Transplanted central marrow ECs incorporate into the adult BM vasculature.

<p>(A) Central marrow ECs, PlGF knockdown central marrow ECs, mock transduced central marrow ECs or central marrow SCs were labeled with CFDA-SE and transplanted into Balb/c mice following 550cGy total body irradiation (1×10⁶ cells per mouse; intravenous injection on day 0 and intraperitoneal injection on day 1 to day 4). The percentage of labeled cells present in the BM and spleen on day 5 post irradiation was determined by flow cytometry. Representative flow plots (left) and percentage of labeled cells present in the BM and spleen (right) are shown (for central marrow ECs and SCs, n = 6 from 2 independent experiments; for PlGF knockdown central marrow ECs and mock transduced central marrow ECs, n = 2). (B) Lodgment of the central marrow ECs in the BM, classified as contiguous with the vascular endothelium (top image) or within the BM space (bottom image). The BM vessel is indicated by an asterix. Bone is outlined by the dashed line. The yellow arrows represent the transplanted central marrow EC labeled with CFDA-SE. Nuclei were visualized with DAPI (blue) present in Vectashield (scale bar: 50 μm; 40 sections per mouse, n = 6 from 2 independent experiments).</p

Reduced PlGF expression diminishes vascular recovery <i>in vivo</i>.

<p>(A) Central marrow ECs, PlGF knockdown central marrow ECs, central marrow SCs or PBS were transplanted into Balb/c mice following 550cGy total body irradiation. On day 20 post irradiation, the tibias were harvested and assessed by immunohistochemistry for VE-cadherin expression (scale bar: 50 μm). (B) Representative images of normal and pathologic vessels (scale bar: 50 μm). (C) Quantitation of normal or pathologic vessels in the BM of the irradiated mice transplanted with the different cell types. Vessels were counted in three 200X fields per section. (*p<0.05, **p<0.01; 40 sections per mouse, n = 9 from 3 independent experiments). (D) BM MNCs from the treated mice were harvested and stained with the antibody to CD31, CD45 and Ter-119. The percentage of BM ECs was determined by CD31⁺CD45⁻Ter119⁻ cells using flow cytometric analysis (*p<0.05; n = 9 from 3 independent experiments; error bars represent standard deviation).</p

Reduced PlGF expression diminishes hematopoietic recovery <i>in vivo</i>.

<p>(A) BM MNCs from Balb/c mice following treatment with central marrow ECs, PlGF knockdown central marrow ECs, central marrow SCs or PBS were evaluated for LT-HSC, ST-HSC and HPC frequencies using flow cytometry (**p<0.01; n = 9 from 3 independent experiments; error bars represent standard deviation). (B) BM MNCs from the same mice were assessed for CAFC frequencies at weeks 2 and 5 (*p<0.05; n = 9 from 3 independent experiments; error bars represent standard deviation).</p

Down-regulation of PlGF in central marrow ECs diminishes their ability to support primitive hematopoietic cells.

<p>(A) Total RNA was obtained from central marrow ECs transduced with shRNA clones targeting PlGF. The mRNA expression level of PlGF was measured using quantitative RT-PCR and normalized to <i>hprt</i> levels (*p<0.05, ***p<0.001; n = 6 from 3 independent experiments; error bars represent standard deviation). (B) Uptake of DiI-Ac-LDL (red) in control-transduced and PlGF knockdown central marrow ECs. Nuclei were visualized with DAPI (Scale bar: 50 µm). (C) The formation of endothelial capillary like tube was evaluated under a phase-contrast microscope on Matrigel following 10 hours of culture (Scale bar: 200 μm). (D, E) LSK cells were seeded in serial dilutions on central marrow ECs, central marrow ECs transduced with E8, E12 or E8 and E12 lentiviral shRNA-PlGF vectors or shRNA-GFP vector and cultured at 33°C and 5% CO₂. CAFCs were scored on week 2 and 5 (*p<0.05, **p<0.01; n = 3 from 3 independent experiments; error bars represent standard deviation).</p

Characteristics of cells grown in endothelial or stromal cell culture conditions.

<p>(A, B) Total RNA was extracted from central marrow EC, endosteal marrow EC, spleen EC, central marrow SC and endosteal marrow SC. The mRNA expression level of Tie-2 and VE-Cadherin was measured using quantitative RT-PCR. The relative expression was normalized to Hypoxanthine-guanine phosphoribosyltransferase (HPRT) levels and calculated from standard curves. (*p<0.05, **p<0.01; n = 6 from 3 independent experiments; error bars represent standard deviation). (C) Cells cultured in endothelial or stromal culture conditions were stained with antibodies to CD31 and VE-Cadherin. Positive signals were visualized with FITC conjugated secondary antibody. Nuclei were visualized with 4,6-diamidino-2-phenylindole (DAPI). (D) Central marrow EC, endosteal marrow EC, spleen EC, central marrow SC and endosteal marrow SC were harvested by enzyme-free cell dissociation solution. The expression of CD31 and CD45 was analyzed by flow cytometry with PE conjugated anti-CD31 and APC-Cy7 conjugated anti-CD45. (E) Cells cultured in endothelial or stromal culture conditions were stained with alkaline phosphatase activity. Positive reaction was observed as dark blue violet in the cells.</p

mRNA expression of VEGFa, VEGFb and PlGF in cultured and fresh ECs and SCs.

<p>Total RNA was extracted from the cultured and freshly isolated central marrow EC, endosteal marrow EC, spleen EC, central marrow SC and endosteal marrow SC. (A) Flow cytometric plots for the purification of ECs and SCs derived from central marrow, endosteal marrow and spleen. SCs and ECs were identified as CD45⁻TER119⁻CD31⁻ and CD45⁻TER119⁻CD31⁺ respectively. The mRNA expression level of VEGFa (B), VEGFb (C) and PlGF (D) was measured using quantitative RT-PCR. The relative expression was normalized to <i>hprt</i> levels and calculated from standard curves (For the cultured cells, *p<0.05, **p<0.01, ***p<0.001; n = 6 from 3 independent experiments; error bars represent standard deviation. For the freshly sorted cells, n = 2).</p

Central marrow ECs demonstrate enhance support of primitive hematopoietic cells.

<p>(A) BM MNCs were seeded in serial dilutions on central marrow EC, central marrow SC, endosteal marrow EC, endosteal marrow SC and spleen EC and cultured at 33°C and 5% CO₂. CAFCs were scored on day 14, 28, 35, 42 and 49. (B) BM LSK cells were seeded in serial dilutions on central marrow EC and central marrow SC and cultured at 33°C and 5% CO2. CAFCs were scored on day 14, 21, 28 and 35 (**p<0.01, *p<0.05 n = 4 from 3 independent experiments; error bars represent standard deviation). (C) 1000 LSK cells were co-cultured with the five supportive cell layers and the number of CD45⁺ cells on day 7 was assessed by flow cytometry (**P<0.01; n = 5 from 2 independent experiments; error bars represent standard deviation). (D) 100 LSK cells were co-cultured with the five supportive cell layers and the number of CFU-Cs was assessed on day 7 (*P<0.05, **P<0.01; n = 5 from 2 independent experiments; error bars represent standard deviation).</p

GRP94-deficient LSK cells displayed increased proliferation and loss of quiescence.

<p>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 ± s.e., **p<0.01, ***p<0.001.</p

<i>Grp94</i> KO mice displayed altered myeloid and lymphoid differentiation.

<p>A). Complete blood count of peripheral blood from WT (n = 31) and cKO (n = 37) mice. B) Representative Wright-Giemsa staining of blood smear with tail peripheral blood from WT and cKO mice. Scale bar represents 500 µm. C) Total thymus cell number (left) and total left and right axillary lymph nodes cell number (right) from WT and cKO mice (n = 7 for each group). D) Representative flow cytometric analysis of splenocytes from WT and cKO mice using lineage markers Gr-1 and CD3 (left), F4/80 and B220 (right). E) Quantitation of (D) from WT (n = 4) and cKO (n = 7) mice. F) Representative flow cytometric analysis with BM cells using lineage markers Gr-1 and B220 (left) and CD4 and CD8a (right). G) Quantitation of (F). Gr-1 and B220 (n = 7 for WT and n = 9 for cKO mice); CD4 and CD8a (n = 7 for each genotype). All data are presented as mean ± s.e., ***p<0.001.</p

Inability of <i>Grp94</i> KO HSCs to express surface integrin α4 and bind to fibronectin.

<p>A) Representative flow cytometric analysis of CD49d and CD49e with BM LSKFlk2⁻ 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 <i>in vitro</i>. 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 ± s.e..</p