GRP94 Regulates Stem Cell-Niche Interactions Figure 7. Grp94 KO HSCs displayed impaired interaction with the niche. A) Scheme of the in vivo competitive lodgment assay. B) Representative flow cytometric analysis of CFSE-labeled WT LK cells and SNARF-labeled cKO LK cells with host BM and spleen cells. C) Summary of LK cells homed to the BM and spleen (n = 4 for BM, n = 2 for spleen), with the level of WT cells in BM and spleen set as 1. D) Bone section of a recipient femur. WT LK cells were labeled with CFSE (green); cKO LK cells were labeled with SNARF (red); and nuclei were stained with DAPI (blue). Solid arrows indicate cells lodged in the endosteal region (within two cell diameters from the endosteal surface), while open arrows indicate cells located in the central marrow. Scale bar represents 1 mm. E) Summary of the percentage of labeled LK cells found at the endosteal region among those homed to BM from 3 independent experiments. All data are presented as mean 6 s.e., *p,0.05. doi:10.1371/journal.pone.0020364.g007 employed is the Grp94; Mx-1-Cre mouse model whereby GRP94 can be acutely eliminated largely in the hematopoietic system in a temporally controlled manner in adult mice. Our results showed that within 3 weeks of depletion of GRP94 in the hematopoietic cells, there was a 2-fold increase in primitive HSCs in the mutant mice. The Grp94 KO LSK cells are more proliferative, likely as a consequence of loss of niche interaction. The latter is supported by our observation that the mutant LSK cells are more mobilized to the spleen and peripheral blood. The mutant LSK cells lack cell surface integrin a4 expression and do not bind to fibronectin as efficiently as the WT LSK cells in vitro, are less able to lodge in the BM niche in vivo, and as a consequence showed no engraftment in competitive repopulation assays. The maintenance of HSCs involves the balance between self- renewal and differentiation, proliferation and quiescence, mobili- zation and homing, as well as their capacity to provide lifelong reconstitution of all blood-cell lineages after transplantation into lethally irradiated recipients [3,12]. Our study demonstrates the unique function of GRP94 in regulating the homeostasis of HSCs and their interaction with the stem cell niche. Currently, the regulation of HSC physiology is mostly attributed to transcription factors, cell-cycle regulators, chromatin-associated proteins, extra- cellular signaling molecules and their receptors [1]. Here we explore the notion that an ER chaperone protein may play a role in the regulation of HSC-niche interactions. The HSC niche is believed to house HSCs, maintain their quiescence and prevent PLoS ONE | www.plosone.org 8 May 2011 | Volume 6 | Issue 5 | e20364