A novel in vitro reporter system for blood stem cell activity

Why did we fund this project?

This award aims to establish an in vitro method to detect mouse haematopoietic stem cell (HSC) function replacing the need to confirm stem cell identity by transplanting cells into recipient mice.

Transplanting HSCs can be used to treat various cancers and diseases of the blood. It is increasingly being explored as a treatment for other conditions such as autoimmune disorders and viral infections. Historically, functional HSCs could not be sustained in vitro for experiments, but in 2019 a protocol was published enabling long term expansion of mouse HSCs. However, the majority of expanded cells are non-functional and require purification to understand which molecules regulate stem cell expansion. The current gold standard assay demonstrates functional HSC activity by transplanting cells into irradiated recipient mice and the previously described expansion protocol required between 300 and 400 recipient mice to confirm HSC functionality. Dr David Kent’s laboratory has established a method using the gene Fgd5 as a reporter to confirm HSC functionality without needing to use recipient mice, however, it currently requires expanded HSCs to be derived from a specific transgenic mouse strain.

To increase the method’s utility, the student will expand HSCs from the reporter mouse and perform molecular profiling to determine differences between HSC “positive” and “negative” cell populations. Genes identified during molecular profiling that distinguish between the two cell populations will then be tested in comparison to Fgd5 to identify a reporter that could be used across all mouse strains. The student will develop skills in cell culture, flow cytometry and transcriptomics.

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.

A rough estimate based on literature searches (2014-2019) suggests that between 30-50 research papers are published per year on mouse HSC expansion and each paper requires animal studies to validate the HSC content of cultures. As an example, the 2019 Nature paper referred to above used between 300-400 recipient mice. While all research papers are not to this scale, the number of mice easily reaches into the thousands every year and the publication of this new expansion method has already driven increased interest in this area due to its substantial clinical importance. Moreover, the ability to generate large numbers of HSCs could permit studies that are currently restricted (i.e., proteomics, metabolomics, epigenetic profiling), thereby eliminating the need for studies that would use typically use large numbers of donor mice (i.e., Cabezas-Wallscheid et al. 2017 used nearly 1000 mice to complete HSC proteomics). If HSCs could be expanded and re-purified, such assays would become much easier and require exponentially fewer mice.