GRP94 Regulates Stem Cell-Niche Interactions Figure 5. Increase extramedullary hematopoiesis in the spleen of Grp94 KO mice. A) Representative spleen size and morphology of WT and cKO mice. B) Average spleen weights of WT (n = 19) and cKO (n = 25) mice. The data are presented as mean 6 s.e., ***p,0.001. C) Representative H&E staining of paraffin sections of spleen from WT and cKO mice. Hematopoietic cells in the red pulp are indicated by the area between the two arrows. Scale bar represents 2 mm. doi:10.1371/journal.pone.0020364.g005 (Figure 7E). These findings suggest that acute deletion of Grp94 in primitive hematopoietic cells impairs the ability of HSCs to home to BM and lodge at the endosteal niche. As a consequence, in the competitive repopulation assay (Figure 8A) cKO LSK cells failed to engraft and reconstitute the hematopoietic system in lethally irradiated recipient mice in the presence of WT competitors (Figure 8B and 8C). GRP94 in HSCs, but not niche cells, is necessary for maintaining HSC homeostasis In the competitive repopulation and competitive lodgment assay, where cKO cells are transplanted into a WT microenvironment, we observed impairment in the function of the cells indicating that Grp94 deletion in HSCs at least partially contributes to the disruption of the HSC-niche interaction. However, in our model system Grp94 was knocked out in the whole BM, therefore the disruption of HSC-niche interactions could also be attributed to the niche cells. To further explore a possible extrinsic role of GRP94 niche cells in HSC homeostasis disturbance in cKO mice, we created a chimeric mouse model with WT hematopoietic cells and a GRP94 microenvironment. WT BM cells were transplanted into lethally irradiated WT or cKO recipient mice. Following 8 weeks to allow for full reconstitution of WT hematopoietic system, Grp94 deletion was induced by pI.pC administration (Figure 9A). According to previous reports, BM stromal cells do not engraft after BM transplantation [48], therefore, Grp94 KO only occurred in the hematopoietic cells. Chimeras with WT hematopoietic cells and cKO microenvironment showed a normal hematology profile and spleen size as WT control chimeras (Figure 9B and 9C). Flow cytometric analysis revealed a similar LSK percentage in BM from cKO chimeras and WT controls (Figure 9D and 9E). These findings provide direct evidence that GRP94 in HSCs is required for their interaction with the niche, and depleting GRP94 in BM microenvironment itself is not sufficient to affect the regulation of HSCs. Grp94 KO HSCs lack cell surface integrin a4 expression and exhibit impaired binding to fibronectin To investigate the potential mechanism by which GRP94 maintains the interaction between HSCs and the niche, we examined the cell surface expression of integrin a4 and a5, which are known to be important for the homing and adhesion of HSCs to the endosteal niche [49,50]. Flow cytometric analysis with mouse BM LSKFlk22 and LSKFlk2+ populations demonstrated significantly lower expression of cell surface CD49d (integrin a4/ b1) on cKO than WT cells, whereas CD49e (integrin a5/b1) expression was comparable between the two groups (Figure 10A). These results establish that GRP94 is required for the expression of integrin a4 on the cell surface of hematopoietic stem and progenitor cells, which is consistent with the recent finding that PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e20364