The life-long production of blood cells is enabled by hematopoietic stem and progenitor cells (HSPC) residing in the bone marrow. An understanding of the genes that control how HSPCs work to sustain the continued production of blood cells will enable new therapies to expand them for life-saving transplantation therapies. The Musashi gene family of RNA binding proteins (RBP) has been implicated to control stem cell function in a variety of adult tissues (e.g. brain and gut) and emerging evidence in mice shows Musashi-2 (Msi2) to be an essential positive regulator of HSPCs, yet no work to date has tested its function in the human system. Here I show that MSI2 is highly expressed in cord blood (CB) HSPCs and that knockdown of MSI2 impairs HSC repopulation in xenotransplanted mice. Conversely, MSI2 overexpression yields multiple pro-self-renewal phenotypes, including 17-fold expansion of short-term repopulating cells as readout at an early post-transplant time point, and upon ex vivo culture, a net 23-fold expansion of long-term hematopoietic stem cells (LT-HSC). I further found that MSI2 overexpression post-transcriptionally downregulates the aryl hydrocarbon signalling pathway, which has been shown when repressed by a small molecule antagonist, greatly supports the expansion of human HSCs. Altogether, this work highlights RBPs and post-transcriptional control to have an integral role in controlling human HSC self-renewal. For the second part of my thesis, I re-explored the role of Msi2 in the mouse hematopoietic system using a newly generated tamoxifen inducible Msi2 knockout mouse. Using this approach I found Msi2 deletion significantly restricted repopulation in competitively transplanted mice and that this effect was due to depletion of early types of multipotent progenitors and not due to a loss of LT-HSCs. As there is currently a discrepancy as to which cells Msi2 precisely regulates in the mouse hematopoietic system, my work provides support that MPPs are the most affected cell type. Altogether, my thesis provides novel insight in to the role and mechanism of action of Musashi-2 in both human and mouse HSPCs.

Doctor of Philosophy (PhD)

Thesis