Aneuploidy is a hallmark of cancer, yet in most cellular contexts it compromises proliferation and developmental fitness. In childhood B-cell acute lymphoblastic leukemia (cB-ALL), hyperdiploidy represents the most frequent cytogenetic abnormality, accounting for ~40% of cases. Converging evidence from monozygotic twin studies and neonatal blood analyses indicates that hyperdiploid clones arise in utero from early hematopoietic stem/progenitor cells (HSPCs), likely through abnormal mitotic events. However, the functional impact of hyperdiploidy on fetal hematopoiesis and its contribution to leukemogenesis remain poorly understood.

To model the two proposed routes of hyperdiploidy emergence in cB-ALL, chromosome missegregation and cytokinesis failure, we transiently treated human fetal liver-derived CD34⁺ HSPCs (hFL-HSPCs) with reversine (MPS1 inhibition) or cytochalasin D (actin polymerization inhibition), respectively. Hyperdiploidy induction was validated by metaphase spreads and single-cell whole-genome sequencing. Functional consequences were assessed using long-term liquid cultures, colony-forming unit (CFU) assays, immunophenotypic differentiation analyses, and intra-bone marrow (BM) transplantation into sublethally irradiated NSG mice.

Although hyperdiploidy was efficiently induced, hyperdiploid hFL-HSPCs displayed impaired proliferative fitness, increased early cell-cycle arrest and modest apoptosis. In vitro, hyperdiploid cells were rapidly outcompeted by isogenic euploid counterparts, leading to restoration of a diploid modal karyotype over time. A transient ploidy-reduction process was observed, with near-tetraploid cells progressively shifting toward lower hyperdiploid states. Notably, hyperdiploidy delayed hematopoietic differentiation, as evidenced by prolonged CD34 expression and increased secondary CFU potential, indicating extended progenitor-like activity. In vivo, treated hFL-HSPCs retained full BM engraftment capacity. Importantly, rare hyperdiploid clones (~5%) persisted long-term in primary recipients, displaying non-random chromosomal gains commonly observed in hyperdiploid cB-ALL, including +10, +18 and +21. Despite their persistence and partial karyotypic resemblance to patient leukemic clones, these cells failed to induce leukemia, even upon secondary transplantation.

Our findings support a two-step model of leukemogenesis in hyperdiploid cB-ALL, in which prenatal mitotic errors generate preleukemic hyperdiploid clones that are tolerated during fetal hematopoiesis but require additional postnatal genetic events for transformation. This human fetal HSPC model provides a physiologically relevant platform to dissect early aneuploidy-driven events and identify cooperating mechanisms underlying childhood leukemia.

PID2022-142966OB-I00 and INVES211226MOLI.