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X. Liu et al.
CD31+Sca1- populations enriched for total EC, arteriolar EC and sinusoidal EC,28 respectively (Figure 1D). The increase of arteriolar and sinusoidal EC was further confirmed by the observation that arteries (Sca-1-staining) and sinusoids (Endoglin-staining) were both significantly increased in Twist1-deleted mice compared with control mice by immunofluorescence of femoral sections (Figure 1E). The increase of EC may be the result of cell proliferation as determined by increased bromodeoxyuridine incorporation into CD45-Ter119-CD31+ stromal cells in Twist1-deleted mice (Figure 1F). We next performed a tube formation assay to determine the effect of TWIST1 on new blood vessel development. Consistent with increased microvessels in knockout mice in vivo, capillary tube formation of Twist1- deleted EC was also increased on matrigel (Figure 1G,H), indicating that Twist1 deletion promotes angiogenesis.
To further evaluate the impact of Twist1 deficiency on cell factors, we performed quantitative real-time poly- merase chain reaction to analyze the expression of key niche factors. The results showed significant decreases in the expression of Cxcl12, Vcam1, Angiopoietin-1 (Angpt1) and Scf, particularly the membrane-bound isoform Scf (m220 Scf), which was found to be extremely important for HSC maintenance, in MSC (5×104 cells) from Twist1-delet- ed mice as compared to control mice (Figure 2A). The expression of Opn, which negatively regulates the HSC pool, was obviously increased in both MSC and OLC (Figure 2B). Enzyme-linked immunoassay demonstrated the reduced protein levels of CXCL12, VCAM1, SCF and elevated level of osteopontin (Figure 2C) in BM super-
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natants of Twist1-deleted mice as compared to those in control mice.
Collectively, Twist1 deletion leads to significant alter- ations in various key niche components, demonstrating its functional importance in the BM microenvironment, and implying its potential regulatory role in HSC maintenance.
Microenvironmental Twist1 deficiency impairs the homing and retention of hematopoietic stem cells but promotes their mobilization
Most HSC are retained in the BM niche in a quiescent, nonmotile mode by adhesion to stromal cells, which are essential for normal hematopoiesis and for protection from myelotoxic injury. Twist1 deletion resulted in decreased expression of BM CXCL12 and VCAM1, which are critical for HSC retention, homing and mobilization,29,30 implying that TWIST1 may have a functional impact on the migra- tion of HSC. We first evaluated homing of normal HSC to the BM of Twist1-deleted mice. Freshly isolated c-Kit+ cells from B6.SJL (CD45.1) mice were injected into lethally irra- diated Twist1-deleted or control mice. Sixteen hours after transplantation, the absolute number of CD45.1+ and CD45.1+ LSK (Lin-Sca-1+c-Kit+) cells was significantly decreased in Twist1-deleted mice compared to the number in controls (Figure 3A), indicating that Twist1 deficiency impairs homing of hematopoietic stem/progenitor cells (HSPC) to the BM microenvironment.
We next determined the contribution of TWIST1 to HSC retention and mobilization by assessing total cells and HSPC in the BM, spleen and peripheral blood of
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Figure 2. Twist1 deletion in the bone marrow microenvironment changes expression of niche factors. (A) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of the expression of C-X-C motif chemokine ligand 12 (Cxcl12), Vascular cell adhesion molecule 1 (Vcam1), Stem cell factor (Scf) and Angiopoietin-1 (Angpt1) in freshly sorted mesenchymal stem cells (MSC), osteolineage cells (OLC) and endothelial cells (EC) from chimeric control (Ctrl) and knockout (KO) mice (n=4). (B) qRT-PCR analysis of the expression of Osteopontin (Opn) in MSC and OLC from chimeric Ctrl and KO mice (n=4). (C) Enzyme-linked immunosorbent assay analysis of BM protein concentrations of CXCL12, VCAM1, SCF and OPN in chimeric Ctrl and KO mice (n=5-8). Column plots show the mean ± standard deviation from three independent experiments. *P<0.05; **P<0.01; ***P<0.001 (Student t test).
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haematologica | 2018; 103(12)