pI.pC injection. These control mouse cohorts are referred to below as WT mice. The status of the Grp94 allele deletion was validated by PCR in isolated BM cells (Figure 1B). Real-time quantitative PCR analysis with mouse BM cells 11 days after 7 shots of pI.pC injection showed that Grp94 transcripts were mostly depleted from the BM cells of the cKO mice (Figure 1C). GRP94 deficiency expands primitive cell pool through loss of quiescence In analyzing the effect of acute Grp94 deletion on primitive hematopoietic cells, we observed that within 3 weeks of GRP94 depletion there was an expansion of the most primitive Lin2 c- Kit+ Sca-1+ Flk22 CD342 (LSKFlk22CD342) LT-HSC-enriched cell population [44] (2-fold, p,0.01) in the BM. Correlating with this was an expansion of the ST-HSC enriched LSKFlk22CD34+ cells, MPP-enriched LSKFlk2+ and LSK populations of cells (p,0.001) (Figure 2A and 2B), indicating that all primitive hematopoietic cells were significantly increased upon acute hematopoietic Grp94 deletion. These findings, coupled with the GRP94 Regulates Stem Cell-Niche Interactions observation that the total BM cell number was not significantly different in the mutant mice (p = 0.373) (Figure 2C), reveal that the total number of primitive hematopoietic cells in cKO mice BM is higher compared to controls. We also analyzed the progenitor cell populations in the BM. While the common myeloid progenitor (CMP) enriched Lin2 c-Kit+ Sca-12 IL-7Ra2 CD34+ FccII/ IIIRlo cells, the common lymphoid progenitor (CLP) enriched Lin2 c-Kitlo Sca-1lo IL-7Ra+ cells, and the megakaryocyte- erythroid progenitor (MEP) enriched Lin2 c-Kit+ Sca-12 IL- 7Ra2 CD342 FccII/IIIRlo cells were all comparable between the cKO mice and their WT littermates, there were significantly more granulocyte-monocyte progenitor (GMP) enriched Lin2 c-Kit+ Sca-12 IL-7Ra2 CD34+ FccII/IIIRhigh cells in the cKO mice (Figure 3A). This correlates with the increased number of colonies (p,0.01 for total colony number; p,0.05 for CFU-G and CFU- GM; p = 0.07 for CFU-M) formed by unfractioned cKO BM cells in CFC assays (Figure 3B). To examine the underlying cause for the increase in primitive hematopoietic cells in the BM of the cKO mice, we analyzed the cell cycle distribution in LSK cells by Hoechst/Pyronin Y staining Figure 2. GRP94 deficiency in the bone marrow expanded the primitive cell pool. A) Representative flow cytometric analysis with BM cells using Lin, c-Kit, Sca-1, Flk2 and CD34. B) Quantitation of flow cytometric analysis of primitive cell proportions. Left panel shows the percentage of LSKFlk22CD342 in BM and right panel shows LSKFlk22CD34+, LSKFlk2+ (n = 5 for WT, n = 8 for cKO) and total LSK (n = 16 for WT, n = 22 for cKO) cells in BM using Lin, c-Kit, Sca-1. C) Total BM cell number from WT (n = 19) and cKO (n = 20) mice (p = 0.373). All data are presented as mean 6 s.e., **p,0.01, ***p,0.001. doi:10.1371/journal.pone.0020364.g002 PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e20364