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Abstract

The bone marrow niche is a complex organ system, which has classically been studied for its role as the seedbed of hematopoiesis. Recent research has highlighted the complexity of the bone marrow at the cellular level unravelling Mesenchymal Stem Cells (MSC) as critical supporting cells for Hematopoietic Stem Cells (HSCs). However, little is known about the role of MSCs in the physiological and pathophysiological states of the bone marrow. Therefore, in the first part of this thesis, we studied the engagement of MSCs in the stress response of the murine bone marrow niche over time. In the second part of this thesis, the HSC expansion potential of MSCs was studied and translated into a human system in order to overcome the limitations of HSC transplantation for regenerative medicine. Taken together, my thesis deals with the “Characterization of Bone Marrow Mesenchymal Stem Cell Niche Dynamics upon Stress, with Focus on Clinical Translation”.

Inflammation is a key component in the complex biological response of the body to harmful stimuli. In the context of the bone marrow, inflammation is an overarching process central to most if not all forms of stress challenges and disease settings. Current research in the field is focused on understanding the response of HSCs to inflammation. While such research provides a descriptive understanding of the HSC niche with its stromal compartment, it falls short in translating this into functional applications confounded by a single marker approach of classifying the niche cell diversity. In this thesis, we utilized the power of single-cell sequencing coupled with a functional proliferation readout to investigate inflammation response overtime of an unbiased bone marrow niche. Further, we identified and described a novel inflammation-responding MSC (iMSC) population which, unlike its stromal counterpart, responded directly and dynamically to IFNα stimulation. We showed that iMSCs uniquely produce key inflammation cytokines and secreted factors at the onset of the IFNα response while they markedly downregulated extracellular matrix (ECM) factors and, thus, facilitated niche remodelling at a late time point. Using ligand-receptor mapping, we further identified pivotal inflammation-specific interactions between iMSCs and HSCs within the bone marrow. Hence, we concluded the first part of my thesis with a novel iMSC signature with direct application in unravelling inflammation dynamics of the bone marrow niche. The proposed iMSC signature has the potential to significantly contribute to our understanding of bone marrow niche perturbations in disease settings, like leukemia and immunodeficiencies.

Bone marrow transplants (BMTs) have highlighted the HSC potential to restore a new functional hematopoietic system in diseased recipients. However, a major roadblock for this scientific breakthrough is our limited potential for ex vivo HSC expansion. In the second part of my thesis, we propose a potent ex vivo HSC expansion system based on bone lining-derived reinvigorating Mesenchymal Stem Cells (rMSCs). Using a functional approach, we created 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. The bulk and single-HSC long-term expansion using rMSCs maintained phenotypic stemness over multiple cell differentiation cycles and provided 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, using our donor-individualized experimental strategy, we isolated and analyzed both plastic-adherent stromal cells (PASCs) and rMSCs from the same patient sample highlighting more favorable gene expression profiles in rMSCs compared to PASCs for HSC expansion. Thus, with the second part of this thesis, we showed 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 patient-individualized rMSC-based HSC expansion system could potentially be used for curative and personalized gene therapy in numerous diseases.

In summary, with my thesis, I deciphered functional subsets of murine stromal cells regulating the in vivo inflammatory response of the bone marrow and promoting ex vivo HSC expansion. Our functional approach of stromal cell characterization revised the current understanding of the bone marrow niche and holds the promise for clinical breakthrough.