A novel in vitro reporter system for blood stem cell activity

Blood stem cells (or Haematopoietic Stem Cells, HSCs) are the fundamental component of regenerative medicine applications involving the blood and immune system. HSC transplantation has a long history in the fields of cancer medicine and gene therapy and is increasingly being explored in a wide array of diseases including viral infections (HIV) and autoimmunity (multiple sclerosis). However, despite significant efforts and investment, researchers have largely failed to maintain fully functional HSCs for substantial periods of time. Recent advances in mouse HSC expansion have put researchers on the cusp of breaking through this decades-old barrier (Wilkinson et al., Nature 2019). The 28-day protocol possesses incredible expansion capacity (~200-fold) of functional HSCs, but the HSCs still represent the vast minority of cells and substantial variability exists between single cell expansion cultures. Further optimisation will enhance HSC expansion capacity, permit molecular and cellular analyses of purified expanded HSCs, and lead to critical improvements in human HSC expansion. This is an area of rapid development with the system already being utilised worldwide. Current gold standard functional assays to precisely identify 'true' HSCs demand in vivo HSC transplantation meaning they are extremely mouse intensive with single manuscripts regularly involving hundreds of recipient animals to demonstrate functional activity. This creates an urgent need for more robust in vitro assays to detect functional HSCs.

This PhD project aims to validate such an in vitro system and will strive to make it universally applicable across mouse strains and, ideally, translate its utility to the human blood stem cell system. Broadly speaking, this project will proceed in two phases. The first is to validate the in vitro reporter system and perform molecular profiling on HSC-containing cultures to both improve our understanding of expanded HSCs and to identify candidate molecules to replace the reporter gene (Fgd5). The second stage is to functionally validate these candidate markers for their ability to replace, or imporve upon, Fgd5 expression, thereby setting the stage for translation of the strategy to human blood stem cell expansion systems.