Fig 1.
(A) Flow cytometry histograms shows that hASC were positive for CD29, CD73, CD90 and CD105, while negative for CD14, CD34, CD146 and HDLA-DR. The percentage of cells staining for each marker (purple area) and the respective isotype-control (green line) are provided. (B) Phase-contrast microscopy imaging of hASCs exposed to adipogenic, osteogenic, or chondrogenic differentiation media. Accumulation of intracellular lipid vacuoles shown by oil red-O staining, calcium-rich extracellular matrix as evidenced by Alizarin red S, and glycosaminoglycans stained with Alcian Blue. (C) Immunocytochemistry detection shows α-SMA and collagen-IV-positive hASCs. Nuclei were stained with Hoechst dye (blue). (D) After normoxic and hypoxic preconditioning, hASC displayed spindle-shaped fibroblast-like morphology. Staining with Calcein-AM indicated that hASC viability remained unaffected by hypoxic preconditioning. Scale bars: 25μm in C and 75μm in D. Values are expressed as means ± SD of at least three independent experiments (n = 8).
Fig 2.
Characterization of HCM and NCM secretomes of human adipose mesenchymal stem cells (hASCs).
(A) Heatmap depicts the overall profile of 55 trophic factors, cytokines, and chemokines relative to protein abundance (mean pixel intensity). Each column represents a different protein, while each row represents a sample. (B) NCM and HCM were analyzed by antibody-based protein array kit. Values are normalized to positive reference spots. Data are representative of three independent experiments, and values are expressed in mean ± SD. (C) STRING analysis uncovering protein-protein interaction (PPI) network of most abundant trophic factors expressed in NCM and HCM visualized by Cytoscape. (D) David Gene Ontology analyses of proteins more abundant in hASC secretome. GO enrichment analysis showed the biological process of most representative trophic factors ranked by p-value. The top 15 processes were selected based on the Benjamini p-value (− Log10 Benjamini p-value are reported as blue bars). Fold enrichment is also reported as orange bars. * p<0.05. N = 8.
Fig 3.
Normoxic- and hypoxic- hASCs secretomes increase cell proliferation and attenuate apoptosis in HUVECs.
(A) HUVECs were cultured for 24h in medium alone (control) or supplemented with either NCM or HCM. TUNEL-positive cells were counted, and the apoptotic index was calculated as the average number of positive cells compared to the total number of cells in at least six visual fields. Values shown are the mean ± SD of at least three independent experiments. (B) HUVECs were pulse-labeled with 10 μM BrdU, cultured for up to 24 hours in the presence of NCM or HCM, and BrdU was visualized by immunocytochemistry and quantified by cell counting. (C) Representative western blots of pAKT and total AKT expression in HUVECs exposed to NCM or HCM. (D) Quantitative data representing the average values of three independent experiments. The Fig 3C was cropped to improve the clarity and conciseness of the presentation, and the full-length blots are presented in S2 Fig. Results indicate mean normalized expression relative to control ± SD. Cell experiment was repeated three times independently. *p<0.05; ** p<0.01 (n = 4).
Fig 4.
Hypoxic- (HCM) and normoxic- (NCM) hASCs secretomes accelerate in vitro cell migration and show in vitro and in vivo pro-angiogenic potential.
(A) Representative image of the migration of HUVEC cells after 0 or 18 h of incubation in HCM, NCM or control. Quantification of the percentage of variation in the wound area (% migration area). Scale bars = 250 μm (B) Representative images of EC-coated beads with NCM or HCM on fibrin gel and stained with calcein. Quantitation of EC sprouting formation and sprout length in arbitrary units formed after 4 days. Scale bars = 100μm (C) Mixture of matrigel-filled plugs containing indicated conditioned medium (NCM or HCM) or PBS (control) were injected subcutaneously in mice (n = 8). After 11 days, hemoglobin content representative of invading vessels was measured. The two-way ANOVA test and the Bonferroni post-test were used to analyze the differences among the groups. Values are expressed as mean ± SD of at least three independent experiments. (C) **p<0.01; ***p<0.001 (n = 12, 6 per group).
Fig 5.
NG2+Nestin+ pericytes surrounding the blood vessels and in close contact with the vascular wall in hASCs secretomes treated animals.
(A) Representative macroscopic images showing cutaneous wounds on days 0 and 7 after injection of control, hypoxic- (HCM) or normoxic- (NCM) secretomes. (B) HCM and NCM accelerated wound closure and microvessel density. Confocal images of the skin wound sections labeled NG2+(red)/Nestin+(green) pericytes and CD31+ blood vessels (blue). Use of pseudocolor (white) to display colocalization of NG2+(red) and nestin+(green) pericytes around CD31+microvessels (blue). (C) The extent of microvessel density was determined by assessing the CD31+ vessel area or NG2+nestin+ area in each of 4 randomly chosen high-power fields within the injury site. Scale bar, 100 μm for images in (B). Results are given as the means ± the SD. *p<0.05, **p<0.01; ***p<0.001 (n = 12, 6 per group).