Human iPSC-derived bone marrow organoids for AML engraftment and immunotherapy testing

Abstract Acute myeloid leukemia (AML) is associated with complex treatment challenges and persistently poor outcomes. Leukemic stem cells are supported by the bone marrow niche microenvironment that may support disease progression and therapy resistance. The molecular factors governing the complex intercellular and intracellular pathways remain poorly understood (Mendez-Ferrer et al., 2020). Current in vitro and in vivo xenograft models of AML immunotherapy have enormous limitations. We have previously developed a human bone marrow organoid (BMO) model system derived from induced pluripotent stem cells (iPSCs) (Frenz-Wiessner et al., 2024). This model recapitulates structural and molecular features of human bone marrow, including a vascular network, mesenchymal stromal populations and a multilineage hematopoietic compartment. We have also shown that iPSC-derived BMO support pre-clinical testing of targeted immunotherapy for Hodgkin's disease (Gottschlich et al., 2025). Here, we investigate whether BMOs can reliably be engrafted with primary AML cells from pediatric and adult patients as well as patient-derived xenograft (PDX) AML cells. We wish to make use of BMOs to assess targeted anti-leukemia immunotherapies, such as T-cell engaging antibodies (TCEs) and chimeric antigen receptor (CAR) T-cells. We seeded 5,000 donor cells per well in 96-well ultra-low attachment plates containing single organoids and monitored engraftment, survival and proliferation of leukemic cells were assessed over 15 days (read-outs were performed on day 2, 5, 10, and 15). Primary AML cells were pre-labeled with CellTrace, whereas PDX cells constitutively expressed mCherry, enabling localization within the organoid structure and quantification of proliferation kinetics over time. To enable tracking of introduced donor cells within the niche architecture, we generated BMOs from a gene-edited iPSC line harbouring an eGFP reporter integrated into the AAVS1 locus. Confocal z-stack imaging revealed AML cell engraftment into the BMOs and AML cell distribution throughout the three-dimensional niche architecture. Stable engraftment over the course of 15 days was shown for all tested donor cells. Flow cytometric analysis showed that both AML and PDX cells remained viable and considerably expanded, with a 5-fold increase of labeled, CD34+ cells by day 15 compared to the initial seeding. In contrast, AML cells maintained on murine MS5 feeder layers declined rapidly within the first two days, with a 10-fold decrease in cell number by day 15. In preliminary studies, we introduced CellTrace-labeled T-cells from a healthy donor into AML-engrafted BMOs. Confocal imaging confirmed successful T-cell engraftment. After addition of TCEs to the culture system, flow cytometry revealed a marked reduction of AML cells in TCE-treated conditions, but no significant reduction in non-targeting antibody controls, suggesting effective T-cell-mediated killing. Taken together, our data indicate that human iPSC-derived BMOs provide a supportive niche for the engraftment, survival, and proliferation of diverse AML subtypes, particularly those challenging to maintain in conventional in vitro systems. This platform may be valuable for testing immunotherapies within a complex human microenvironment and enabling interrogation of leukemia-niche interactions in future studies.