## 4 Discussion
However, recent scientific advancements have challenged this conventional understanding and shed light on the importance of maintaining nicotinamide adenine dinucleotide (NAD) homeostasis. NAD is a coenzyme involved in numerous cellular processes, including energy metabolism, DNA repair, and the regulation of gene expression. It plays a fundamental role in cellular functions and has been found to decline with age [42]. This decline in NAD levels is linked to age-related impairments in tissue-resident stem cells. Recent studies have revealed that replenishing NAD levels can have a significant impact on the functionality of stem cells, offering potential therapeutic benefits [23; 24; 25; 26]. By restoring NAD homeostasis, it is possible to counteract the age-related functional decline observed in various tissue-resident stem cells. Replenishing NAD levels using NR has shown promising results in improving the regenerative potential of stem cells in different tissues, including skeletal muscle, brain, and skin [23; 26]. The current data reveal that NR treatment reduces terminal adipocyte differentiation of ASCs. Although we observed a higher transcription of key adipogeneic transcription factors, the terminal outcome is lower adipogenesis. Our study highlighted this interesting mechanistic regulation in the adipogeneic transcriptional players in the presence of NR. A previous study using NAD showed a reduction in terminal differentiation in hASCs [14]. Although NAD treatment showed lower PPARy expression at day 8 the levels were significantly higher at an earlier time point at day 4 [14]. NAD-mediated SIRT1 regulation contributed to terminal differentiation blockade [14]. Our results demonstrated that a pre-treatment of hASCs with NR is sufficient to reduce the differentiation cascade. The mechanism behind NAD's rejuvenating effects on stem cells is multifaceted. NAD is crucial for maintaining the balance between cellular energy production and consumption. It activates enzymes such as sirtuins, which are involved in regulating cellular metabolism, stress response, and longevity. Additionally, NAD is involved in DNA repair processes, which play a critical role in preserving the genomic integrity of stem cells. By replenishing NAD levels, it is possible to enhance the metabolic and functional properties of stem cells. This can lead to improved cellular energy production, increased DNA repair capacity, and enhanced cellular resilience to stressors. As a result, stem cells regain their regenerative potential and can contribute more effectively to tissue repair and rejuvenation. Our study showed, for the first time, the increase in the stemness signature of hASCs by NR treatment. We observed an increase in the expression of _BMP7_, _DPP4_, _DLK1_, and _CD34_. NR treatment also resulted in an increase in the surface expression of _CD34_ on hASCs. Studies have shown that adipocyte progenitor cells having higher stemness features resist differentiation pressure [33]. It is most likely that the NR-mediated increase in stemness for hASCs contributed to the resistance to differentiation. Our results demonstrated that NR treatment results in a decrease in mitochondrial activity reflected by TMRM staining and ROS activity. It is most likely that NR treatment results in increased NAD\({}^{+}\) levels that drive the ROS production via a SIRT3-dependent deacetylation and the stimulation of superoxide dismutase (_SOD2_) (Figure 5) [43; 44]. The exact mechanism of how NR enhances the stemness features in hASCs needs further investigation. The emerging field of NAD therapeutics offers a promising avenue for combating age-related decline in stem cell functionality. By targeting NAD homeostasis, researchers aim to develop interventions that can restore the youthful characteristics of stem cells and enhance their regenerative abilities. The failure of adipose tissue regeneration with age is a key contributor to the development of age-associated metabolic disorders.