Activation of circulating myeloid cells by induced UCBMSCs.

<p><b>A</b>) Characterization of myeloid (CD45⁺) cells derived from human peripheral venous blood and their progeny in cell culture. Representative flow cytometric dot blots showing co-expression of CD133 and VEGFR-2 in isolated cell fraction. Quadrants were first placed on FL1/FL2 dot plot based on negative control staining for the subsequent detection of dual labelled cells. Representative images showing a colony-forming unit-Hill grown over human fibronectin (top left) and detection of CD34 (top right), VEGFR-2 (bottom left) and vWF (bottom right) after 14 days in culture. Histogram shows the transcriptional levels of CD45, CD31 and CD34 genes in the extracted myeloid cell subset quantified by qRT-PCR. Data are expressed as mean ± S.D of Ct values and each value was normalized to the corresponding GAPDH value. N = 5 and bars = 20 µm. <b>B</b>) Histogram showing results from transwell chemotaxis assays used to quantify SDF-1α-mediated migration of circulating myeloid cells. Human recombinant SDF-1α was used as positive control. N = 3. *<i>P</i> = 0.02 and ^†<i>P</i> = 0.016. <b>C</b>) Representative images displaying Alexa488-Ac-LDL-labelled circulating myeloid cells in Matrigel-free conditions, and a representative ‘hybrid’ cell network comprising EGM-2-induced UCBMSCs and Alexa488-Ac-LDL⁺myeloid cells after co-culture in Matrigel during 7 h. Note that fluorescent, circulating myeloid cells with characteristic amoeboid morphology were not visible as single cells, but were closely associated with or aligned along the cell network developed by induced UCBMSCs. Bars = 20 µm (left) and 10 µm (right). A minimum of 10 microscopic fields per experiment (N = 3) were registered under a confocal microscope. CM = conditioned medium.