Congenic interval of CD45/Ly-5 congenic mice contains multiple genes that may influence hematopoietic stem cell engraftment

Abstract

The B6.SJL-Ptprc(d)Pep3(b)/BoyJ (B6.SJL) congenic mouse strain, a valuable and widely used tool in murine bone marrow transplantation studies, has long been considered equivalent to the parental C57B/L6 (B6) strain with the exception of a small congenic interval on chromosome 1 harboring an alternative CD45/Ly-5 alloantigen (Ly-5.1). In this study we compared functional properties of stem and stromal cells between the strains, and delineated the boundary of the B6.SJL congenic interval. We identified a 25% reduction in homing efficiency, 3.8-fold reduction in transplantable long-term hematopoietic stem cells (LT-HSCs), a 5-fold reduction in LT-HSCs capable of 24-hour homing, and a cell-intrinsic engraftment defect of 30% to 50% in B6.SJL-derived bone marrow cells relative to B6-derived cells. These functional differences were independent of stem cell number, cycling, or apoptosis. Genotypic analysis revealed a 42.1-mbp congenic interval in B6.SJL including 306 genes, and at least 124 genetic polymorphisms. Moreover, expression profiling revealed 288 genes differentially expressed between nonhematopoietic stromal cells of the 2 strains. These results indicate that polymorphisms between the B6 and SJL genotype within the B6.SJL congenic interval influence HSC engraftment and result in transcriptional variation within bone marrow stroma.

Figure 1

B6-derived Ly-5.2 cells exhibit competitive advantage in B6 recipients. (A top panel) Ly-5.2 cells increase significantly (#P < .0001) and Ly-5.1 cells decrease significantly (#P = .001) from 5 to 30 weeks after transplant in B6 recipients. Within the Ly-5⁺ population, the percentage of Ly-5.2 (mean ± SD) cells is significantly higher (*P = .001) than the percentage of Ly-5.1 (mean ± SD) cells at 30 weeks after transplant in B6 recipients. (Bottom panel) Ly-5.2 cells increase significantly (#P = .002) in B6.SJL recipients from 5 to 30 weeks after transplant. Within the Ly-5⁺ population, the percentage of Ly-5.1 cells (mean ± SD) is significantly higher (*P = .001) than the percentage of Ly-5.2 cells (mean ± SD) at 5 weeks after transplant in B6 recipients. (B) Within the Ly-5.1 population, the percentage of Mac-1/Gr-1 cells (mean ± SD) decreased significantly (##P = .009) in B6 and B6.SJL recipients, the percentage of B220 cells (mean ± SD) decreased significantly in B6 (#P = .03) but not B6.SJL (P = .5) recipients, and the percentage of Thy-1.2 cells dropped slightly (slope = −0.069) but not significantly (P = .6) in both strains. Cross-strain comparisons revealed a significant difference in Thy-1.2 cells at 5 (*P = .03) and 10 (*P = .02) weeks after transplant. Data were pooled from a total of 3 to 8 mice per strain at each time point.

Figure 2

Ly-5.1 cells are underrepresented in all hematopoietic organs of B6 recipients. (A) In Ly-5–positive cells, the percentage of Ly-5.1 cells (mean ± SD) was significantly lower than the percentage of Ly-5.2 cells (mean ± SD) in the peripheral blood (P = .001), spleen (P = .007), and bone marrow (P = .002) of B6 mice 30 weeks after transplant.

Figure 3

B6 and B6.SJL strains possess equivalent patterns of cell cycling, apoptosis, and CAFCs. (A) There were no significant differences between B6 and B6.SJL cells in any phase of the cell cycle or apoptosis in any bone marrow population tested. Values represent the mean ± SD calculated from 3 independent experiments each using 2 mice per strain. Statistical comparisons were made using the unpaired Student t test assuming unequal variance. (B) There were no significant differences in in vitro defined bone marrow HSC number (CAFCd28 and CAFCd35) between B6 and B6.SJL mice (*P < .05). Data were combined from 3 independent CAFC assays each using marrow pooled from 2 to 3 mice per strain.

Figure 4

B6.SJL congenic interval spans 106 to 148 mbp on mouse chromosome 1. Schematic representation of B6.SJL chromosome 1: the SJL donor-interval is designated in white, the distance to the nearest informative B6 marker is light gray, and the B6 genotype is represented in darker gray.

Figure 5

Differentially expressed genes between B6 and B6.SJL stroma are distributed throughout the mouse genome. (A) Using a statistical threshold of P < .05, differentially expressed genes are concentrated on mouse chromosomes 11 and 17, and genomic distribution cannot be attributed only to chromosome size. (B) Using a statistical threshold of P < .01, differentially expressed genes are enriched on chromosomes 1 and 7 when displayed relative to chromosome number (top), and on chromosome 17 when displayed relative to chromosome size (middle). Correlation between chromosome size and the number of differentially expressed genes does not account entirely for genomic distribution.