Methods to Detect and Compare Cellular and Mitochondrial Changes in Senescent and Healthy Mesenchymal Stem Cells

Cellular senescence is a multifaceted process marked by irreversible cell cycle arrest in response to stressors such as DNA damage, oxidative stress, and telomere shortening, leading to significant cellular and mitochondrial alterations. These changes impact mesenchymal stem cell (MSC) function, affecting their differentiation, self-renewal, and regenerative abilities. Senescent MSCs adopt the senescence-associated secretory phenotype (SASP), characterized by the secretion of pro-inflammatory factors that propagate senescence to neighboring cells. Key features of senescent MSCs include altered morphology, reduced proliferative and differentiation capacity, and changes in their secretome. Mitochondrial dysfunction plays a central role in this process, impairing stemness, increasing oxidative stress, and contributing to cellular aging by generating reactive oxygen species (ROS). The chapter provides an overview of various methods to analyze senescent cells, including techniques to detect changes in cell proliferation, DNA damage, apoptosis, and mitochondrial function. It also highlights assays for mitochondrial alterations such as fluorescent staining, membrane potential analysis, and mitophagy evaluation. These tools are essential for understanding the complex mechanisms of cellular senescence and mitochondrial dysfunction, offering insights into aging and potential therapeutic strategies.