Inc., Warrington, PA) was added to the cells at a final concentration of 0.75 mg/ml and incubated at 4uC for 30 min. Cell cycle status was examined using a BD LSR II flow cytometer. Apoptosis assay 16106 whole BM cells were stained with primitive hematopoi- etic cell antibodies (Lin, c-Kit and Sca-1) as described above. The stained cells were resuspended in 150 mL 16Annexin V binding buffer (BD Pharmingen) and incubated with Annexin V and 7AAD (both from BD Pharmingen) for 15 min. Cell apoptosis was examined within an hour using a BD LSR II flow cytometer. Colony-forming cell (CFC) assay Freshly isolated BM cells were mixed thoroughly with 300 mL Iscove’s MDM with 2% FBS and 4 mL defined methylcellulose medium M3434 (StemCell Technologies). Cells were then plated in triplicate in six-well plates with 1.1 mL volume at a density of 16104 BM cells per well. The colonies were then scored at day 10 under an inverted microscope and reported as colony number in specific lineages according to the morphology of colonies described in the manufacturer’s instructions. Real-time quantitative RT-PCR To detect Grp94 knockout efficiency, RNA was extracted from mouse whole BM cells and reverse-transcription was performed as previously described [59]. cDNA samples were analyzed in triplicate with the SYBR Green Supermix (Quanta Biosciences, Gaithersburg, MD) according to manufacturer’s instructions. The following primers were used: Grp94, 59-TGG GCC TCT GCT GTG TCC TGC-39 and 59-GGC TTT TAC CCA GGT CCT CTT CCA CTG T-39; 18S RNA, 59-ACG GCC GGT ACA GTG AAA C-39 and 59-GAG GGA GCT CAC CGG G-39. Competitive repopulation assay LSK cells were sorted using a FACSAria flow cytometer and 1000 WT or cKO LSK cells (CD45.2) were mixed with 250,000 BM MNCs (CD45.1) which is equivalent to approximately 500 LSK cells, and injected into the tail vein of B6.SJL mice that were lethally irradiated at 9.5Gy approximately 24 hr prior to transplantation. Engraftment levels and multilineage reconstitu- tion were measured in peripheral blood samples obtained from the tail vein of hosts starting week 4. In vivo homing Sorted WT and cKO Lin2 c-Kit+ (LK) cells (,56105 to 16106) were labeled with a green fluorescent dye, carboxyfluorescein diacetate succinimidyl ester (CFSE), and a red fluorescent dye, SNARF-1 (both from Invitrogen), respectively, according to References 1. Zon LI (2008) Intrinsic and extrinsic control of haematopoietic stem-cell self- renewal. Nature 453: 306–313. 2. Wagers AJ, Christensen JL, Weissman IL (2002) Cell fate determination from stem cells. Gene Ther 9: 606–612. 3. Wilson A, Trumpp A (2006) Bone-marrow haematopoietic-stem-cell niches. Nat Rev Immunol 6: 93–106. 4. Jones DL, Wagers AJ (2008) No place like home: anatomy and function of the stem cell niche. Nat Rev Mol Cell Biol 9: 11–21. 5. Lymperi S, Ferraro F, Scadden DT (2010) The HSC niche concept has turned 31. Has our knowledge matured? Ann N Y Acad Sci 1192: 12–18. 6. Kiel MJ, Morrison SJ (2008) Uncertainty in the niches that maintain haematopoietic stem cells. Nat Rev Immunol 8: 290–301. 7. Garrett RW, Emerson SG (2009) Bone and blood vessels: the hard and the soft of hematopoietic stem cell niches. Cell Stem Cell 4: 503–506. 8. Nilsson SK, Johnston HM, Coverdale JA (2001) Spatial localization of transplanted hemopoietic stem cells: inferences for the localization of stem cell niches. Blood 97: 2293–2299. GRP94 Regulates Stem Cell-Niche Interactions manufacturer’s instructions. Labeled cells were injected into the tail vein of non-irradiated Grp94F/F C57BL6; 129/Sv background littermates. Mice were then sacrificed after 16 hr, and the number of labeled cells was measured in the BM and spleen through the detection of CFSE+ and SNARF+ cells by flow cytometry. In vivo lodgment LK cells were labeled with 5 mM CFSE or 5 mM SNARF cell- labeling solutions and injected into the tail vein of non-irradiated mice, as described above. Tibias and femurs were dissected from recipient mice 16 hr after injection, decalcified for 3 days in Immunocal (Decal Chemical Corporation, Tallman, NY), and embedded in paraffin blocks after processing. A total of 180 bone sections of 5 mm thickness from three recipients were cut and mounted with Vectashield containing 49,6-diamidino-2-phenylin- dole (DAPI) (Vector Laboratories, Burlingame, CA). To assess the lodgment of injected cells to the endosteal niche, the numbers of CFSE+ (green) or SNARF+ (red) cells within two cell diameters from the endosteal surface were counted. Adhesion assay 56102 LSK cells were added to wells coated with fibronectin (1.5 mg/cm2, BD Biosciences, Bedford, MA) in cell culture treated 96-well plates and incubated in minimum essential medium (MEM) alpha medium with 10% FBS for 3 hr at 37uC/5% CO2 in a humidified atmosphere. Non-adherent cells were washed off with DPBS and adherent cells were visually counted microscop- ically. To control for nonspecific binding, adhesion to 1% bovine serum albumin (Sigma-Aldrich) was quantified. Statistics Statistical significance was assayed by Student’s t test. Acknowledgments We thank Dr. Louis Dubeau, Min Wei, Xiaoxia Jiang and Lee lab members for helpful discussions. We thank the Longo lab for the use of the hematology analyzer, and the USC Norris Cancer Center Flow Cytometry Core, the Translational Pathology Core, the Cell and Tissue Imaging Core. We also thank the USC Center for Stem Cell and Regenerative Medicine Histology Core and the USC Radiation Safety Department for technical assistance. Author Contributions Conceived and designed the experiments: BL GBA ASL. Performed the experiments: BL BSL SHL SW HZ MW. Analyzed the data: BL BSL SHL SYC GBA ASL. Contributed reagents/materials/analysis tools: MW SYC GBA ASL. Wrote the paper: BL GBA ASL. 9. Calvi LM, Adams GB, Weibrecht KW, Weber JM, Olson DP, et al. (2003) Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425: 841–846. 10. Zhang J, Niu C, Ye L, Huang H, He X, et al. (2003) Identification of the haematopoietic stem cell niche and control of the niche size. Nature 425: 836–841. 11. Yin T, Li L (2006) The stem cell niches in bone. J Clin Invest 116: 1195–1201. 12. Cheng T (2008) Toward ‘SMART’ stem cells. Gene Ther 15: 67–73. 13. Arai F, Hirao A, Ohmura M, Sato H, Matsuoka S, et al. (2004) Tie2/ angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 118: 149–161. 14. Stier S, Ko Y, Forkert R, Lutz C, Neuhaus T, et al. (2005) Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size. J Exp Med 201: 1781–1791. 15. Nilsson SK, Johnston HM, Whitty GA, Williams B, Webb RJ, et al. (2005) Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells. Blood 106: 1232–1239. PLoS ONE | www.plosone.org 13 May 2011 | Volume 6 | Issue 5 | e20364