The effect of Poly I:C induced inflammation on hematopoietic stem and progenitor cell behaviour in the zebrafish hematopoietic transplant model

Abstract

Hematopoietic stem cells are a small but significant population of cells fundamental for generating and maintaining the hematopoietic system. These cells are used in the treatment of cancer and auto-immune patients. Studies in mammals suggest that inflammation and infection can modulate the biology of these cells, affecting their location, self-renewal capacity and directing differentiation. The aim of this work was to study the effect of repeated stimulation on the hematopoietic stem and progenitor (HSPCs) population in zebrafish (Danio rerio) to benefit from the live imaging potential of this model organism. It was hypothesised that early post-transplant HSPC behaviour (e.g. lodgement in the niche, self-renewal, mobilisation and differentiation) could be observed and would be indicative of the success or failure of HCT. Double transgenic Tg (cd41:GFP; lysc:dsRed) donors, in which HSPCs express green fluorescent protein (GFP+) and myeloid cells express red fluorescent protein (dsRed+) were used. HSPCs were sorted from donor whole kidney marrow (WKM) and transplanted into irradiated optically-transparent recipients, which were then imaged using wide-field microscopy, individually tracked for survival and hematopoietic reconstitution was assessed after 28 days by flow cytometry. Indeed, initial experiments showed that early observations of cells in the WKM correlated with hematopoietic recovery and survival of recipients, although the strength of the correlation was not sufficiently powerful for predicting recipient outcome.

This refinement of the HCT protocol lead to the potentiality of studying the behaviour of HSPCs in the context of inflammation. Inflammation was initiated with repeated intra-peritoneal injections of the viral mimic Polyinosinic:polycytidylic acid (poly I:C) in either the donor or recipient prior to transplant. Poly I:C injection of donors prior to transplant causes HSPCs to colonise the recipient WKM at a greater rate than HSPCs from sham (PBS-injected) donors. However, this did not appear to affect recipient survival or WKM reconstitution at 28 days. Poly I:C injection of recipients prior to transplant did not affect early post-transplant repopulation of the WKM, myelopoiesis, recipient survival, or WKM reconstitution at 28 days. Future work will use competitive transplants to confirm these findings and will explore alternative inflammation models. Furthermore, the confounding factor of irradiation-caused inflammation will be mitigated by transplanting HSPCs into optically-transparent bloodless recipients. Overall, this thesis has demonstrated that the zebrafish can provide valuable in vivo data for studying HSPC behaviour in the recipient post-transplant.