Research project objectives:

Endothelial cells (ECs) are a critical component of a hematopoietic niche in the bone marrow, that protects hematopoietic stem cells (HSCs) from premature exhaustion. It is still not clear, however, which cells compose the niche and which are the conductors orchestrating the niche function. There are many indications that quiescent HSCs are harbored by arterioles or transitional capillaries containing type H endothelium, but direct imaging shows that quiescent HSCs are adherent to sinusoidal venules. How to explain such a discrepancy? Based on our initial data, we hypothesize that sinusoids contain distinct endothelial subpopulations, not recognized so far, that constitute HSC niche and regulate HSC outcomes. Our proposal is designed to characterize such putative subsets.

Research project methodology:

We will combine three components: i) comprehensive and unbiased transcriptome profiling at the single cell level; ii) breakthrough mouse models introduced recently, suitable to identify and target in-vivo the stringently defined HSCs (developed by prof. Irving Weissman group), and iii) brand-new type of transgenic mice, the model we are currently developing, to detect in-vivo the cells involved in cell-cell contact. Fluorescent reporter proteins will enable detection of HSCs or interacting cells. We will visualize the fluorescent cells in-situ in the cleared bone marrow tissue using confocal microscopy, phenotype them using flow cytometry or collect them by FACS-sorting to perform functional assays or transcriptome profiling. scRNA-seq will aim to measure cell-to- cell heterogeneity and to classify the responses triggered by the direct contact of stem cell with the niche cell. To evaluate the functional significance of these results, we will apply a conditional CreERT-lox system to generate mice for ablation of specific cells (by diphtheria toxin gene) and deletion of particular genes. To analyze the plasticity of ECs and their interactions with mesenchymal compartment, we will use ectopic bone organoids.

Impact of the research project:

The direct result of our application should be the development of cell type-specific mouse models allowing for in-situ detection of intercellular contacts and isolation of the cells involved. We hope to i) identify cells within the hematopoietic niche directly contacting the HSCs; ii) characterize molecular pathways mediating the interaction between HSCs and their niche; iii) shed light on origin and plasticity of ECs within the niche; iv) demonstrate the role of the mesenchymal compartment in aging of ECs. Finally, we want to understand whether endothelial cell subsets directly interacting with HSCs are intrinsically predefined or rather are induced by extrinsic signals from mesenchymal or hematopoietic compartments. Research on this interplay is in its infancy. The long-term goal of the current application is to clarify how distinct cell types that constitute specific hematopoietic niches regulate HSC fate decisions. As a composite outcome, we want to generate a comprehensive cellular and molecular atlas of hematopoietic niches. We anticipate that this dataset will drive next studies on modulation of hematopoietic niche
from a clinical perspective.
 

Pioneering nature of the research project:

The proposed tracing system is a thoroughly novel technology – no similar approach is currently available. We forecast that this technique may find an application in stem cell biology, but also in tumor immunology. Using these models we have a chance to provide data unavailable so far. Our results will be an important supplement of the already published single-cell expression data from mouse bone marrow, as they will characterize the functionally, not only phenotypically, defined cells. In longer perspective, identifying the HSC specific mechanisms, if they are also present in the human HSC niche, may lay the groundwork for the development of new clinical strategies for the mobilization and transplantation of HSCs.