Identification of a reporter strategy for functional haematopoietic stem cells during in vitro expansion

Haematopoietic stem cells (HSCs) are the peak of the haematopoietic hierarchy and can both self-renew and differentiate into all mature blood cells. HSC transplantation has long been used clinically for cancer treatment, gene therapy, and, increasingly, autoimmune conditions (multiple sclerosis) and viruses (HIV). Despite decades of research, efficient HSC expansion in vitro has been extremely difficult to achieve. A recent breakthrough in mouse HSC expansion (allowing up to 899-fold increases in HSC numbers over 28 days) is hugely significant, yet HSCs remain the minority of cells in the culture, and significant HSC heterogeneity exists – problematic when initiating cultures with single cells. Optimising the expansion protocol to increase HSC self-renewal divisions and HSC content of the cultures will allow molecular and cellular analysis of these expanded cells. Further understanding these cells will ideally enable application of refined expansion protocols to human HSCs. The current gold-standard for HSC identification is transplantation yet this is expensive and time-intensive. Replacing this with a universal HSC reporter strategy, able to identify HSCs to the same level of efficiency and removing the need for reporter mice for HSC identification would be extremely beneficial. This would open up reporter strategies to alternative mouse strains, including disease models, and also to human HSCs. In this thesis, I found that ESAM can replace the Fgd5+/ZsGreen reporter mouse as an efficient reporter for functional HSCs in culture. We used 28 day expansion screens to test candidate molecules able to predict HSC content of clones and identified the surface marker Siglec F as a candidate for distinguishing between the LT-HSC and progenitor populations. Finally, we tested the addition of novel compounds to expansion cultures to improve HSC self-renewal divisions, and found FSTL1 as a molecule that potentially promotes more HSCs to successfully expand in culture.