American Society of Hematology

Figure 4

$Figure 4. Angptl3-producing BM endothelial cells support HSC activity. (A) Angptl3 is highly expressed in BM endothelium and CD45−SSEA4+ cells. Indicated populations of BM cells were collected by flow cytometry and Angptl3 expression was measured by real-time RT-PCR. The cells sorted for analysis were gated from the CD45− fraction. (*significantly different between 2 groups, P < .05, n = 6). The levels of Angptl3 mRNA in each population were normalized to the level of β-actin transcripts present in the same sample (Mean ± s.e.m). (B) BM Angptl3+ endothelium is close to HSCs. Top, HSCs were associated with sinusoidal endothelial cells in mouse BM. BM was stained to reveal CD150+CD48−CD41−Lin− HSCs (red, with arrowhead); MECA-32+ sinusoidal endothelial cells (blue, with star); and CD41+, CD48+, or Lin+ differentiated hematopoietic cells (green or orange). Middle, most sinusoidal endothelial cells in mouse BM were Angptl3+, and some Angptl3+ cells were close to the sinusoidal endothelium. Most sinusoidal endothelial cells were also positive for Angptl3 (arrowhead); many Angptl3+ cells (green, with arrows) were also in contact with MECA-32+ sinusoidal endothelial cells (red). Nuclei were counterstained with DAPI (blue). Bottom, HSCs were associated with Angptl3+ cells in mouse BM; 31 of 51 (60.8%) HSCs are adjacent to Angptl3+ cells ( ≤ 2 cell distances). CD150+CD48−CD41−Lin− HSCs (red, with arrowhead), Angptl3+ cells (blue), and CD41+, CD48+, or Lin+ differentiated hematopoietic cells (green or orange). One megakaryocyte is marked by an arrow. Star markers indicate the possible vascular niche. Scale bar applies to all images. (C) Angptl3-deficient BM CD45− cells and CD45−CD31+ cells had decreased ability to support expansion of HSCs. One hundred and fifty BM CD45.1 Lin−Sca-1+Kit+Flk-2−CD34− cells were cocultured with 450 CD45− BM stromal cells isolated from CD45.2 WT mice (black bar), 450 CD45−CD31+ BM endothelial cells isolated from CD45.2 WT mice (purple bar), or the same number of CD45− or CD45−CD31+ cells from Angptl3-null mice (orange or green bars, respectively) in serum-containing StemSpan supplemented with SCF, TPO, and FGF-1. After 5 days, the cocultured cells were cotransplanted with 1 × 105 CD45.2 competitors into lethally irradiated CD45.2 recipient mice. The figure shows the engraftment at 3, 7, and 12 weeks after transplantation (*significantly different between 2 groups. P < .05, n = 7-8).$

Angptl3-producing BM endothelial cells support HSC activity. (A) Angptl3 is highly expressed in BM endothelium and CD45⁻SSEA4⁺ cells. Indicated populations of BM cells were collected by flow cytometry and Angptl3 expression was measured by real-time RT-PCR. The cells sorted for analysis were gated from the CD45⁻ fraction. (*significantly different between 2 groups, P < .05, n = 6). The levels of Angptl3 mRNA in each population were normalized to the level of β-actin transcripts present in the same sample (Mean ± s.e.m). (B) BM Angptl3⁺ endothelium is close to HSCs. Top, HSCs were associated with sinusoidal endothelial cells in mouse BM. BM was stained to reveal CD150⁺CD48⁻CD41⁻Lin⁻ HSCs (red, with arrowhead); MECA-32⁺ sinusoidal endothelial cells (blue, with star); and CD41⁺, CD48⁺, or Lin⁺ differentiated hematopoietic cells (green or orange). Middle, most sinusoidal endothelial cells in mouse BM were Angptl3⁺, and some Angptl3⁺ cells were close to the sinusoidal endothelium. Most sinusoidal endothelial cells were also positive for Angptl3 (arrowhead); many Angptl3⁺ cells (green, with arrows) were also in contact with MECA-32⁺ sinusoidal endothelial cells (red). Nuclei were counterstained with DAPI (blue). Bottom, HSCs were associated with Angptl3⁺ cells in mouse BM; 31 of 51 (60.8%) HSCs are adjacent to Angptl3⁺ cells ( ≤ 2 cell distances). CD150⁺CD48⁻CD41⁻Lin⁻ HSCs (red, with arrowhead), Angptl3⁺ cells (blue), and CD41⁺, CD48⁺, or Lin⁺ differentiated hematopoietic cells (green or orange). One megakaryocyte is marked by an arrow. Star markers indicate the possible vascular niche. Scale bar applies to all images. (C) Angptl3-deficient BM CD45⁻ cells and CD45⁻CD31⁺ cells had decreased ability to support expansion of HSCs. One hundred and fifty BM CD45.1 Lin⁻Sca-1⁺Kit⁺Flk-2⁻CD34⁻ cells were cocultured with 450 CD45⁻ BM stromal cells isolated from CD45.2 WT mice (black bar), 450 CD45⁻CD31⁺ BM endothelial cells isolated from CD45.2 WT mice (purple bar), or the same number of CD45⁻ or CD45⁻CD31⁺ cells from Angptl3-null mice (orange or green bars, respectively) in serum-containing StemSpan supplemented with SCF, TPO, and FGF-1. After 5 days, the cocultured cells were cotransplanted with 1 × 10⁵ CD45.2 competitors into lethally irradiated CD45.2 recipient mice. The figure shows the engraftment at 3, 7, and 12 weeks after transplantation (*significantly different between 2 groups. P < .05, n = 7-8).

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