Fig. 2

Dynamic-sprouting angiogenesis synchronized with MSC migration and adhesion. A Representative 2D fluorescent microscopic images in time series during mono- and co-cultures for 10 days. Monoculture did not show the clear angiogenic sprout, whereas co-culture with MSCs accelerated the formation of mature sprouts. MSCs (green) are encapsulated in the collagen gel toward microvessels (red) and attach to the vessel walls’ surface at an early stage. The elongation of sprouts was promoted after day 4. B Spatial correlation analysis between HUVECs and MSCs. The distribution of HUVECs and MSCs from one co-culture sample is shown as density maps and scatter plots by analyzing the co-culture images shown in (A). Pearson’s correlation coefficients, r, were calculated for each day and are shown in the plots. The correlation was constantly high (> 0.7) after day 4. C–E Orientation analysis of MSCs around the microvessels. Scale bars: 100 μm. C Definitions of MSC location and orientation around microvessels to analyze their behavior around microvessels. MSCs beside the vessel walls within a marginal region were considered “neighbor” cells, while MSCs outside of the margin were considered “distant” cells. D Image processing in 2D to quantify the location and orientation of MSCs during co-culture using one sample. After the red channel images were processed, the microvessel’s surface edges were extracted and smoothed (red or thick white lines). After the green channel images were processed, the location and orientation of MSCs were quantified. Please note that the areas in the two pictures are from the same ROI on day 1 and day 10. See “Materials and methods” section. Scale bars: 100 μm. E Orientation analysis of “neighbor” or “distant” MSCs by fitting ellipses to the binarized MSC objects. Histograms with thick black lines or filled with green represent “neighbor” or “distant” MSCs, respectively. The numbers in the directional charts represent the number of objects that were detected by the particle analysis. The tilt angles of “neighbor” MSCs were constantly smaller than those of “distant” MSCs through co-culture. Statistical significances between “distant” and “neighbor” MSCs were evaluated using a set of time-lapse images from one HUVEC-CapSC co-culture sample with two-way ANOVA followed by the Bonferroni’s post hoc tests using this fluorescent image set (n = 1). *p < 0.05; **p < 0.01