Text

Objectives and questions: Mitochondrial quality control is crucial for human mesenchymal stem cells (hMSCs) osteogenic differentiation, with the PTEN induced kinase 1 (PINK1) - Parkin (PARK2) pathway maintaining mitochondrial integrity by mitophagy. Defective mitophagy leads to damaged mitochondria, disrupting metabolism and osteoblast maturation. Dysregulates hMCS osteogenesis and contributes to bone-loss disorders such as osteoporosis, yet the role of PINK1-PARK2-mediated mitophagy remains elusive. In this project, we investigate the role of PINK1 and PARK2 during osteogenic differentiation of hMSCs.

Material and methods: hMSCs were transfected with siRNA targeting PINK1, PARK2, PINK1+PARK2 or non-targeting control, followed by osteogenic differentiation for 21 days. Differentiation was evaluated on days (d) 7,14, and 21 by measuring ALP activity, performing Alizarin Red staining, and conducting qRT-qPCR analysis of PINK1, PARK2, ALPL, IBSP and SP7 expression. Senescence-associated β-galactosidase (SA-β-gal) activity was assessed on day 7 after transfection without differentiation. Statistical analysis was performed using one-way ANOVA for staining assays and gene expression analysis.

Results: qRT-PCR confirmed stable knockdown (KD) of PINK1, PARK2 and PINK1+PARK2 at all time points. ALP activity was significantly reduced at day 7 in all KD conditions (p=<0.0001). Alizarin Red staining at days 14 and 21 revealed the most pronounced reduction in mineralization following PARK2 KD (d14: p=0.0038; d21: p=0.0004). ALPL decreased in all KD groups at day 7, reaching statistical significance in PINK1 (p=0.0273) and PARK2 (p=0.0056) KD cells. In PINK1 KD cells, ALPL expression returned to control levels by day 14, whereas it remained reduced in PARK2 and double KD cells. No differences were detectable at day 21. SP7 expression was consistently increased at all time points in PARK2 KD cells (d7 p=0.0001; d14 p=0.0003; d21 p=0.04), while PINK1 and double KD showed no change. IBSP expression was significantly decreased in PINK1 (p=<0.0001) and PARK2 (p=0.0007) KD conditions at day 7 and returned to control levels by day 14 and 21. KD of PINK1 and/or PARK2 resulted in increased SA-β-gal activity in transfected hMSCs and accumulation of (intracellular) lipid droplets.

Discussion and conclusions: PINK1, PARK2, and double knockdown impaired early osteogenesis, with PARK2 having the strongest and most sustained effect. Reduced ALP activity and ALPL expression in all knockdowns indicate a role for both genes in osteogenic initiation, while recovery in PINK1 knockdown suggests only a transient delay. In contrast, persistent loss of ALP activity and mineralization in PARK2 knockdown points to a more pronounced role of PARK2 in osteogenic differentiation. These findings also suggest increased cellular stress and mitochondrial ROS, favoring adipogenic shift and senescence in hMSCs after PINK1 and/or PARK2 knockdown.