Ex Vivo Modeling of Leukemic Stem Cell Targets in Relapse/Refractory Pediatric Leukemia
Presenter Status
Post-Doctorial Research
Abstract Type
Translational Research
Primary Mentor or Principal Investigator
John M Perry
Presentation Type
Poster
Start Date
19-5-2026 11:00 AM
End Date
19-5-2026 12:00 PM
Abstract Text
Background:
Despite advancements in therapeutic strategies for leukemia, relapse and refractory (R/R) cases remain a significant clinical challenge. LSCs are believed to play a crucial role in disease recurrence, as they remain in a dormant state within the bone marrow and evade conventional treatments. Strategies to effectively target LSCs are still in their infancy, necessitating deeper investigation into their biology and the pathways driving therapy resistance and relapse. 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 relapse, as well as facilitate drug screening and identification of therapeutic targets.
Objectives/Goal: Our goal is 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
Methods/Design:
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 (Scl-Cre) 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. Bone marrow cells from Scl-Cre⁺ Pten^fl/fl dBC^fl/wt mice were harvested and cultured in StemSpan™ SFEM 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⁻).
Results:
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 LSCs decreased slightly (0.14% vs. 0.25%). Ongoing work focuses on optimizing 4-OHT dosage and culture duration while maintaining cell morphology.
Conclusion:
The findings suggest that LSC enrichment peaks around day 13, making this time point optimal for LSC culture and expansion.
Ex Vivo Modeling of Leukemic Stem Cell Targets in Relapse/Refractory Pediatric Leukemia
Background:
Despite advancements in therapeutic strategies for leukemia, relapse and refractory (R/R) cases remain a significant clinical challenge. LSCs are believed to play a crucial role in disease recurrence, as they remain in a dormant state within the bone marrow and evade conventional treatments. Strategies to effectively target LSCs are still in their infancy, necessitating deeper investigation into their biology and the pathways driving therapy resistance and relapse. 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 relapse, as well as facilitate drug screening and identification of therapeutic targets.
Objectives/Goal: Our goal is 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
Methods/Design:
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 (Scl-Cre) 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. Bone marrow cells from Scl-Cre⁺ Pten^fl/fl dBC^fl/wt mice were harvested and cultured in StemSpan™ SFEM 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⁻).
Results:
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 LSCs decreased slightly (0.14% vs. 0.25%). Ongoing work focuses on optimizing 4-OHT dosage and culture duration while maintaining cell morphology.
Conclusion:
The findings suggest that LSC enrichment peaks around day 13, making this time point optimal for LSC culture and expansion.
Comments
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Poster Board Number: 37