Molecular mechanisms of stem cell migration, homing and engraftment

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Human endothelial stem/progenitor cells, angiogenic factors and vascular repair

Neovascularization or new blood vessel formation is of utmost importance not only for tissue and organ development and for tissue repair and regeneration, but also for pathological processes, such as tumour development. Despite this, the endothelial lineage, its origin, and the regulation of endothelial development and function either intrinsically from stem cells or extrinsically by proangiogenic supporting cells and other elements within local and specific microenvironmental niches are still not fully understood. There can be no doubt that for most tissues and organs, revascularization represents the holy grail for tissue repair, with autologous endothelial stem/progenitor cells, their proangiogenic counterparts and the products of these cells all being attractive targets for therapeutic intervention. Historically, a great deal of controversy has surrounded the identification and origin of cells and factors that contribute to revascularization, the use of such cells or their products as biomarkers to predict and monitor tissue damage and repair or tumour progression and therapeutic responses, and indeed their efficacy in revascularizing and repairing damaged tissues. Here, we will review the role of endothelial progenitor cells and of supporting proangiogenic cells and their products, principally in humans, as diagnostic and therapeutic agents for wound repair and tissue regeneration

Immunofluorescence vessel imaging.

<p>(a) Representative photomicrographs of the matrigel implants containing eGFP-UCB ECFC derived cells and SS-AF-MSCs. (b-e) Representative photomicrographs of matrigel implant sections containing non eGFP tagged UCB ECFC derived cells and SS-AF-MSCs after staining with (b) DAPI (blue), (c) hCD31 (green), (d) following tomato lectin perfusion (red), and (e) mCD31 (white). (f) Co-localization of eGFP (green) with hCD31 (white) staining in matrigel implant sections containing eGFP-UCB ECFC derived cells and SS-AF-MSCs. (g) Representative photomicrographs of matrigel implant sections containing SS-AF-MSCs only stained for hCD31 (green) and mCD31 (white) antigens, but where hCD31 was not detected. (h) Representative photomicrographs of matrigel implant sections containing UCB ECFC derived cells only, stained for hCD31 (green) and mCD31 (white) antigens.</p

Analysis of angiogenic factors secreted by SS-AF-MSCs, BM-MSCs and hDFs in vitro using proteome arrays.

<p>(a-c) Representative proteome profiler arrays for (a) SS-AF-MSCs, (b) BMSCs and (c) hDFs respectively; (d) corresponding names of each molecule within the array summarized in tabular form.</p

Quantitating vessel formation in in vivo studies.

<p>(a) Histological evaluation of vessels containing SS-AF-MSCs and UCB ECFC derived cells, harvested 14 days post-implantation and stained (i) with hematoxylin/eosin and for (ii-iii) human CD31 antigen (brown stain). High-power view of a vessel containing red blood cells (arrowed) from (ii) is shown in (iii). (b) Microvessel density in matrigel implants containing combined SS-AF-MSCs, BMSCs or hDFs with UCB ECFC derived cells, SS-AF-MSCs only, BMSCs only, hDFs only or UCB ECFC derived cells only. Vessel number (vessels/mm²) was estimated using Image J 1.38× software. Statistical analysis was performed using Student’s <i>t</i> test. (c) Vessel diameter estimation in matrigel implants containing SS-AF-MSCs, BMSCs or hDFs and UCB ECFC derived cells, SS-AF-MSCs only, or UCB ECFC derived cells only using Image J 1.38× software, (*p<0.05 Student’s <i>t</i> test). A minimum of 15 fields of view (40x) were analyzed from each photograph. Error bars indicate S.D. of the mean for 10 photographs from each group.</p

A novel function for the haemopoietic supportive murine bone marrow MS-5 mesenchymal stromal cell line in promoting human vasculogenesis and angiogenesis

The bone marrow contains specific microenvironmental stem cell niches that maintain haemopoiesis. CXCL12-expressing mesenchymal stromal cells are closely associated with the bone marrow sinusoidal endothelia, forming key elements of the haemopoietic stem cell niche, yet their ability to regulate endothelial function is not clearly defined. Given that the murine nestin+ cell line, MS-5, provides a clonal surrogate bone marrow stromal niche capable of regulating both murine and human primitive haemopoietic stem/progenitor cell (HSC/HPC) fate in vitro, we hypothesized that MS-5 cells might also support new blood vessel formation and function. Here, for the first time, we demonstrate that this is indeed the case. Using proteome arrays, we identified HSC/HPC active angiogenic factors that are preferentially secreted by haemopoietic supportive nestin+MS-5 cells, including CXCL12 (SDF-1), NOV (CCN3), HGF, Angiopoietin-1 and CCL2 (MCP-1). Concentrating on CXCL12, we confirmed its presence in MS-5 conditioned media and demonstrated that its antagonist in receptor binding, AMD-3100, which mobilizes HSC/HPCs and endothelial progenitors from bone marrow, could significantly reduce MS-5 mediated human vasculogenesis in vitro, principally by regulating human endothelial cell migration. Thus, the clonal nestin+MS-5 murine bone marrow stromal cell line not only promotes human haemopoiesis but also induces human vasculogenesis, with CXCL12 playing important roles in both processes

Analysis of angiogenic factors secreted by UCB ECFC derived cells in vitro.

<p>(a) Representative proteome profiler array for UCB ECFC derived cell-CM; (b) corresponding names of each molecule within the array summarized in tabular form; (c) Relative expression levels of angiogenic factors in UCB ECFC derived cell-CM. Values are normalized to positive controls. Values are means ± S.D. for three independent experiments, (*p<0.05 Student’s <i>t</i> test).</p

Role of IL-8, PDGF-AB/BB and MMP9 molecules from conditioned media in migration, proliferation and ability of tubule formation of UCB ECFC derived cells.

<p>(a) Histograms showing the migration of UCB ECFC derived cells towards SS-AF-MSC-conditioned medium (CM), EGM-2 medium, control medium (EBM-2, 0.5% (v/v) FCS), SS-AF-MSC-CM+IL8 neutralizing Ab, SS-AF-MSC-CM +PDGF-AB/BB neutralizing Ab or SS-AF-MSC-CM+MMP9 inhibitor (inh). Values are means ± S.D. for three independent experiments (*p<0.05 Student’s <i>t</i> test). (b) Examination of the proliferation rate in vitro of UCB ECFC derived cells under the same conditions. Control medium with recombinant (rec) IL-8 or PDGF-AB/BB was also included. Values are means ± S.D. for three independent experiments, (*p<0.05 Student’s <i>t</i> test). (c) In vitro angiogenesis matrigel assay for UCB ECFC derived cells under the respective conditions for estimation of the number of (i) tubules and (ii) junctions formed. Error bars indicate S.D. of the mean for 10 (5x) photographs from each group (*p<0.05 Student’s <i>t</i> test).</p