Research Paper Volume 8, Issue 1 pp 16—30

Vesicular Galectin-3 levels decrease with donor age and contribute to the reduced osteo-inductive potential of human plasma derived extracellular vesicles

Figure 2. Galectin-3 and osteogenic differentiation capacity. (A) Spearman correlation of Galectin-3 protein levels before induction of osteogenic differentiation to corresponding mineralization capacity of ASCs. (B) Comparison of the mineralization capacity of ASCs from different donors after induction of osteogenesis to the corresponding donors age reveals a not significant trend towards impaired osteogenic differentiation capacity of donors older than 40. N=9 (C) Spearman correlation of Galectin-3 protein levels before induction of osteogenic differentiation to the age of the ASC donors. (D) Relative fold change of Galectin-3 mRNA levels over a time course of 21 days of differentiated und undifferentiated ASCs was evaluated by qPCR and normalized to GAPDH. Galectin-3 mRNA transcription was significantly increased and peaked at day 9 in ASCs which were induced to undergo osteogenesis (grey squares) compared to undifferentiated ASCs (black dots). N=4 (E) Detection of total Galectin-3 and GAPDH protein levels by Western blot in protein lysates derived from ASCs transfected with siRNA against Galectin-3 (siGal3) or with the corresponding non-targeting control (siC). (F) Mineralization of siGal3 or siC transfected ASCs was evaluated by Alizarin Red staining. The released dye was quantified by microplate reader at 425nm. Osteogenic differentiation was reduced in siGal3 transfected ASCs compared to control transfected cells. (G) ALP activity was quantified by microplate reader at 405nm. Activity was significantly decreased in siGal3 transfected ASCs compared to non-targeting control transfected cells. (H) Relative fold change of ON mRNA levels of transfected ASCs was evaluated by qPCR and normalized to GAPDH. ON mRNA levels were significantly decreased in siGal3 transfected ASCs compared to non-targeting control transfected cells. (I) Detection of Galectin-3 protein levels normalized to GAPDH protein levels by Western blot in protein lysates derived from ASCs transfected with Galectin-3 overexpression construct (Gal3) or with the corresponding empty vector control (C). (J) Mineralization of Gal3 or control transfected ASCs was evaluated by Alizarin Red staining. The released dye was quantified by microplate reader at 425nm. Osteogenic differentiation was enhanced in Gal3 transfected ASCs compared to control transfected cells (C). (K) ALP activity was quantified by microplate reader at 405nm. Activity was significantly increased in Gal3 transfected ASCs compared to control transfected cells. (L) Relative fold change of ON mRNA levels of ASCs was evaluated by qPCR and normalized to GAPDH. ON mRNA levels were significantly increased in Gal3 transfected ASCs compared to control transfected cells. (D, F-H, J-L) *: p<0.05, **: p<0.01, ***: p<0.001 in comparison to indicated group. Data are presented as mean values ± SD and were statistically analysed using unpaired t test, n=4.