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Updated: Oct 17, 2025
In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells
Published on: June 12, 2017
Autophagy stimulation delayed biological aging and decreased cardiac differentiation in rabbit mesenchymal stem cells
Mehdi Hassanpour1,2, Omid Cheraghi3, Reza Rahbarghazi4
1Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.
Abstract:
Cardiovascular disease (CVD) is a type of disease that affects the function of cardiac-vascular tissues. This study aimed to consider the possible effects of autophagy, as an intrinsic catabolic pathway of cells, on the differentiation and aging process of mesenchymal stem cells (MSCs). In this study, bone marrow-derived MSCs were obtained from rabbit bone marrow aspirates. The stemness feature was confirmed by using flow cytometry analysis Cells at passage three were treated with 50 μM Metformin and 15μM hydroxychloroquine (HCQ) for 72 hours. The intracellular accumulation of autophagolysosomes was imaged using LysoTracker staining. Protein levels of autophagy (LC3II/I ratio), aging (Klotho, PARP-1, and Sirt-1) effectors, and cardiomyocyte-like phenotype (α-actinin) were studied by western blotting. Based on our findings, flow cytometry analysis showed that the obtained cells expressed CD44 and CD133 strongly, and CD31 and CD34 dimly, showing a typical characteristic of MSCs. Our data confirmed an increased LC3II/I ratio in the metformin-received group compared to the untreated and HCQ-treated cells (P < 0.05). Besides, we showed that the incubation of rabbit MSCs with HCQ increased cellular aging by induction of PARP-1 while Metformin increased rejuvenating factor Sirt-1 comparing with the normal group (P < 0.05). Western blotting data showed that the autophagy stimulation response in rabbit MSCs postponed the biological aging and decreased the differentiation potential to the cardiac cells by diminishing α-actinin comparing with control cells (P < 0.05). In summary, for the informants in this study, it could be noted that autophagy inhibition/stimulation could alter rabbit MSCs aging and differentiation capacity.
Insights
Autophagy modulation impacts mesenchymal stem cell (MSC) aging and cardiac differentiation. Inhibiting autophagy accelerates aging, while stimulating it rejuvenates cells but reduces their cardiac differentiation potential.
Area of Science:
- Cell Biology
- Stem Cell Research
- Cardiovascular Disease Research
Background:
- Mesenchymal stem cells (MSCs) are crucial for tissue repair and regeneration.
- Autophagy, a cellular degradation process, plays a role in cell fate.
- Understanding autophagy's role in MSCs is vital for regenerative medicine and cardiovascular disease (CVD) therapies.
Purpose of the Study:
- To investigate the effects of autophagy modulation on mesenchymal stem cell (MSC) aging.
- To examine how autophagy influences the differentiation potential of MSCs towards a cardiomyocyte-like phenotype.
- To explore the therapeutic implications for cardiovascular disease (CVD) by modulating MSCs.
Main Methods:
- Bone marrow-derived MSCs were isolated from rabbits and characterized via flow cytometry.
- Cells were treated with Metformin (autophagy stimulant) and hydroxychloroquine (HCQ, autophagy inhibitor).
- Autophagy markers (LC3II/I), aging markers (Klotho, PARP-1, Sirt-1), and cardiac differentiation marker (α-actinin) were assessed using western blotting and LysoTracker staining.
Main Results:
- Metformin increased the autophagy marker LC3II/I ratio and the anti-aging factor Sirt-1.
- HCQ treatment increased the aging marker PARP-1.
- Autophagy stimulation with Metformin delayed MSC aging but reduced their potential for cardiac differentiation, indicated by decreased α-actinin expression.
Conclusions:
- Autophagy modulation significantly affects MSC aging and differentiation.
- Inhibiting autophagy accelerates MSC aging.
- Stimulating autophagy rejuvenates MSCs but impairs their ability to differentiate into cardiac cells, suggesting a complex role in cardiovascular applications.
