Cenzano-Armendáriz, I. (Itziar)

Prósper-Cardoso, F.L. (Felipe Luis)

Saez, B. (Borja)

Haematopoiesis encompasses a complex process tightly regulated by mechanisms depending not only on haematopoietic stem cells (HSC) but also on the bone marrow (BM) microenvironment that provides the niche that nurtures HSCs. This HSC niche is a specialised microenvironment where a complex and dynamic network of interactions across a milieu of cell types maintains HSCs function. During the last years, it became progressively clearer that changes in the HSC niche are responsible for specific alterations of HSC behaviour. Thus, a comprehensive cell atlas of the BM microenvironment is required to elucidate the regulation of normal and malignant human haematopoiesis. Such a goal has been partially accomplished using the most sophisticated and advanced single-cell sequencing and imaging techniques developed in the last decades. These findings, along with the knowledge generated in previous experimental studies, have provided an exponential impact on the understanding of the astonishing complexity of this biological system, refining our simplistic paradigms. Despite these efforts, the field is still open to deeper molecular characterisation due to its inherent complexity and technical challenges. Here, we develop a tailored bioinformatic pipeline to effectively integrate and cluster single-cell RNA sequencing (scRNA-seq) datasets from public and proprietary sources. As a result, we delineated multiple intermediate cell states in the endothelial and mesenchymal BM compartments, each of which exhibited distinctive gene expression patterns. These cell states may be primed to perform specialized functions within the murine microenvironment and suggests a higher level of specialisation of the cellular circuits than previously anticipated. Furthermore, this deep characterisation allows inferring conserved features in human BM aspirates, meaning that the layers of microenvironmental regulation of haematopoiesis may also be shared between species. However, although these results and some recent evidence suggest that niche heterogeneity exists, many aspects of niche biology in humans remain poorly defined. Based on scRNA-seq data from BM orthopaedic surgeries, we dissect the human microenvironment at a thus far unreached molecular level and generate a comprehensive transcriptional fingerprint of distinct endothelium and mesenchymal subsets. Moreover, to our knowledge, the study of healthy ageing represents one of the first that describes the aged-associated transcriptional remodelling of the human BM microenvironment. Finally, to further expand the role of the microenvironment in regulating haematopoiesis, we examined the BM niche in the context of disease. Specifically, focusing on patients with MDS and SF3B1 mutations, we revealed MDS-related transcriptional alterations in the both the endothelial and mesenchymal compartments. These gained insights offer a roadmap for understanding the transformation of the BM microenvironment in disease and paving the way for the development of niche-targeted preventive and therapeutic strategies. Overall, the knowledge and methodology proposed in this thesis are a stepping-stone toward a comprehensive cell atlas of the human BM microenvironment in health, ageing and disease

Deep deconvolution of the haematopoietic stem cell regulatory microenvironment in health, ageing and malignant transformation.

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Universidad de Navarra