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

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

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

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

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

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