Lymphoprep

Bone marrow (BM) or peripheral blood (PB) samples from patients with AML were collected after written informed consent in accordance with the Declaration of Helsinki. Mononuclear cells (MNCs) were isolated using Lymphoprep (GE Healthcare Bio-Sciences AB) and samples were viably frozen. Clinical and genetic characteristics of the NPM1-mutated samples from patients were characterized by flow cytometry are summarized in supplemental Table 1. In addition, BM samples from healthy donors were prepared in the same way. The study was approved by a regional ethics committee.

Cord blood was collected with written informed consent from the mother before delivery at the Japanese Red Cross Cord Blood Bank. Cord blood experiments were approved by the ethical committees of the Japanese Red Cross Cord Blood Bank and National Center for Global Health and Medicine. The sample was processed using Lymphoprep (GE Healthcare) density gradient medium to isolate mononuclear cells. CD34 enrichment was performed by magnetic cell separation using a MACS CD34 Microbead Kit (Miltenyi Biotec). CD34⁺ enriched cells were frozen until use. Frozen cells were thawed in vials in a 37°C water bath and transferred to a 15 mL tube. RPMI-1640 medium containing 10% fetal calf serum (FCS) was slowly added to the suspension while gently swirling to fill the tube. The suspension was centrifuged at 200 × g for 15 min at room temperature. Supernatants were aspirated and the wash step was repeated. An antibody cocktail (50 μL of phosphate-buffered saline (PBS) containing 2% FCS plus 10 μL of anti-CD34-FITC, 2 μL of anti-CD38-PerCP-Cy5.5, 5 μL of anti-CD90-PE-Cy7, and 10 μL of anti-CD45RA-PE) was added to the suspension and kept on ice for 30 min. Cells were washed once with PBS containing 2% FCS and resuspended in PBS containing 2% FCS supplemented with 0.1% propidium iodide (PI). Cells were sorted into StemSpan SFEM-I using a FACSAria IIIu instrument.