Alteration of fatty acid oxidation by increased CPT1A on replicative senescence of placenta-derived mesenchymal stem cells

Abstract

Background: Human placenta-derived mesenchymal stem cells (PD-MSCs) are powerful sources for cell therapy in regenerative medicine. However, a limited lifespan by senescence through mechanisms that are well unknown is the greatest obstacle. In the present study, we first demonstrated the characterization of replicative senescent PD-MSCs and their possible mitochondrial functional alterations.

Methods: Human PD-MSCs were cultured to senescent cells for a long period of time. The cells of before passage number 8 were early cells and after passage number 14 were late cells. Also, immortalized cells of PD-MSCs (overexpressed hTERT gene into PD-MSCs) after passage number 14 were positive control of non-senescent cells. The characterization and mitochondria analysis of PD-MSCs were explored with long-term cultivation.

Results: Long-term cultivation of PD-MSCs exhibited increases of senescent markers such as SA-β-gal and p21 including apoptotic factor, and decreases of proliferation, differentiation potential, and survival factor. Mitochondrial dysfunction was also observed in membrane potential and metabolic flexibility with enlarged mitochondrial mass. Interestingly, we founded that fatty acid oxidation (FAO) is an important metabolism in PD-MSCs, and carnitine palmitoyltransferase1A (CPT1A) overexpressed in senescent PD-MSCs. The inhibition of CPT1A induced a change of energy metabolism and reversed senescence of PD-MSCs.

Conclusions: These findings suggest that alteration of FAO by increased CPT1A plays an important role in mitochondrial dysfunction and senescence of PD-MSCs during long-term cultivation.

Keywords: CPT1A; Fatty acid; Mitochondria; Placenta-derived mesenchymal stem cell; Senescence.

Fig. 1

Characterization of senescent PD-MSCs during long-term cultivation. a Expression of stemness related genes was analyzed by qRT-PCR in early and late passage PD-MSCs. b In vitro proliferation of early and late passage PD-MSCs for 4 days were analyzed by Ez-Cytox cell viability assay kit. c Quantitation of cells in the respective phases of cell cycle by FACS analysis. d Expressions of p21 protein were assayed by Western blotting in early and late passage PD-MSCs. e Senescence of PD-MSCs was detected by senescence-associated-β-galactosidase staining (magnification, × 20). f The enlarged cell size was significantly counted and observed in late passage PD-MSCs by Image J program (magnification, × 200). The data were representative of three independent experiments and expressed as means ± S.D. An asterisk indicates P < 0.05 versus early passage

Fig. 2

Differentiation potential of senescent PD-MSCs. a After early and late passage PD-MSCs and hTERT+ were differentiated for 2 weeks, the expression of genes related to osteogenesis and adipogenesis was measured by RT-PCR. b Osteogenic and adipogenic lineages were determined by von Kossa (magnification, × 100) and Oil-Red O staining (magnification, × 200), respectively. The data were representative of three independent experiments

Fig. 3

Effect of cell survival and death pathway in senescent PD-MSCs during long-term cultivation. a The expression of p-AKT/AKT and p-ERK1/2 gene related to survival pathway were analyzed by Western blot in early and late passage PD-MSCs. b The expression of Bax and Bcl2 gene related to pro-/anti-apoptosis regulator were analyzed by Western blot in early and late passage PD-MSCs. c The gene expression of p-mTOR/mTOR, PI3K-p100/-p85, ATG 5–12, and LC3 I/II related to autophagy pathway was analyzed by Western blot in early and late passage PD-MSCs. The data were representative of three independent experiments and presented by Image J software and expressed as means ± S.D. An asterisk indicates P < 0.05 versus early passage

Fig. 4

Effect of mitochondrial dysfunction in senescent PD-MSCs during long-term cultivation. a Representative confocal images showed mitochondrial superoxide levels (MitoSox Red) and mitochondrial content (MitoTracker Green) in early passage, late passage, and hTERT+ PD-MSCs (magnification, × 200). b The ROS levels of early and late passage PD-MSCs were measured with the fluorescent dyes DCFDA. c The mitochondrial mass of early and late passage PD-MSCs was analyzed by using the NAO, respectively. d The mitochondrial membrane potential of early and late passage PD-MSCs was detected by JC-1 fluorescent dye which is measured as the ratio of the J-aggregated (red) to the JC-1 monomeric (green) forms. e Relative ATP levels of early and late passage PD-MSCs were analyzed by ATP production assay kit. f XF analyses revealed the oxygen consumption rates of fatty acid, glucose, and glutamine pathways in early and late passage PD-MSCs. The data were representative of three independent experiments and expressed as means ± S.D. An asterisk indicates P < 0.05 versus early passage

Fig. 5

Effect of increased CPT1A in senescent PD-MSCs during long-term cultivation. a The levels of CPT1A and ACC mRNA were determined by using qRT-PCR. b Protein levels of p-AMPK/AMPK, p-ACC/ACC, PPARα, and CPT1A related to fatty acid pathway of early and late passage PD-MSCs were analyzed by Western blotting. c SA-β-galactosidase activity of late passage PD-MSCs treated with CPT1A siRNA was assayed by SA-β-galactosidase staining (scale bar, × 40). The SA-β-galactosidase stained cells were counted by Image J software. The data were representative of three independent experiments and expressed as means ± S.D. An asterisk indicates P < 0.05 versus early passage and control group

Fig. 6

Effect of downregulated CPT1A on mitochondrial metabolism in senescent PD-MSCs. a To analyzed mitochondrial function of PD-MSCs with CPT1A siRNA, the mitochondrial mass, b the ROS levels, c mitochondrial membrane potential, and d ATP production were analyzed in late passage PD-MSCs treated with CPT1A siRNA such as CPT1A inhibitor compared to control. The glycolytic ability of senescent PD-MSCs treated with etomoxir such as CPT1A inhibitor was determined by XF24 analyzer. e The extracellular acidification rate (ECAR) of early and late passage PD-MSCs was analyzed by using glycolysis-XF assay kit and f also determined in late passage PD-MSCs treated with etomoxir. g The mitochondrial oxygen consumption rate (OCR) of early and late passage was determined by using mitochondrial stress-XF assay kit and h also determined after the treatment with etomoxir. i The oxygen consumption rates (OCR) of fatty acid, glucose, and glutamine pathways were analyzed in early passage PD-MSCs and j late passage PD-MSCs treated with siRNA and etomoxir. The data were representative of three independent experiments and expressed as means ± S.D. An asterisk indicates P < 0.05 versus early passage. Red, early; blue, late; pink, hTERT+. Red, control; blue, etomoxir; pink, CPT1A siRNA