UBC Theses and Dissertations

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UBC Theses and Dissertations

Development of an ex vivo assay to examine transcription factors required for endothelial to hematopoietic transition Fentiman, Amanda Lorraine


Hematopoietic stem cells (HSCs) arise from a specialized population of endothelial cells, termed hemogenic endothelium (HE). HE was first identified in the murine dorsal aorta (DA) at embryonic day (E) 10.5. This process is known as endothelial to hematopoietic transition (EHT). Our aim was to identify genes crucial for the development of embryonic HSCs from the endothelium through the process of EHT. We accomplished this through the use of a transgenic mouse model which expresses GFP under the control of an intronic enhancer of the HE gene Runx1. The expression of this enhancer was combined with endothelial and hematopoietic markers to sort specialized endothelial cell populations from the DA of E10.5 embryos. RNA-seq data was generated from these sorted cell populations and 9 possible upstream transcription factors were identified. These candidate transcription factors include both known and novel regulators of EHT, including a novel regulator Meis1. Additionally, endothelial cell populations isolated from E9.5 embryos were sorted and cultured to develop an ex vivo co-culture assay that supports differentiation of pre-HE cells to HSCs. The effect of oxygen tension on endothelial and hematopoietic cell growth was investigated as a means to better support endothelial and hematopoietic cells. Oxygen transition during culture was found to significantly increase the proportion of wells which produced hematopoietic cells, and may aid in HE cell maintenance in culture. To examine the possibility of using this model to study regulators of EHT, the effect of blocking Notch signalling with a ɣ-secretase inhibitor added to our ex vivo culture was examined. Inhibition of Notch signalling did not significantly affect the generation of hematopoietic cells in our assay. Additionally, we evaluated the hematopoietic activity of tissue isolated from E9.5 Meis1fl/fl VeCre null embryos in this assay. No differences in hematopoietic cell generation were observed between wild-type and Meis1fl/fl VeCre null tissues in culture. Characterization of these embryos at E14.5 suggests that there exists a potential defect in Meis1fl/fl VeCre null embryos in later stages of embryonic hematopoiesis. This project contributes to the further understanding of genes important in EHT, while potentially defining transcriptional networks involved in HSC development.

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