B Dontje

Expression quantitative trait loci are highly sensitive to cellular differentiation state

Genetical genomics is a strategy for mapping gene expression variation to expression quantitative trait loci (eQTLs). We performed a genetical genomics experiment in four functionally distinct but developmentally closely related hematopoietic cell populations isolated from the BXD panel of recombinant inbred mouse strains. This analysis allowed us to analyze eQTL robustness/sensitivity across different cellular differentiation states. Although we identified a large number (365) of “static” eQTLs that were consistently active in all four cell types, we found a much larger number (1,283) of “dynamic” eQTLs showing cell-type–dependence. Of these, 140, 45, 531, and 295 were preferentially active in stem, progenitor, erythroid, and myeloid cells, respectively. A detailed investigation of those dynamic eQTLs showed that in many cases the eQTL specificity was associated with expression changes in the target gene. We found no evidence for target genes that were regulated by distinct eQTLs in different cell types, suggesting that large-scale changes within functional regulatory networks are uncommon. Our results demonstrate that heritable differences in gene expression are highly sensitive to the developmental stage of the cell population under study. Therefore, future genetical genomics studies should aim at studying multiple well-defined and highly purified cell types in order to construct as comprehensive a picture of the changing functional regulatory relationships as possible.

Engraftment of syngeneic bone marrow is not more efficient after intrafemoral transplantation than after traditional intravenous administration

OBJECTIVE Hematopoietic stem cells are key elements for life-long production of mature blood cells. The success of clinical stem cell transplantation may be improved when the number of stem cells that engraft after transplantation can be increased. Here, we investigated in a syngeneic mouse model whether engraftment and reconstitution can be improved by transplantation directly into the bone marrow. MATERIALS AND METHODS In this study, we directly compared syngeneic transplantation of hematopoietic stem cells into the bone marrow with intravenous administration and assessed reconstitution kinetics and engraftment by bioluminescent imaging and chimerism determination. RESULTS Surprisingly, only about 10% of cells injected directly into the femur (intrafemoral, IF) could be retrieved within 5 minutes after injection. Only in the first 48 hours after transplantation, engraftment in IF-transplanted animals was higher compared with intravenous injection. However, at all later time points no differences could be detected using whole body bioluminescence or measuring blood cell reconstitution. Most importantly, we found that IF-transplanted cells did not outcompete cells transplanted intravenously when cotransplanted in the same recipient. CONCLUSIONS In conclusion, IF transplantation in a murine syngeneic setting revealed no enhanced engraftment. Previous reports on IF transplantation may have relied on escape from immune rejection in xenogeneic or allogeneic models. Therefore, we conclude that stem cells can find the proper microenvironment irrespective of the route of administration.

Differential role for very late antigen-5 in mobilization and homing of hematopoietic stem cells

The role of very late antigen-5 (VLA-5) in homing and mobilization of hematopoietic stem cells from normal bone marrow (NBM) and bone marrow (MBM) and peripheral blood (MPB) from mobilized mice was investigated. We found a decreased number of VLA-5-expressing cells in the lineage-negative fraction of MPB. However, virtually all stem/progenitor cells were present in the VLA-5+ fraction and hence mobilization of hematopoietic stem cell subsets does not coincide with a downregulation of VLA-5. Stem/progenitor cells from MPB and MBM demonstrated enhanced stromal-derived factor-alpha-induced migration. This enhanced migration correlates with an improved hematopoietic reconstitution potential, with the migrated MPB cells showing the fastest reconstitution. Interestingly, homing of MPB, MBM and NBM stem/progenitor cells in bone marrow and spleen did not differ and is therefore not responsible for the differences in hematopoietic reconstitution. The observed increase in VLA-5+ cells in the recipients after transplantation can most probably be attributed to selective homing of VLA-5+ cells instead of an upregulation of VLA-5. Treatment with an antibody to VLA-5 partially inhibited bone marrow homing of progenitor cells, whereas homing in the spleen was hardly affected. These data indicate a differential role for VLA-5 in the movement of stem cells from and toward bone marrow.

Identification of quantitative trait loci regulating haematopoietic parameters in B6AKRF2 mice.

The haematopoietic system is a complex organised tissue with a hierarchical structure. Identification of organisational pathways within the haematopoietic system is relevant for a better understanding of haematopoiesis in health and disease. We have analysed numerous haematopoietic parameters in two panels of a total of 157 genetically distinct B6AKRF2 mice, derived from an intercross between AKR and C57Bl/6 mice, strains known to differ in various stem cell traits. The major objective of our study was to assess the extent to which various haematopoietic parameters, such as stem cell numbers, progenitor cell cycling, progenitor cell mobilisation and neutrophil numbers in blood and bone marrow are coregulated. The genotypes of these mice were used to search for genetic loci that regulate these parameters. We found significant quantitative trait loci (QTL) associated with the number of stem cells (CAFC‐35) in the bone marrow and the number of neutrophils in the blood. However, most haematopoietic parameters appeared to be controlled by non‐heritable (epigenetic) factors, or by multiple QTLs. Our study reveals striking differences in structure of the haematopoietic hierarchy between individual mice. Surprisingly, stem and progenitor cell pool size and proliferation rate, as well as peripheral blood cell counts are all independently regulated.