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International 3Rs Prize now open for applications. £30k prize (£2k personal award) for outstanding science with demonstrable 3Rs impacts.

NC3Rs | 20 Years: Pioneering Better Science
PhD Studentship

Development of novel tools to assay haematopoietic stem cell activity ex vivo

Portrait of Professor Marella de Bruijn and Associate Professor Adam Wilkinson

At a glance

In progress
Award date
January 2023 - January 2026
Grant amount
Principal investigator
Professor Marella de Bruijn
University of Oxford


  • Replacement


Why did we fund this project?

This award aims to develop an in vitro assay to quantify haematopoietic stem cell (HSC) activity replacing the need to assess HSC activity by transplanting cells into irradiated mice.

HSC transplantation can be used to treat various cancers and diseases of the blood. It is also increasingly being explored as a treatment for other conditions such as autoimmune disorders and viral infections. Finding suitable donors can be a barrier to treatment so research is ongoing to promote HSC expansion and activity, for example using genetic modification. Associate Professor Wilkinson (Co-Investigator) and colleagues have developed a protocol enabling long term expansion of mouse HSCs ex vivo but the activity of these cells needs to be confirmed for each batch of cells produced. The current gold standard assay to test patient or animal-derived HSC activity is to transplant cells into irradiated recipient mice with long term activity confirmed by serial transplantation. The student, with Professor Marella de Bruijn and Associate Professor Wilkinson, will now develop an in vitro assay of HSC activity to quantify both self-renewal potential and multi-lineage differentiation potential, the two key functional outputs of HSCs.

Application abstract

The overall goal of this PhD studentship is to develop a culture-based method to replace the use of in vivo haematopoietic stem cell (HSC) transplantation assays.

HSCs are a rare haematopoietic cell type that have unique capacities for self-renewal and multi-lineage haematopoietic differentiation. These stem cells thereby support life-long blood production. They are also used in the clinic, in stem cell transplantation therapy, to treat and cure patients suffering from a range of blood diseases such as leukaemias and anaemias. Stem cell transplantation is based on the ability of donor HSCs to stably reconstitute the entire blood system of the patient following bone marrow conditioning by radiation and/or chemotherapy. Although this therapy has been used for over 50 years, there are still hurdles in its application, e.g. due to limits on suitable bone marrow donors available and toxicity of the conditioning. Therefore, much research focuses on better understanding HSC biology to promote their expansion and fitness, with the ultimate aim to improve existing, and develop new, therapeutic strategies for blood-related disorders. Most of these studies are performed in mice.

Analogous to clinical stem cell transplantation, HSC transplantation is used in experimental research to test the functional activity of HSCs. This assay relies on intravenous injection of test (donor) HSCs into lethally-irradiated recipient mice, moderate procedure, and subsequent evaluation of donor blood system chimerism within the recipient. This is a widely used assay in experimental haematology because there are currently no alternative methods to assay the self-renewal and multi-lineage differentiation potentials of HSCs ex vivo.

Using a new culture method that stably induces HSC self-renewal ex vivo, we plan to establish a novel quantitative assay of HSC activity. The proposed assay will quantify both self-renewal potential and multi-lineage haematopoietic differentiation potential, the two key functional activities of the HSC. Based on our own work so far, we estimate this assay could reduce the number of mice used in HSC transplantation assays by 70% in our own laboratory. Given the number of mice in HSC transplantation assays globally, we conservatively estimate that the new culture assay could reduce animal usage on this moderate transplant procedure by ~10,000-20,000 mice per year. The proposed research project has two specific aims: (1) to establish a quantitative culture-based assay to assess HSC fitness throughout life; (2) to demonstrate that the method can be used to test the activity of genetically-modified HSCs.

Alongside these research aims, this proposal also aims to provide excellent training opportunities in state-of-the art technologies for a PhD student, with an emphasis on experimental design, research integrity and reproducibility, which are all critical to the 3Rs. Finally, in order to maximise the 3Rs impact of this assay we will disseminate our findings to the research community through publication and presentation at local, national and international meetings (NC3R and field specific), protocol publication, organisation of workshops, and through other relevant communication channels.