Stemreginin 1

Umbilical cord blood (UCB) was collected with written consent from full‐term deliveries at the Women's Health Unit, Lyell McEwin Hospital (Adelaide, South Australia), or the Department of Obstetrics and Gynaecology, Medical University of Graz (Austria), with institutional review board approval (IRB approval: 31‐322 ex 18/19; HREC/20/WCHN/65). Samples were processed using a Ficoll‐Paque (GE Healthcare) density gradient to isolate mononuclear cells (800 g, room temperature, 30 min, deceleration off), followed by red cell lysis (ammonium–chloride–potassium lysing buffer) to remove remaining red blood cells. CD34 enrichment was performed by magnetic cell separation using CD34 Microbead kit (Miltenyi Biotech). Alternatively, purified CD34⁺ cells from cord blood were purchased from Lonza. CD34⁺ cells with a purity above 90% were either cryopreserved or directly cultured in serum‐free stem cell retention media (StemSpan SFEMII, Stemcell Technologies, Vancouver, BC, Canada) supplemented with human recombinant SCF, TPO, FLT3, IL‐6 (all 20 ng/ml), UM729 (1.75 µM) and StemReginin 1 (SR1, 300 nM). All cytokines and SR1 were purchased from Peprotech (Rocky Hill, NJ, USA), whereas UM729 was purchased from Stemcell Technologies.

Primary AML cells for scRNA-seq were cultured on hMSC feeders as described above in the following media: SFEMII (StemCell Technologies), 1 μM UM729 (StemCell Technologies), 750 nM StemReginin 1 (StemCell Technologies) supplemented with 150 ng/mL SCF, 100 ng/mL TPO, 10 ng/mL IL-3, 10 ng/mL G-CSF (Perpro tech). After 1 passage (1 week) in culture cells were treated for 24 h with 10 μM CBFβi or 0.1% DMSO in the absence of UM729 and StemReginin 1. After treatment cells were sorted for CD45 using magnetic beads (Miltenyi Biotec). Cells were loaded on a Chromium Single Cell Instrument (10X Genomics), to recover 5000 single cells. Library generation was performed using the Chromium single cell 3′ library and gel bead kit v3.1. Illumina sequencing was performed on a NovaSeq 6000 S1 run in paired-end mode for 150 cycles at a depth of 20000 reads per cell.