## 4 Discussion  
Adipose stem cells (ASCs) play a pivotal role in the regeneration of adipose tissue by engaging in self-renewal and differentiation processes, which contribute to the maintenance of the tissue's stem cell pool. ASCs possess the remarkable ability to proliferate and differentiate into adipocytes, allowing them to replace damaged or depleted adipose tissue. However, the aging process and obesity have been found to negatively impact the stemness properties of ASCs [28]. As individuals age, there is a gradual decline in the regenerative capacity of ASCs. This decline is characterized by a reduction in their ability to self-renew and differentiate into adipocytes. The loss of stemness properties in aged ASCs is often associated with decreased proliferation potential, altered gene expression profiles, and impaired differentiation capabilities [15]. These age-related changes contribute to the overall decline in adipose  
Figure 4: (**A**–E) hASCs were treated with control media or 2 mM NR for 7 days. Cells were lysed for RNA extraction and reverse-transcribed for real-time PCR quantification of _BMP7_ (**A**), _DPP4_ (**B**), _DLKI_ (**C**), _CD34_ (**D**), and _CD36_ (**E**) genes. (**F**) hASCs were treated with NR or control and analyzed by flow cytometry for the surface expression of _CD34_. (**G**) Control or NR-treated ASCs were stained with TMRM and analyzed by flow cytometry. (**H**) NR-treated ASCs were stained with DCFDA dye and analyzed by flow cytometry. * \(p\) value \(<\) 0.05, ** \(p\)** \(<\) 0.005, ** \(p\)** \(<\) 0.0005, ** \(p\)** \(<\) 0.00005.  
tissue function and may contribute to metabolic disorders associated with aging [17]. Similarly, obesity has been linked to the alteration of ASCs' stemness properties. Excessive accumulation of adipose tissue due to obesity disrupts the normal cellular microenvironment and leads to adipose tissue dysfunction. Excessive calories in the form of fat and carbohydrates result in adipose tissue expansion via an increase in adipocyte number (hyperplasia) and size (hypertrophy) [34]. A major mechanism of size expansion is the uptake of lipids through fatty acid scavenger, CD36, and the conversion of glucose to carbohydrates through lipogenesis [35; 36]. Long-term overnutrition results in fragile adipocytes and exhausted senescent ASCs that lead to adipose tissue and systemic inflammation and consequently metabolic disorder [37; 38; 36]. In this context, ASCs undergo phenotypic and functional changes that compromise their ability to effectively regenerate adipose tissue [8]. The process of differentiating ASCs into adipocytes is accompanied by increased energy demands and metabolic rates, leading to elevated mitochondrial activity and the generation of reactive oxygen species (ROS). Higher levels of ROS can drive stem cells towards differentiation, leading to senescence and cell death [39]. In our study, using hASCs as an adipocyte differentiation model, we also observed an increase in the mitochondrial activity and ROS production upon differentiation of ASCs to adipocytes. The exact mechanism of how oxidative stress inhibits adipocyte differentiation is not known, but most of the studies link impaired adipogenesis to mitochondrial dysfunction [17; 40]. Chronic inflammation and oxidative stress result in a reduction in NAD+ biosynthesis and mitochondrial levels, leading to dysfunction and ROS production. Increased ROS levels further result in the downregulation of mitochondrial complex I and II [17; 41]. In addition, oxidative stress inhibits mitotic expansion, a critical step involved in adipocyte differentiation via the repression of E2F target genes cyclin A and MCM7, most likely through an epigenetic modification [17]. In the past, the aging process has been thought to result in an irreversible decline in the functionality of stem cells. Stem cells are crucial for tissue regeneration and repair, but as organisms age, their regenerative capacity diminishes.