Establishing a Novel High-Throughput Conditioning-Free HSC Transplant Model in Zebrafish

Abstract

Hematopoietic stem cell (HSC) transplantation is a widely successful treatment, but the risk of mortality remains. Preclinical modeling is critical to uncover the biological components that underlie poor patient outcomes. Zebrafish is a vertebrate animal with high genetic and cellular conservation with human hematopoiesis with many advantages to discover regulators of HSC transplant biology, such as high fecundity and short generation time. We developed a novel HSC transplant model using bloodless runx1 W84X mutant zebrafish which are devoid of endogenous HSCs. Most embryos die 8-12 days post fertilization (dpf) due to the absence of definitive hematopoiesis. We hypothesized that the empty HSC niche and lack of an immune system would prevent graft rejection, making robust HSC engraftment possible. We transplanted donor marrow ubiquitously expressing green fluorescent protein (ubi:GFP) via intravascular injection into runx1 homozygous mutants and heterozygotes at 2dpf. Sham-injected and uninjected embryos served as negative controls. We transplanted an average of 100-200 recipients per experimental day. Survival was significantly improved in transplanted runx1 mutants, with 64% surviving in the transplanted cohort compared to 5% in the sham-injected controls, suggesting HSC transplantation likely supplied these fish with a functional hematopoietic compartment critical for survival. Chimerism was quantified by flow cytometry 8 weeks post-transplant. Successful engraftment was defined as >5% GFP+ myeloid cells. Over 99% of animals meeting this criterion also showed robust multi-lineage engraftment. Over 70% of runx1 mutant recipients were engrafted compared to only 3% of the runx1 heterozygotes. The myeloid chimerism of engrafted mutant fish was 84% (+/-25%), while the engrafted heterozygous control had only 21% myeloid chimerism. Transplanted fish remain robustly engrafted >6 months post-transplant. The runx1 mutants supported HSC self-renewal, as the GFP+ marrow cells from primary recipients were able to robustly engraft secondary runx1 mutant hosts. We also demonstrated that competitive transplantation in runx1 mutants can be used to measure HSC frequency, a critical feature needed to functionally and quantitatively assess HSC potential following genetic or pharmacological perturbations. These data demonstrate that the runx1 mutant zebrafish is an advantageous HSC transplant host that allows quantification of long-term serially-repopulating true HSCs. The advantages of our zebrafish transplant model allow the real-time visualization of stem cell trafficking and homing in a healthy, uninjured niche and the ability to perform large-scale screens to identify drugs that modify engraftment. This will provide unprecedented insight into both the donor and host factors needed for robust HSC engraftment that will be helpful in improving human HSC graft outcomes.