GRP94 Regulates Stem Cell-Niche Interactions Figure 4. GRP94-deficient LSK cells displayed increased proliferation and loss of quiescence. A) Representative flow cytometric analysis of LSK cell cycle status by Hoechst and Pyronin Y staining. To examine early effects of GRP94 depletion on HSC proliferation, BM was extracted from WT and cKO mice 3 days after 4 shots of pI.pC injection every other day. B) Summary of cell cycle distribution of LSK cells from WT and cKO mice (n = 7). C) Summary of flow cytometric analysis of apoptotic LSK cells using Annexin V and 7AAD (n = 5 for WT, n = 8 for cKO) (p = 0.324). All data are presented as mean 6 s.e., **p,0.01, ***p,0.001. doi:10.1371/journal.pone.0020364.g004 (Figure 5A and 5B) with signs of increased extramedullary hematopoiesis as indicated by the increased hematopoietic cells in the red pulps (Figure 5C). Flow cytometric analysis showed that there was a significant increase (2.5-fold, p,0.001) in the percentage of LSK cells in the spleen of cKO mice compared to WT mice (Figure 6A). The number of circulating LSK cells in the peripheral blood of cKO mice were also greatly increased (17-fold, p,0.001) compared to control (Figure 6B). We also observed increased Lin2 Sca-12 c-Kit+ progenitor cells in the peripheral circulation. These results suggest that primitive cell mobilization was increased as a consequence of Grp94 deletion. Since the HSC niche has been proposed to maintain stem cells in the BM in a quiescent state, we hypothesized that the increase in proliferation of the Grp94 KO HSCs may have been mediated by a loss of interaction with the niche, which thus also led to increased HSC mobilization and enlargement of the spleen by extramedullary hematopoiesis in cKO mice. Therefore, we directly examined the capability of Grp94 KO progenitors to home and lodge in the endosteal niche. To do this, we performed a competitive lodgment assay (Figure 7A), in which the lodging of primitive cells is dependent upon their functional interaction with the niche in vivo. An equal number of WT and cKO Lin2 c-Kit+ (LK) cells were labeled with fluorescent vital dyes CFSE and SNARF respectively, and co-injected into non-irradiated WT littermates. In these experiments we used LK cells, as in agreement with previous studies, the use of a purified HSC population would result in too few cells to be reliably detected [47]. We observed that fewer labeled cells from mutant mice homed to BM, while more of them migrated to the spleen of recipient mice (p,0.05) (Figure 7B and 7C). We cut a total of 180 sections of femurs and tibias from a total of three recipient mice in three independent experiments, and visualized the localization of cells as shown in Figure 7D. The endosteal region is defined as the area within 2 cell diameters from endosteal surface. Among all the 400–1000 cells that homed to the BM (numbers varied each time according to the number of cells injected), about 13.5% of WT LK cells lodged at the endosteal region, while only 5% of the cKO cells that homed to the BM were found at the endosteal surface (p,0.05) PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e20364