anti-CD117 (BioLegend, #313232) and anti-CD68 (BD Biosciences, #564943). Subsequently, cells were finally washed with PBS and then analyzed by flow cytometry (BD Biosciences, USA). A total of 500,000 events were recorded and analyzed using FlowJo software, version 10.6.2 (Tree Star, USA). The full gating strategy is shown in Supplementary Fig. 6.

1 × 10⁶ healthy donor or GS-2 iPSCs were co-cultured with Op9 stromal cells (kindly provided by Prof. Dr. Juan Carlos Zúñiga-Pflücker, Sunnybrook Research Institute, Toronto, Canada [20 (link)]) in different HSC differentiation media for 8 days. A total of 7 clones (2 from healthy donor iPSCs, 5 derived from 3 different GS-2 patients) were tested for HSC differentiation potential. HSC differentiation media consisted of serum-free StemMACS HSC expansion medium with 1× hematopoietic expansion cocktail (STF: SCF, TPO, Flt3-ligand), 50 ng/mL BMP4 (Bone Morphogenic Protein 4 (R&D Systems, 314-BP-050), 5 μM ATRA (all-trans retinoic acid, Sigma-Aldrich, R2625) and 1 μM dexamethasone. Differentiated CD34+ HSC were further expanded with StemMACS HSC expansion medium with 1× STF for 1–2 weeks. Immunophenotype of the differentiated cells was measured using anti-CD43 (BioLegend, 343206), anti-CD45 (BD Biosciences, 560976), anti-CD34 (eBioscience, 17-0349-42), and anti-CD38 (BD Biosciences, 555459) antibodies. To assess the colony-forming capacity of HSCs differentiated from iPSC, the cells were cultured in Methocult H4434 classic (Stem Cell Technologies, 04444) for 10–14 days. After counting, colonies were picked and stained with anti-CD45, anti-CD16 (BioLegend, 302012), anti-CD14 (BD Biosciences, 555399), and anti-CD33 (BioLegend, 366608) to confirm myeloid differentiation.