Functional Characterisation of Endomucin in Haematopoietic Stem Cells

Haematopoietic stem cells (HSCs) maintain the homeostasis of the blood system through proliferation and differentiation and they self-renew in order to preserve the stem cell pool and prevent its exhaustion. The isolation of a homogeneous population of HSCs is not only crucial for the study of differentiation processes, but also for the understanding of a variety of diseases such as leukaemia. The current method for isolating HSCs is based on the use of cell surface markers in flow cytometry, but we lack specific markers to isolate very pure blood stem cells. Therefore, new cell surface proteins with an
expression pattern restricted to HSCs need to be further investigated. Such a potential marker is Endomucin (EMCN), a transmembrane protein expressed both in murine and human HSC populations. The aim of this research project was to characterise EMCN as a potential marker for HSCs and to investigate its function in haematopoiesis.
First, the expression of EMCN was characterised in murine haematopoiesis. Flow
cytometry analysis showed that the highest levels of EMCN can be detected in long-term repopulating HSCs (LT-HSCs), while it was absent in their more differentiated progeny.
Transplantation assays further demonstrated that using EMCN in addition to "standard" HSC markers enriches LT-HSCs with increased self-renewal capacity. Secondly, functional and transcriptional analyses indicate that EMCN+ LT-HSCs tend to be more quiescent compared to more activated EMCN􀀀 LT-HSCs. In conclusion, the addition of EMCN to previously defined HSC markers allows to enrich for dormant HSCs with increased self-renewal capacity.
The function of EMCN in haematopoiesis was examined using a conventional EMCN knock-out mouse model. Loss of Emcn had no effect on the transcriptome of LT-HSCs or immune-phenotype of blood populations during steady-state haematopoiesis. In addition, Emcn deficiency in HSCs did not influence their bone marrow reconstitution capacity after transplantation. Stimulation of dormant LT-HSCs to proliferate had a mild, but significant effect on the maintenance of LT-HSCs in the bone marrow. Taken together, EMCN surface expression in HSCs is dispensable during steady-state haematopoiesis,
but may be involved in the maintenance of dormant LT-HSCs. Interestingly, Emcn deficiency in endothelial cells (ECs) affected long-term repopulation of wildtype HSCs.
In conclusion, the data presented in this thesis show that EMCN is a promising marker to be used in addition to previously established markers for the isolation of LT-HSCs using flow cytometry. Though negligible during steady-state haematopoiesis, EMCN may be involved in maintaining the quiescence of LT-HSCs in the bone marrow. EMCN in ECs is required for properBMrepopulation during repopulation experiments, indicating that surface expression in ECs promotes HSC homing or maintenance after transplantation.