Characterization of immune-matched hematopoietic transplantation in zebrafish

Abstract

Evaluating hematopoietic stem cell (HSC) function in vivo requires a long-term transplantation assay. Although zebrafish are a powerful model for discovering the genetics of hematopoiesis, hematopoietic transplantation approaches have been underdeveloped. Here we established a long-term reconstitution assay in adult zebrafish. Primary and secondary recipients showed multilineage engraftment at 3 months after transplantation. Limiting dilution data suggest that at least 1 in 65 000 zebrafish marrow cells contain repopulating activity, consistent with mammalian HSC frequencies. We defined zebrafish haplotypes at the proposed major histocompatibility complex locus on chromosome 19 and tested functional significance through hematopoietic transplantation. Matching donors and recipients dramatically increased engraftment and percentage donor chimerism compared with unmatched fish. These data constitute the first functional test of zebrafish histocompatibility genes, enabling the development of matched hematopoietic transplantations. This lays the foundation for competitive transplantation experiments with mutant zebrafish HSCs and chemicals to test for effects on engraftment, thereby providing a model for human hematopoietic diseases and treatments not previously available.

Figure 1

Transplantation of GFP-labeled transgenic whole kidney marrow shows long-term engraftment in adult zebrafish. (A) FACS analysis of control Tg(β-actin:GFP) WKM cells showing the forward scatter vs side scatter profile from a representative donor animal. The erythroid gate is marked in red, the myeloid gate is green, the precursor gate is black, and the lymphoid gate is blue. (B) Histograms for GFP expression of cells within the myeloid, lymphoid, and precursor lineage gates for a representative Tg(β-actin:GFP) donor fish. The percentage of GFP⁺ cells in each lineage gate is shown. (C) Forward scatter (FSC) vs side scatter (SSC) profile of marrow from an animal 3 months after transplantation with 500 × 10³ marrow cells showing full reconstitution with donor cells. (D) Histograms for GFP expression of cells within the myeloid, lymphoid, and precursor gates for a representative transplant recipient fish analyzed 3 months after transplantation showing multilineage engraftment with GFP⁺ donor cells. (E) Kaplan-Meier survival curves of adult zebrafish transplanted with 500 × 10³ whole kidney marrow cells after graded doses of total body irradiation. (F) Percentage GFP⁺ cells in the myeloid and lymphoid populations of control Tg(β-actin:GFP) animals (left), in myeloid cells of transplant recipients (middle), and in lymphoid cells of transplant recipients (right). Each diamond represents an individual animal. Each host was transplanted with 500 × 10³ WKM cells after exposure to 20, 25, or 30 Gy of total body irradiation. Percentage of GFP⁺ cells in the myeloid gate (middle) and lymphoid gate (right) at 90 days after transplantation is shown. Percentages plotted correspond to raw data numbers. Red lines indicate the lower threshold for successful myeloid (> 4%) and lymphoid (> 0.6%) engraftment as determined by negative control animals. (G) Percentage GFP⁺ cells in the myeloid and lymphoid populations of WKM from secondary transplant recipients. Each unique symbol represents an individual animal.

Figure 2

Limiting dilution analysis reveals that survival and engraftment are tightly linked. Data from Table 1 were graphed for each cell dose in an HSC limiting dilution transplantation experiment (bars) and the SE calculated and shown. Recipients were injected with increasing numbers of marrow cells, and a constant number of peripheral blood carrier cells (10⁵ per recipient). After 3 months, recipients were killed and the marrow was dissected for FACS analysis. GFP⁺ expression in the myeloid and lymphoid gates was used to determine donor engraftment. The percentage of animals surviving 90 days (A) and the percentage of animals engrafted (B) are shown. Transplanted marrow cell dose is depicted on the x-axis. The solid black line depicts the statistical fitted model.

Figure 3

Four haplotypes identified at the MHC locus on chromosome 19. (A) Haplotype A is found on Zv8 chromosome AB and is formed by alignment of 5 bacterial artificial chromosomes: AL672176, AL672164, AL672185, AL672151, and AL672216. Haplotype B is found on Zv8 chromosome 19 and is formed by alignment of 2 bacterial artificial chromosomes (BX927188 and BX510994) and 6 pieces of shotgun sequences from Zv8_scaffold2271. MHC class I U genes are in red, and flanking genes are in black. Haplotype A contains 4 MHC class I U genes and 18 flanking genes, whereas haplotype B contains 2 MHC class I U genes and 17 flanking genes. Both haplotypes are defined by the same flanking genes, such as col11a2, daxx, and flot1, in a region 350 kb in length. The megabase pair (Mb) positions under the haplotypes are from Ensembl Zebrafish genome browser. Haplotypes C and D are identified by direct sequencing of PCR amplified DNA sequences and are defined by the 2 MHC class I U genes (LOC751750 and mhc1uxa2) that independently segregated in the family of F1 siblings. (B) MHC genotypes for the wild-type AB male and Tg(β-actin:GFP) female parents are shown, as well as the 4 genotypes identified in their F1 progeny.

Figure 4

Increased percentage donor chimerism in MHC-matched transplant recipients. The percentage of GFP⁺ cells within the myeloid and lymphoid gates was plotted for each individual transplant recipient. Each recipient animal received 50 to 75 × 10³ WKM cells in addition to 2 × 10⁵ peripheral blood carrier cells. Each unique symbol represents a single transplant recipient. Sixteen recipients were evaluated 16 weeks after transplantation, including all the unmatched recipients. Five of the matched recipients were evaluated at 14 weeks. The red lines indicate the thresholds for myeloid (> 4%) and lymphoid (> 0.6%) engraftment. Mean percentage of GFP⁺ cells ± SEM is shown for the engrafted animals in each group. The level of donor chimerism in the engrafted matched recipients was significantly higher than for the engrafted unmatched recipients (P = .0002 and P = .05 for the myeloid and lymphoid gates, respectively).