S249: EX VIVO HEMATOPOIETIC STEM CELL (HSC) EXPANSION USING BONE-LINING REINVIGORATING MESENCHYMAL STROMAL CELLS (RMSCS)

Abstract

Background: Bone marrow transplants (BMTs) have highlighted the Hematopoietic Stem Cell (HSC) potential to restore a new functional hematopoietic system in diseased recipients. However, a major roadblock for the clinical application and translational research on HSCs is our limited potential for ex vivo HSC expansion. Aims: To develop an approach for improved ex vivo HSC expansion using novel bone-lining reinvigorating Mesenchymal Stromal Cells (rMSC)-based culture system. Methods: Using a functional approach, we developed a robust pipeline for the fluorescence-activated cell sorting (FACS)-based isolation and ex vivo expansion of rMSCs from both murine and patient-individualized human samples. We tested the long-term HSC expansion potential in the rMSC-based co-culture system, phenotypically using FACS based proliferation analysis and functionally using limiting dilution and colony forming assays. Further, we performed transplantation experiments to study the hematopoietic reconstitution ability of the ex vivo expanded HSCs. Moreover, we expanded a single HSC over multiple cell divisions using our culture system to demonstrate bona fide HSC self-renewal potential of the rMSC-based system. Results: We propose a potent ex vivo HSC expansion system based on novel bone lining-derived reinvigorating Mesenchymal Stromal Cells (rMSCs). Both bulk- and single-HSCs expanded long-term using the rMSC co-culture system maintained phenotypic stemness over multiple cell differentiation cycles and possessed functional bone marrow reconstitution capabilities upon transplant. Notably, our rMSC co-culture system outperformed existing alternatives for HSC expansion including systems using stromal cells, non-cellular coating factors, or different medium compositions. Further, our results highlight the reliable isolation and robust culture of human rMSCs using our experimental strategy, and the potential to also utilize the rMSC co-culture system for the ex vivo expansion of human HSCs. Summary/Conclusion: We could demonstrate that our rMSC-based system for HSC expansion can play a pivotal role in research to reduce the number of mice used for ex vivo experiments. Moreover, our data also shows that the rMSC-based co-culture can be used for human HSC expansion, opening possibilities of applying this potent system for research on numerous diseases including immunodeficiencies and leukaemia.