Fig. 4

Angiogenic activity of PlaMSC-exo. a Endothelial tube formation assay revealed reduced angiogenic activity of PlaMSC-CM after depletion of the exosome fraction. White, black, and gray bars show D-MEM, PlaMSC-CM, and PlaMSC-CM without exosomes (w/o exo), respectively. b PlaMSC-exo enhanced angiogenesis with comparable efficiency to that of PlaMSC-CM. White, gray, and black bars shows D-MEM, PlaMSC-CM, and PlaMSC-exo, respectively. c Migration of endothelial cells enhanced by PlaMSC-exo. Endothelial cell migration was evaluated using a scratch wound healing assay. Pictures of the wound area in the presence or absence of PlaMSC-exo (equivalent to 0, 0.2, 1.0, or 5.0 μg of protein) taken under a microscope every 3 h for 12 h. Representative data from three independent experiments. d Scratch wound healing assay showing PlaMSC-exo enhanced endothelial cell migration. Wound area filling by migrating cells was quantitated 12 h after treatment at three selected points (upper, mid, and lower portion of the wound). Quantitative data from the scratch wound healing assay shown in pixels. Data expressed as mean ± SE (0 μg, n = 4; 0.2 μg, 1.0 μg, and 5.0 μg, n = 5 in each experiment). *P < 0.05, **P < 0.01, determined by Dunnett’s tests. NS not significant. e Fluorescent microscope image showing incorporation of PKH67-labeled PlaMSC-exo (upper panel). HUVECs were exposed to PlaMSC-exo or control solution for 24 h and fixed, which was subjected to flow cytometric analysis of the incorporation of PlaMSC-exo by endothelial cells (lower panel). Mean frequency of fluorescent signals indicated. f PlaMSC-exo enhance expression of angiogenesis-related genes in HUVECs. HUVECs were exposed to PlaMSC-exo (black bar) or PBS (white bar) for 72 h, and then harvested for isolation of total RNA, which was subjected to qRT-PCR analyses of angiogenesis-related gene expression. Each experiment independently repeated three times. Expression level of each mRNA normalized to that of GAPDH mRNA. Data expressed as mean ± SE. *P < 0.05, determined by Student’s t tests. D-MEM Dulbecco’s modified Eagle’s medium, PlaMSC-CM conditioned medium from MSCs isolated from human term placental tissue, MSC mesenchymal stem cell, PlaMSC-exo exosomes derived from MSCs isolated from human term placental tissue, Ang-2 human angiopoietin-2, VEGFR2 human vascular endothelial growth factor receptor 2