Modulation of osteoarthritis-related microRNAs using locked nucleic acid-antisense oligonucleotides boosts chondrogenic lineage commitment of mesenchymal progenitors

Figure 1 Lipid-assisted delivery of 3’-fluorescein-labelled locked nucleic acid-antisense oligonucleotide microRNA inhibitors into rat bone-marrow mesenchymal stem cells. A: Representative flow-cytometry profiles of rat bone-marrow mesenchymal stem cells 48 hours after lipofection with locked nucleic acid-antisense oligonucleotide microRNA inhibitors, used either individually or in predefined combinations. Left panels show the FSC/SSC gating and example dot plots; right panels display the corresponding fluorescence histograms for the 3’-fluorescein (FAM) label. The green numbers indicate the percentage of FAM-positive cells for each transfection condition. Controls include non-transfected cells and cells transfected with a non-specific power inhibitor. Data illustrate high intracellular uptake across conditions; B: Phase-contrast and fluorescence micrographs of adherent rat bone-marrow mesenchymal stem cells 48 hours post-transfection (20 ×). Merged images showing predominantly nuclear signal with a minor cytoplasmic component, consistent with efficient intracellular trafficking of the labelled locked nucleic acid-antisense oligonucleotides; C: Relative expression of the indicated microRNAs measured by quantitative real-time polymerase chain reaction 48 hours after transfection. Data are normalised to U6 and expressed as 2^-ΔΔCt relative to CTRL (power inhibitor). Bars represent mean (n = 3 independent experiments) and error bars = SEM. Statistical analysis: Two-tailed Student’s t-test vs CTRL; ^aP < 0.05, ^bP < 0.01, ^cP < 0.005.