*2.5. Prolonged Treatment with Histochrome Attenuates Cellular Senescence in hCPCs*

Several antioxidants have been reported to be associated with inhibition of cellular senescence [29–31]. To examine whether histochrome affects cellular senescence, we investigated the long-term effect of histochrome treatment on an in vitro culture of hCPCs. Evaluation of senescence-associated β galactosidase (SA-β-gal) activity in senescent hCPCs (passage 13) revealed a significant increase in the number of SA-β-gal positive cells (sene; 40.7% ± 3.07%) (Figure 5). However, increased SA-β-gal positive cells were significantly downregulated (sene+Histochrome; 31.3% ± 2.87%) on prolong treatment of hCPCs with histochrome. This suggested that long-term treatment of histochrome alleviates replicative cellular senescence in hCPCs.

**Figure 5.** Effect of prolonged treatment with histochrome on hCPCs senescence. (**A**) Representing images of senescence-β-galactosidase (SA- β-gal) stained hCPCs (Scalebar = 50 μm). (**B**) SA- β-gal positive cells were quantified and presented as a graph (\*\*\* *p* < 0.001, versus Young; ### *p* < 0.001, versus Sene) Error bars indicate S.E.M. Abbreviation: Sene, senescent hCPCs (passage 13); sene + histochrome, prolonged treatment with histochrome (till passage 13).

#### **3. Discussion**

To enhance the engraftment rate of transplanted cells, several studies have been performed focusing on cell priming, using physical stimulations such as heat shock, and chemical stimulations using natural products and growth factors. Recently, stem cell priming using natural products has been proposed as a new strategy to enhance the cell activity and promote stable and efficient cell functioning. In our previous study, we demonstrated that pretreatment with fucoidan, a marine-sulfated polysaccharide derived from seaweeds, inhibits cellular senescence and promotes neo-vasculogenic potential [32].

In this study, we identified a novel priming factor for enhancing cell therapy potentials against oxidative stress. A recent study has revealed that echinochrome A promotes cardiomyocyte differentiation of mouse embryonic stem cells via direct binding to serine-threonine kinase PKCι and inhibition of its activity [33]. We are the first to report the effects of histochrome on hCPCs. In the present study, we demonstrated that pretreatment with histochrome enhanced the survival of hCPCs against oxidative stress. Further, pretreatment with different concentrations (0 μM, 5 μM, 10 μM, 15 μM, and 20 μM) of histochrome did not alter the expression of multipotent cardiac stem cell markers such as c-kit (Histo 0 μM; 99.9% ± 0.1%, Histo 5 μM; 99.9% ± 0.1%, Histo 10 μM; 99.8% ± 0.1%, and Histo 20 μM; 99.9% ± 0.1%, respectively); CD29 (Histo 0 μM; 99.9% ± 0.1%, Histo 5 μM; 99.6% ± 0.1%, Histo 10 μM; 99.1% ± 0.2%, and Histo 20 μM; 93.7% ± 5.6%, respectively); CD166 (Histo 0 μM; 88.9% ± 1.5%, Histo 5 μM; 80.2% ± 2.7%, Histo 10 μM; 80.9% ± 1.5%, and Histo 20 μM; 79.8% ± 2.1%, respectively); CD105 (Histo 0 μM; 95.0% ± 0.8%, Histo 5 μM; 98.5% ± 0.1%, Histo 10 μM; 97.8% ± 0.1%, and Histo 20 μM; 99.7% ± 0.1%, respectively); and CD44 (Histo 0 μM; 97.8% ± 1.0%, Histo 5 μM; 99.5% ± 0.1%, Histo 10 μM; 99.4% ± 0.2%, and Histo 20 μM; 99.6% ± 0.1%, respectively). In contrast, histochrome-treated hCPCs showed negative expression of hematopoietic markers such as CD34 (Histo 0 μM; 0.7% ± 0.01%, Histo 5 μM; 1.1% ± 0.1%, Histo 10 μM; 0.6% ± 0.1%, and Histo 20 μM; 1.0% ± 0.2%, respectively) and CD45 (Histo 0 μM; 0.1% ± 0.1%, Histo 5 μM; 0.1% ± 0.06%, Histo 10 μM; 0%, and Histo 20 μM; 0%, respectively).

In ischemic heart disease, ROS is produced upon reperfusion [34]. Excessive ROS formation promotes cell death which induces development and progression of cardiovascular disease [35]. The generation of ROS is associated with alteration in electrophysiology leading to myocardial ischemia [36]. In addition, mitochondrial ROS scavenging has been reported for its potential to prevent cardiac failure [37]. It is necessary to target intracellular ROS and mitochondrial ROS to prevent cell death caused by oxidative stress. Histochrome showed high intracellular ROS scavenging activity and reduced production of mitochondrial superoxide in hCPCs. Furthermore, pretreatment with histochrome revealed an anti-apoptotic effect against H2O2-induced cell death.

The Bcl-2-family members (Bcl-2, Bcl-xL, Bax) are predominant regulators of cell cycle progression and apoptosis [38]. Specifically, Bcl-2 and Bcl-xL are well-known negative regulators of apoptosis which promote cell survival [39]. On the other hand, the apoptosis regulator Bax inhibits cell survival [40] and cell cycle progression [41]. In this study, pretreatment with histochrome upregulated the expression of Bcl-2 and Bcl-xl and downregulated that of Bax under the H2O2-induced oxidative stress condition. DNA damage induces the formation of double stranded breaks (DSBs) and stimulates phosphorylation of histone H2AX [42]. Subsequently, γ-H2A.X is used as a reference marker for DNA damage. In our study, pretreatment of hCPCs with histochrome reduced the expression of the DNA damage marker, γH2A.X foci under the oxidative stress condition. Thus, we concluded that histochrome prevents ROS-mediated DNA damage in hCPCs (Figure 6).

**Figure 6.** Schematic representation of cytoprotective effects of histochrome against H2O2-induced cell death via reduction of DNA damage and activation of survival signaling.

Any cell undergoes a limited number of divisions and thus, following a certain number of cell cycles, it enters an irreversible cell cycle arrest which is referred to as cellular senescence [43]. Stem cell therapy for regeneration of tissues requires over hundreds of millions of cells [44]. Repetitive in vitro cultures are essential and cellular senescence is a major threat in such stem cell therapies. Thus, preventing cellular senescence is a promising strategy in stem cell therapy. SA-β-gal assay revealed that histochrome delayed the progression of cellular senescence in hCPCs. Thus, our study suggests that histochrome can be a potential effective strategy to overcome cellular senescence.

Overall, our present study demonstrated that histochrome protects hCPCs against oxidative stress by regulating cell survival signaling. Furthermore, histochrome prevents cellular senescence of hCPCs. Thus, our study presents a simple and effective strategy to improve cell survival in post-transplanted CPCs under ischemic oxidative stress conditions and improve the efficiency in myocardial regeneration.
