An irradiated marrow niche reveals a small noncollagenous protein mediator of homing, dermatopontin.

Hematopoietic cell homing after hematopoietic cell transplant (HCT) is governed by several pathways involving marrow niche cells that are evoked after pre-HCT conditioning. To understand the factors that play a role in homing, we performed expression analysis on zebrafish marrow niche cells following conditioning. We determined that the noncollagenous protein extracellular matrix related protein dermatopontin (Dpt) was upregulated sevenfold in response to irradiation. Studies in mice revealed DPT induction with radiation and lipopolysaccharide exposure. Interestingly, we found that coincubation of zebrafish or murine hematopoietic cells with recombinant DPT impedes hematopoietic stem and progenitor cell homing by 50% and 86%, respectively. Similarly, this translated into a 24% reduction in long-term engraftment (vs control; P = .01). We found DPT to interact with VLA-4 and block hematopoietic cell-endothelial cell adhesion and transendothelial migration. Finally, a DPT-knockout mouse displayed a 60% increase in the homing of hematopoietic cells vs wild-type mice (P = .03) with a slight improvement in long-term lin-SCA1+cKIT+-SLAM cell engraftment (twofold; P = .04). These data show that the extracellular matrix-related protein DPT increases with radiation and transiently impedes the transendothelial migration of hematopoietic cells to the marrow.

Dermatopontin in Bone Marrow Extracellular Matrix Regulates Adherence but Is Dispensable for Murine Hematopoietic Cell Maintenance.

The hematopoietic marrow microenvironment is composed of multiple cell types embedded in an extracellular matrix (ECM). We have explored marrow ECM using mass spectrometry and found dermatopontin (DPT), a small non-collagenous ECM protein, to be present. We found that DPT cooperates with other ECM proteins to promote hematopoietic cell adherence in vitro on plastic as well as OP9 stromal cells. We generated constitutional DPT-/- mice that were viable and had no peripheral lympho-hematopoietic abnormalities. The composition of the marrow of wild-type and DPT-/- mice was equivalent in terms of cellularity, CFU-C, LSK (Lineage-, SCA-1+, KIT+), and LSK-SLAM (LSK, CD48-, CD150+) frequencies. These data suggest that DPT fosters adherence but is not required for steady-state hematopoiesis in vivo. There are likely overlapping cellular adhesion mechanisms that can compensate to maintain the hematopoietic niche in the absence of DPT.

A Functional Bioluminescent Zebrafish Screen for Enhancing Hematopoietic Cell Homing.

To discover small molecules that modulate hematopoietic cell homing after adoptive transfer, we created a transgenic zebrafish expressing firefly luciferase downstream of the ubiquitin promoter (ubi:luc) to serve as a hematopoietic donor. Bioluminescence imaging (BLI) was used to detect and follow ubi:luc hematopoietic cells that homed to the marrow as early as 1 day post-transplant. BLI was able to detect the biological effect of prostaglandin E2 on early homing/engraftment of donor hematopoietic cells. This system was utilized in a functional screen of small molecules to enhance homing/engraftment. We discovered a phytosterol, ergosterol, that could increase hematopoietic cell homing in zebrafish and mice. In addition, ergosterol increased CXCR4 expression and promoted expansion of Lin-SCA-1+KIT+ cells in vitro. We have demonstrated the utility of in vivo BLI to non-invasively monitor donor hematopoietic cell activity in adult zebrafish as a functional screen for mediators of cellular homing.