GRP94 Regulates Stem Cell-Niche Interactions Figure 10. Inability of Grp94 KO HSCs to express surface integrin a4 and bind to fibronectin. A) Representative flow cytometric analysis of CD49d and CD49e with BM LSKFlk22 and LSKFlk2+ cells from WT and cKO mice. Grey-filled histogram represents isotype control staining; dashed green line represents WT cells; solid red line indicates cKO cells. B) The percentage of WT and cKO LSK cells bound to fibronectin in vitro. The number of cells binding to BSA was subtracted from that binding to fibronectin, the results then were normalized against the number of WT cells bound to BSA. The experiments were performed twice in duplicate; each replicate contained pooled BM from 2 to 4 WT or cKO mice. The data are presented as mean 6 s.e.. doi:10.1371/journal.pone.0020364.g010 regulating HSC homeostasis is not known, and awaits further investigation. In comparing our Mx-1-Cre-mediated acute GRP94 KO model with the chimera model using tamoxifen-inducible GRP94 KO following BM transplantation [40], similar hematopoietic pheno- types, including general leukocytosis, increased spleen cellularity, decreased thymus and lymph node cellularities, increased myeloid cells and decreased lymphoid cells in the BM and spleen were observed in both models. The increase of LSK cells in the BM, spleen and peripheral blood and loss of integrin a4 expression upon GRP94 depletion are also in agreement with the previous report [40], which focuses on the role of GRP94 in lymphogenesis, specifically pro-B to pre-B transition in B cell development, as well as T cell development beyond CD42 CD82 double negative stage. Our work here investigates the unique function of GRP94 in the homeostasis of HSCs and their interaction with the stem cell niche. With our model, we provide new evidence that the expansion of primitive hematopoietic cells is attributed to the change of cell cycle status upon Grp94 deletion and that the change of HSC cell cycle status and localization is due to loss of interaction with the niche, revealed by the impaired homing and lodging ability of GRP94-deficient HSCs in vivo. We further determined that GRP94 in the niche cells is not required for maintaining the interaction with HSCs. In summary, we identified GRP94 as a novel regulator of HSCs physiology and their interaction with the niche. It is tempting to speculate that agents that can alter GRP94 expression or activity may be explored for therapeutic intervention, such as increasing the proportion of HSCs in peripheral blood as a source for blood stem cell therapy, or as an alternative method for non- myeloablative conditioning through emptying the HSC niche [55] and these warrant future investigation. Furthermore, overexpression of GRP94 has been associated with cellular transformation, tumorigenicity and decreased sensitivity to anti- cancer treatment [56]. With cancer stem cells implicated as the cancer initiating cells responsible for tumorigenesis and contribute to cancer resistance [57], it would be interesting to determine in future studies whether GRP94 regulates cancer stem cell niche interaction and thus contributes to tumorigenicity and self renewal of resistant cancer cells. Materials and Methods Ethics statement All protocols for animal use were reviewed and approved by the USC Institutional Animal Care and Use Committee. The animal assurance number is A3518-01. The protocol number is 9964. Mice Grp94F/F mice in a mixed C57BL/6; 129/Sv background were generated as previously described [42]. Grp94F/F mice were crossed with the transgenic Mx-1-Cre mice on a C57BL/6 background (Jackson Laboratory) to generate Grp94F/F; Mx-1-Cre mice. Littermates that were negative for Cre transgene were used as controls. Genotyping was performed by PCR using genomic DNA extracted from mouse tail biopsies as previously described [58]. To induce the genomic deletion, 5.5 to 6.5 week-old male and female mice were injected intraperitoneally with pI.pC (25 mg/g mouse body weight) every other day. Mice were analyzed 11 days post 7 injections, except with modifications as specifically mentioned in the figure legends. Complete blood count Peripheral blood was collected via tail bleeding and analyzed using an auto hematology analyzer BC-2800 vet (Mindray, Redmond, WA) according to manufacturer’s instructions. Flow cytometry BM cells were flushed from long bones (tibias and femurs) with Dulbecco’s phosphate-buffered saline without calcium and magnesium (DPBS), and then filtered through nylon screen (70 micron, BD Biosciences) to obtain single cell suspension. PLoS ONE | www.plosone.org 11 May 2011 | Volume 6 | Issue 5 | e20364