Abstract Despite advancements in therapies for leukemia, relapses and refractory cases remain a significant clinical challenge largely due to persisted dormant leukemic stem cells (LSCs) and evade treatment. LSCs represent a rare cell population, making their isolation, culture, and expansion difficult and limiting our ability to study their functional properties. Developing an ex vivo model to culture and expand LSCs would enable detailed analysis of their role in drug resistance and relapses. Traditional 2D cultures fail to replicate the complexity of the bone marrow (BM) microenvironment, and animal models are limited by interspecies differences. To overcome these limitations, we are developing BM organoids derived from iPSCs, which are expected to better mimic the native BM architecture and function.In this study, we are establishing an ex vivo system for LSC culture and expansion. In previous studies, we demonstrated that mice with hematopoietic stem cell-specific (HSC-SCL-Cre-ERT) loss of the tumor suppressor Pten and activation of the oncogene beta-catenin develop leukemia by expansion of normal stem cells with transformation to LSCs. BM cells from HSC-SCL-Cre-ERT+ Ptenfl/fl dBCfl/wt mice were harvested and cultured in media supplemented with mTPO and mSCF and treated with 1 μM 4-OHT during serial passaging to induce recombination. Cultures were analyzed at multiple time points (3, 6, 13, and 18 days) using flow cytometry to quantify hematopoietic stem cells (HSCs: Lin- CD3- cKit+ Sca1+), LSCs (Lin- CD3+ cKit+), and blast cells (Lin- CD3+ cKit-). Preliminary results indicate that HSCs and blast cells increased by day 18 (62.1% and 0.65%, respectively) compared to day 13 (19.3% and 0.22%), whereas LSC frequency declined (0.14% vs. 0.25%). These findings suggest that LSC enrichment peaks around day 13, making this time point optimal for LSC culture. Ongoing work focuses long-term transformation of pre-leukemic HSCs to LSCs with subsequent expansion and maintenance of LSC phenotype and function. Concurrently, we are developing BM organoids. iPSCs were cultured to form embryoid bodies EBs), after which mesodermal induction and subsequent promotion of hemogenic endothelium Sprouting in hydrogel matrix was stimulated by supplementing different cytokines at different stages of culture. Finally, sprouted EBs were harvested and individually seeded into low-attachment 96-well plates. Mature BM organoids were analyzed using flowcytometry, immunofluorescence and PCR. Following organoid formation, LSCs will be engrafted to enable chemoresistance screening. Together, these approaches aim to establish a robust platform for LSC expansion to support immunocompetent functional assays, including co-culture studies, and develop new treatments targeting therapy-resistant leukemia. Citation Format: Sashi Kandel, Jacqelyn M. Nemechek, Tykeem Manor, John N. Maina, John M. Perry. Establishing ex vivo models for targeting leukemic stem cells to investigate relapse and refractory pediatric leukemias abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6594.
Kandel et al. (Fri,) studied this question.