Refinement of hematopoietic cell transplantation using optical imaging in zebrafish

Hematopoietic stem cells (HSC) are self-renewing progenitor cells that give rise to all blood cells and immune cells. Despite the importance of these cells, many questions remain about their basic biology including:

• Where do transplanted HSCs migrate to following transplantation and in particular are there multiple niches?
• Is it important for HSC to establish in their niche within a particular time frame for successful engraftment?
• Do HSC differentiate in the primary niche or migrate to secondary sites of hematopoiesis (e.g. the spleen)?
• Are myeloid and lymphoid lineage signals detected by HSCs in different locations?

HSCs are very rare, making their study challenging. One way to address whether this cell compartment is affected under certain conditions (e.g. under inflammatory conditions) is to compare their capacity to engraft after transplantation in limit dilution assays. For this, animals are transplanted with decreasing numbers of putative HSCs and the survival of the animal is measured as the ultimate readout. Comparing survival between groups gives an indication on the fitness of the HSC in the original marrow and/or how it is affected by the recipient environment.

To use survival of the animal as a readout of successful engraftment carries a severe severity banding in the UK. Our aim is to develop alternative methods to study HSC biology based primarily on live optical imaging and using transgenic zebrafish lines in which a variety of cell lineages (including HSCs) are fluorophore tagged as donors and recipients which are either irradiated or bloodless (c-myb mutants). This approach will not only reduce the severity, thereby refining our protocols, but reduce the number of animals needed and allow higher data quality to be obtained. Our primary objective is to develop this refined protocol with a secondary objective to understand the effects of a donor and/or host inflammatory environment on HSC behavior.

• Further Funding: NC3Rs Studentship, Live imaging of mucosal and vascular inflammation in zebrafish in response to a high cholesterol diet, September 2013, £90,000
• Further Funding: NC3Rs Strategic Grant, Developing a model for the study of respiratory inflammation in the zebrafish, March 2015, £98,384