Long-Term Hematopoietic Transplantation in Zebrafish Using Immune-Matched Marrow Cells.

Abstract 3535

Poster Board III-472

Transplantation of hematopoietic cells, tumors or any allogeneic tissue has been hindered in the zebrafish due to poor understanding of the functional major histocompatibility complex (MHC) genes and inability to immune-match donors and recipients. Despite this, the zebrafish has been used prominently for the study of hematopoiesis, due to the advantages of large scale forward and reverse genetic and chemical screening methodologies leading to the discovery of novel genetic mechanisms. We created a quantitative long-term hematopoietic reconstitution assay to assess hematopoietic stem cell (HSC) function in adult zebrafish. We identified a sublethal radiation dose of 25Gy which was optimal for hematopoietic reconstitution while minimizing the mortality presumably due to radiation damage of other organs. At 3 months post-transplant, primary and secondary recipients showed multi-lineage engraftment, as measured by flow cytometric analysis of GFP-expressing donor cells. Statistical analyses of limiting dilution data suggest that at least 1 out of 65,000 nucleated zebrafish marrow cells contain repopulating activity, consistent with mammalian HSC frequencies. We defined zebrafish haplotypes at the proposed core MHC locus on chromosome 19, testing the functional significance of these haplotypes by performing matched and mismatched hematopoietic transplants. Utilizing a single family for subsequent experiments, we identified the four parental MHC haplotypes by sequencing PCR products amplified for specific MHC genes. MHC-typed recipient fish were transplanted with whole kidney marrow cells from sibling donors, demonstrating that matching the donor and recipient MHC haplotypes at the chromosome 19 locus increases engraftment and percentage of donor chimerism in recipients compared to MHC-mismatched donors and recipients. At 3 months post-transplant, MHC-matched recipients had multilineage engraftment in 13 of 15 fish with mean donor chimerism of 47.86% (range 5.56- 93.44%) for myeloid cells and 10.51% (range 0.92-77.57%) for lymphoid cells. Engrafted animals receiving MHC-mismatched donor marrow (n= 4 of 6) had only 6.45% mean myeloid donor chimerism (range 4.58 to 8.58%) and 1.28% mean lymphoid donor chimerism (range 0.83 to 1.41%). This new assay allows long term HSCs to be distinguished from other hematopoietic progenitor cells and provides the first functional test of zebrafish MHC genes. Our data indicate the zebrafish MHC locus at chromosome 19 is functionally important for immune matching in the setting of transplantation. This method will facilitate MHC-matched long-term transplantation experiments in zebrafish that have previously not been possible, including competitive transplantation experiments with zebrafish mutants already identified in prior genetic screens, and long-term tumor transplantation assays.

Note: The first two authors contributed equally to this work.