FACS and HSPC Culture

Bone marrow mononuclear cells were stained for FACS after thawing. HSPCs were identified using the following surface markers: Lin⁻CD11c⁻CD16⁻CD34⁺, CD38⁻/CD45RA⁻ (Supplementary Fig. S1). We defined (t-)MN blasts from both first and second diagnoses based on diagnostic immunophenotyping data if available. In most cases, these blasts were CD33, CD38, and/or CD34 positive. ALL blasts from the first diagnosis were defined based on diagnostic immunophenotyping data if available (mostly these were CD10, CD19, or CD7 positive).

Blasts and HSPCs were purified on an SH800S Cell Sorter (Sony, RRID:SCR_018066). First, blasts were sorted in bulk for DNA isolation, after which HSPCs were index sorted in a flat-bottom, 384-well plate prepared with 75 μL HSPC culture medium per well. HSPC culture medium consisted of StemSpan SFEM medium (STEMCELL Technologies) supplemented with SCF (100 ng/mL), FLT3 ligand (100 ng/mL), IL6 (20 ng/mL), IL3 (10 ng/mL), TPO (50 ng/mL), UM729 (500 nmol/L), and Stemregenin (750 nmol/L).

For five samples (UPN001DX2, UPN023DX1, UPN002DX1, UPN005DX1, and UPN004DX1), the obtained sample was depleted for monocytic, pro–T cell, or pro–B cell blasts (marked by anti-CD14, CD7, and CD10, respectively) using the EasySep anti-APC kit, following the manufacturer's instructions. After blast deletion, we plated mesenchymal stromal cells (MSC) and sorted HSPCs following the same procedure as with all other samples.

HSPCs were cultured for 4 to 7 weeks at 37°C in 5% CO₂ before collection. MSCs were cultured from a fraction of bone marrow cells by plating bulk cells in 12-well culture dishes with DMEM-F12 medium (Gibco) supplemented with 10% FBS. The medium was refreshed every other day to remove nonadherent cells, and MSCs could be harvested when confluent (after approximately 2 to 3 weeks).