Molecular Characterisation of Expanded Mouse Haematopoietic Stem Cells Using a Novel in Vitro Reporter Strategy

• Main Author: Che, Lok Chi James
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2020
• Subjects: Blood; Stem cells

Haematopoietic stem cells (HSCs) are responsible for the lifelong maintenance of the blood forming system which produces trillions of blood cells daily. They are able to achieve this because of two defining properties: 1) they can give rise to progeny which eventually form all of the blood cell types in an organism and 2) they can create equally potent daughter cells. This latter property of self-renewal has the potential to be harnessed to create unlimited numbers of HSCs outside the body, which would be highly beneficial to cellular and gene therapy. As a result, decades of research have focused on improving in vitro HSC expansion efficiency with most studies failing to expand functional HSCs in sufficiently large quantities. Recent efforts in mouse HSC biology achieved a more than 200-fold expansion of functional HSCs; however, single cell cultures in these conditions displayed a large amount of heterogeneity. Using a recently generated HSC reporter mouse, I devised a novel in vitro reporter strategy capable of reading out functional HSC activity in vitro and also discovered a previously unreported population of lymphoid cells marked by the reporter (Chapter 3). I showed that the in vitro reporter strategy could be used to screen for molecules that promote HSC expansion and could prospectively identify single-cell derived cultures that contained large numbers of functional HSCs (Chapter 4). This permitted us to undertake gene expression profiling to determine the molecular identity of expanded HSCs using RNA sequencing. Comparing the transcriptome of these cells and the secretome of these heterogeneous clonal cultures, I identified potentially novel regulators for promoting the expansion of HSCs (Chapter 5).