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Volume 6, Issue 3, Pages 284-291 (March 2016) Extracellular Vesicles Derived from Osteogenically Induced Human Bone Marrow Mesenchymal Stem Cells Can Modulate Lineage Commitment Margarida Martins, Diana Ribeiro, Albino Martins, Rui Luís Reis, Nuno Meleiro Neves Stem Cell Reports Volume 6, Issue 3, Pages 284-291 (March 2016) DOI: 10.1016/j.stemcr.2016.01.001 Copyright © 2016 The Authors Terms and Conditions

Stem Cell Reports 2016 6, 284-291DOI: (10.1016/j.stemcr.2016.01.001) Copyright © 2016 The Authors Terms and Conditions

Figure 1 Characterization of Osteogenically Induced hBMSC-EVs EVs were isolated from the CM of hBMSCs under osteogenic medium (OM) or RUNX2 transcriptionally activated in basal medium (BM, RUNX2). (A and B) EV size and charge. Representative size distribution profile highlighting the mean particle diameters (A); and EV size within the peak with main intensity (left axis) and charge (right axis), shown as the mean ± SEM (three donors, three technical replicates) (B). (C) EV morphology. Representative atomic force micrographs (top) and graphical representation of individual EV size (a–c) (bottom). (D) EV enrichment in CD9, CD63, and CD81 analyzed by flow cytometry. Representative histograms of labeled (open peak) and unlabeled (filled peak) EVs and the median fluorescence intensity ratio (median fluorescence intensity of the detected molecule divided by that of the non-labeled control), shown as the median (interquartile range [IQR]) (three donors, two technical replicates). (E) EV yield in terms of normalized CD63-positive EVs (left axis) and normalized total EV protein (right axis), shown as the mean ± SEM (three donors, two and three technical replicates, respectively). a denotes significant differences compared with BM. Stem Cell Reports 2016 6, 284-291DOI: (10.1016/j.stemcr.2016.01.001) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Effect of Osteogenically Induced hBMSC-EVs on the Biochemical Parameters of Homotypic Cells EVs derived from hBMSCs under osteogenic medium (OM), OM-EVs, or RUNX2 transcriptionally activated in basal medium (BM), RUNX2-EVs, were transferred to uncommitted hBMSCs without further supplementation. Cell lysates (A) DNA concentration, (B) normalized protein, (C) normalized ALP activity are shown as Tukey boxplots (three donors, three technical replicates). The black dots denote outliers; letters denote significant differences compared with: a, BM; b, OM; c, RUNX2; β, OM-EVs. Stem Cell Reports 2016 6, 284-291DOI: (10.1016/j.stemcr.2016.01.001) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Effect of Osteogenically Induced hBMSC-EVs on Osteogenic Lineage-Related Markers of Homotypic Cells EVs derived from hBMSCs under osteogenic medium (OM), OM-EVs, or RUNX2 transcriptionally activated in basal medium (BM), RUNX2-EVs, were transferred to uncommitted hBMSCs without further supplementation. BM was set as control. qPCR data for relative gene expression of (A and B) transcription factors, (C) effector, and (D–G) downstream factors, are shown as means ± SEM (three donors, three technical replicates). Letters denote significant differences compared with: a, BM; b, OM; c, RUNX2. Stem Cell Reports 2016 6, 284-291DOI: (10.1016/j.stemcr.2016.01.001) Copyright © 2016 The Authors Terms and Conditions