Publication
The in vitro expansion of hematopoietic stem cells (HSC) for clinical applications is hampered by a rapid loss of HSC blood reconstitution capability in culture. While these rare cells can be stimulated to massively proliferate, cell divisions mostly result in differentiation caused by the lack of interactions with the native microenvironment, termed niche, in the bone marrow. Indeed, an increasing body of literature highlights the crucial role of instructive niche signals directing HSC fate in vivo. But how HSC fate, and in particular the delicate choice between self-renewal and differentiation divisions, is controlled by niche signaling cues remains unknown. Therefore, the goal of this thesis was to systematically characterize HSC fate decisions in vitro, and to utilize this knowledge to design artificial niches that maintain stemness. Differentiating HSCs first give rise to several transient populations of highly proliferative multipotent progenitors. Because reliable markers that distinguish HSCs from the earliest multipotent progenitors in vitro are lacking, it is currently not possible to discriminate between HSC self-renewal and commitment fate choices. The development and application of novel tools to probe HSC fate at the single cell level is therefore crucial, even more so because HSC populations are highly heterogeneous. In the first part of this thesis, a micro-engineered single cell analysis platform was employed to track in high-throughput the fate of individual HSCs by time-lapse microscopy. Single cell imaging revealed a surprising direct generation of megakaryocytes, cells that produce blood platelets, from phenotypic HSCs in the complete absence of a cell division. Megakaryocytic differentiation has previously been shown to occur very early in hematopoiesis and recent studies revealed the existence of a subpopulation of HSCs that is predetermined to undergo megakaryocytic differentiation. Our results suggest that some megakaryocyte progenitors may not be directly derived from HSCs but rather share the same cell surface marker repertoire such that they are indistinguishable from HSCs by state-of-the-art purification strategies. In order to discriminate between HSC self-renewal and commitment at single cell level, in the second part of this thesis gene expression signatures associated with the stem cell and multipotent progenitor cell states were established. Twelve differentially expressed genes marking the quiescent HSC state were identified, including four genes encoding cellcell interaction signals in the niche. Single cell multigene expression analysis performed on daughter cells, derived from single HSCs in serum-free culture, showed a rapid loss of the HSC identity with increasing number of cell divisions. In order to prevent such a dramatic loss of stemness in vitro, biomimetic microenvironments were engineered to display ligands of the newly identified niche components. Exposure of single HSCs to these artificial niches r