Fig 1.
Schematic illustration of the experimental set-up.
Fig 2.
Characterisation of exosomes by transmission electron microscopy (A) and nanoparticle tracking analysis (B).
(A) A1, A4 and A7: Survey transmission electron micrographs of exosomes from the Exo_P6, Exo_D3 and Exo_D21 groups. Exosomes are approximately 30–150 nm and mainly display a rounded shape. A4-A9: Details of selected exosomes without (A2, A5 and A8) or with (A3, A5 and A9) labelling of 10 nm gold-labelled anti-CD63 antibodies. Scale bar: top panel (A1, A4 and A7), 100 nm; bottom panel (A2-A3, A5-A6 and A8-A9), 50 nm. (B) B1-B3: Distribution of particles from all nine captures of three biological replicates in each group of exosomes. B4-B6: Average concentration/size distribution of all nine captures in each group of exosomes. B7-B8: Mean and mode of particle size of each group of exosomes. B9: Mean concentration of particles in each group of exosomes. Error bars in B7-B9 denote standard error mean, SEM; n = 3 biological replicates.
Fig 3.
Evaluation of osteogenic differentiation of hMSCs after exosome treatment.
(A) ALP activity 14 d after treatment with exosomes or the appropriate controls; (B) Quantification of calcium in ECM after 21 d; (C) Quantification of phosphate in ECM after 21 d. In (A-C), bars indicate mean values whereas error bars denote standard errors of the mean (SEM). Small letters a, b and c represent statistical significance when compared with NCtrl_D0, NCtrl_D14/D21 and Sexo_Ctrl respectively, based on Bonferroni-corrected p value < 0.05. n = 3 independent experiments; (D) Alizarin red staining after 21 d.
Fig 4.
Internalisation of exosomes in hMSCs.
Confocal micrographs of hMSCs incubated for 24h with A) PBS (negative control); B) Exo_P6; C) Exo_D3 and D) Exo_D21. PKH67-stained exosomes are detected mainly in the cytoplasm of some of the cells. The intensity varied between individual cells. No PKH67-stained material was found in the negative control. A1-D1, blue channel; A2-D2, green channel; A3-D3, transmission (TD) channel; A4-D4, merged channels. Blue, DAPI-stained nucleus; Green, PKH67-stained exosomes. Scale bar: 20 μm.
Fig 5.
MicroRNA profiles of exosomes and hMSCs during expansion and osteogenic differentiation.
(A) Altered microRNA profiles of exosomes and hMSCs during expansion (P6) and osteogenic differentiation (D3 and D21) of hMSCs and (B) Altered microRNA profiles of exosomes derived from the expansion (P6) and osteogenic differentiation (D3 and D21) of hMSCs. The heat map diagram shows the result of the two-way hierarchical clustering of microRNAs and samples. The clustering is performed on all samples and on the top 50 microRNAs with the highest standard deviation. The normalised (dCq) values have been used for the analysis. Each row represents one microRNA and each column represents one sample. The microRNA clustering tree is shown on the left. The colour scale shown at the bottom illustrates the relative expression level of a microRNA across all samples: red colour represents an expression level above the mean, green colour represents expression lower than the mean.
Table 1.
Differentially expressed microRNAs in exosomes derived from hMSCs during expansion and osteogenic differentiation.
Table 2.
Summary of biological pathways enriched by differentially expressed microRNAs.
Table 3.
Enriched KEGG biological pathways related to osteogenic differentiation and general regulation mechanism.