Introduction

Dietary fat is a contributing factor to chronic diseases such as diabetes, obesity, and arteriosclerosis (1). Research indicates that FAs, such as omega-3 and omega-6, are advantageous for preventing and treating conditions such as Alzheimer's, cancer, and cardiovascular disease (2,3) by activating specific protein-targeting biogenesis pathways in the cytosol that mitigate numerous human diseases (4). These FAs are allocated to cells and incorporated into cellular membranes, impacting metabolism and viability (5). They participate in multiple mitochondrial functions, encompassing calcium homeostasis, gene expression, respiratory function, reactive oxygen species (ROS) generation, and apoptosis (3). The protective benefits of FAs depend on their structural characteristics, cellular uptake, metabolic processes, competition with intracellular reserves, and the intrinsic properties of their metabolites (6,7). In addition, FAs increased ROS and nitric oxide production and elevated intracellular calcium levels. They promoted endothelial NO synthase (eNOS) in mESCs (8). Mitochondria are cell powerhouses responsible for ATP synthesis and regulation of cellular metabolism, and they are engaged in cellular functions such as proliferation, differentiation, the cell cycle, reprogramming, aging, and apoptosis (9). They undergo constant fission and fusion events, which regulate mitochondrial size, number, and function (10,11). Mitochondrial biogenesis indicates the maturation and division of pre-existing mitochondria to generate new ones. It is influenced by cellular stimuli and increased energy needs, implicating diverse biological processes, such as the synthesis of both inner and outer mitochondrial membranes, mitochondrial proteins, oxidative phosphorylation, mtDNA replication, and mitochondrial fusion and fission (12-15). Mitochondrial dynamics, which encompass mitochondrial fission, fusion, biogenesis, and autophagy (mitophagy), are a dynamic, actively controlled trait that affects mitochondrial morphology (16). Alterations in MD have been linked to the control of calcium homeostasis, oxidative metabolism, and necrotic or apoptotic cell death (17). Differentiated cells and mESCs have variable mitochondrial dynamics. Mitochondria in stem cells are often described as spherical, punctate, fragmented, perinuclear, and having rarer cristae (18-20). It is commonly acknowledged that stem cells have immature mitochondria with low quantities of ATP, ROS, and OXPHOS (21). This mitochondrial condition is consistent with the general role of stem cells, which is to maintain the nuclear, epigenomic, and mitochondrial genomes of differentiated cells. Thus, mESCs are protected from ROS-induced genotoxicity by their immature mitochondria, which may have more severe and pervasive effects on mESCs than differentiated cells. Changes in mitochondrial morphology, notably the appearance of larger, elongated, and tubular forms, coincide with an increase in mitochondrial content during cell differentiation into final cell types. Differentiated cells have tightly packed mitochondria, some of which are widely dispersed throughout the cytoplasm and have many branches. ROS, OXPHOS, and mitochondrial ATP levels in differentiated cells also increase with maturity. It has been shown that when many mESC populations differentiate, their cellular metabolism shifts from glycolysis to oxidative metabolism (9,20,22-24).

In light of the established roles of FAs in membrane remodeling and the signaling pathways that influence mitochondrial function, the present study investigates the regulation of mitochondrial biogenesis in mESCs exposed to two different FAs: EPA, an omega-3, and LA, an omega-6, both of which are unsaturated FAs.

Materials and methods

Ethical approval

The College of Veterinary Medicine at the University of Mosul provided ethical approval for the research project UM.VET.2024.107. on August 18, 2024.

Cell culture

In summary, Iscove's basal media was used to grow mESCs on feeder layers derived from mouse embryonic fibroblasts. In humidified conditions with 5% CO₂ at a temperature of 37°C, the growth medium was supported with 15% heat-inactivated foetal calf serum, 1mM Pyruvic acid sodium salt, 2 glutamic acid γ-amide, 1% nonessential amino acids, 100 μM β-mercaptoethanol, 0.4% penicillin/streptomycin, and 1,000 U/ml Leukemia inhibitory factor (LIF). To create three-day-old, three-dimensional (3D) spherical structures of embryoid body tissue, the attached cells were separated using 0.05% trypsin-EDTA. Following dissociation, 250 ml siliconized spinner flasks containing 125 ml of Iscove's medium supplemented as previously mentioned, but with LIF excluded, were used to seed single cells at a concentration of 3 × 10⁶ cells/ml. Finally, 250 ml was achieved by adding 125 ml of media after 48 hours. An Integra Biosciences stirrer device was used to agitate the medium in the spinner flask at 20 rpm. The rotation direction was reversed every 1440° to improve uniform aggregation. Every day, 125 millilitres of cell culture media were swapped out (8,25)

Single-cell preparation and FAs exposure

On the third day of cell culture, embryoid bodies were formed and subsequently enzymatically digested for 30 minutes at 37°C in 1X phosphate-buffered saline (PBS) containing 2 mg/ml collagenase B to produce single-cell preparations. Dissociated single cells were grown in Iscove's media and supplemented with additional nutrients after being seeded onto gelatin-coated coverslips on 24-well cell culture plates. Cells were treated with physiological concentrations of EPA or LA (50 µM) for 48 hours. EPA and LA were dissolved in DMSO (final DMSO concentration ≤ 0.1% in the culture). This concentration of EPA and LA has previously been shown to have a physiological impact on mESCs' differentiation (8). Alternatively, they were exposed to DMSO at suitable concentrations as a vehicle control. The cell culture medium enriched with EPA and LA was refreshed every 48 hours (Figure 1) (8,26).

Figure 1: A schematic overview of the experimental setup.

Live cell staining and image acquisition by confocal microscopy

MitoTracker Green FM (MTG) (Ca. no.: M7514; Thermo Fisher Scientific) was used to stain the mitochondria in live cells. It was done according to the manufacturer's protocol. After 48 hours, individual cells were adhered and subsequently incubated under standard conditions in serum-free Iscove's basal medium with 50 nM MTG for 30 minutes. Thereafter, the coverslips were transferred to an incubation slide chamber containing freshly prepared Iscove's basal medium free of serum before placement on the stage of the confocal laser scanning microscope (Wetzlar, Germany) SP8 FALCON, which is outfitted with an HCX PL APO 63x/1.2 water immersion objective. The images were captured at 512 x 512 pixels, with a magnification of 63x and a digital zoom of 4X. Mitochondria were quantified as previously described, and fluorescence recordings were obtained using a confocal laser-scanning microscope mounted on an inverted microscope (8,27-29).

Utilising the image analysis software ImageJ (ImageJ 1.54 f USA), the mitochondrial configurations within the 2D projection were quantified by the mitochondrial image analysis plugin. This procedure assessed mitochondrial elements, which were clearly delineated. The 2D analysis commenced with processing and thresholding, followed by employing the resultant binary images as input for the analyze particles command. This command facilitates the quantification of various parameters, including the number of mitochondria, area, perimeter, form factor, aspect ratio, branch length, branches per mitochondrion, and branch junctions per mitochondrion. Considered as the "length-to-width ratio," the aspect ratio is computed as the quotient of the major and minor axes.

Furthermore, reflecting the complexity and branching properties of mitochondria, the form factor is represented as the square of the circumference divided by 4π multiplied by the surface area (21). The skeletonize 2D function was used on the threshold image to generate a structure map, which was subsequently analyzed for quantity within the skeletonized network (30-32). Our method for analyzing mitochondrial morphologies enables large-scale measurement of cells within a single experiment by combining automated imaging, computational high-content analysis, and machine-learning-derived classifiers.

Statistical analysis

The PRISM statistics program (GraphPad version 8.0) was used for statistical analysis. The statistics are presented as mean ± SD, representing the average of at least 3 separate cell cultures. One-way ANOVA was utilized for statistical evaluation when applicable. The significance level of *P ≤ 0.05 was established.

Results

The study investigates confocal imaging of mitochondria in live mESCs under either standard or treatment conditions. Thus, 8 -10 images were taken, particularly with 50 µM of LA or EPA of FAs over a duration of 48 h during the differentiation of mESCs. This study meticulously uses fluorescent dyes to tag mitochondria. It optimizes photographic capture to achieve high resolution and superior image quality. Following a comprehensive image thresholding procedure, the next phase involves quantifying the morphological characteristics of the identified mitochondrial structures (Figure 2a). A 2D analysis was performed, and mitochondrial counts were assessed. The results indicate that the mitochondrial count exhibited a relative increase across all experimental groups (control, vehicle, 50 µM LA, 50 µM EPA), demonstrating significant variation as illustrated in Figure 2 b. The group treated with 50 µM LA and EPA shows a noticeable increase compared with the control and vehicle groups, suggesting a potential stimulatory effect on mitochondrial count. Furthermore, the vehicle group displayed no significant change.

Figure 2: The effect of LA and EPA on mitochondrial morphology in mESCs. Embryoid bodies were enzymatically dissociated, followed by the cultivation of single cells on gelatin-coated coverslips under 50 µM LA or EPA conditions, and were either left untreated or exposed to a vehicle (DMSO). Modifications in mitochondrial morphology were monitored using MTG dye. a) A representative image of MTG labeled mitochondrial. The scale bars represent 10 μm. The data were analyzed for parameters such as b) mitochondrial count, c) area, d) perimeter, e) form factor, f) aspect ratio, g) branch length, h) branches per mitochondrion, and i) branch junctions per mitochondrion. The data presented are representative of the 8 to 10 analyzed images obtained from distinct and separate plates across 3 different studies. *P<0.05 denotes statistically significant variations relative to the untreated control.

However, mitochondrial dimensions are defined by the evaluation of area and perimeter. The mean area is modestly increased across all groups, with significant disparities observed between the control, vehicle, 50 μM LA, and 50 μM EPA groups. Additionally, perimeter values increased significantly in the EPA and LA groups compared with the control, with EPA showing the largest increase (Figures 2c and 2d). Furthermore, mitochondrial shape is characterized using form factor and aspect ratio. The form factor shows an improvement in results with only 50 µM EPA treatment, highlighting noticeable differences among the groups. An increased form factor indicates enhanced elongation and branching typical of active mitochondria. The aspect ratio appears to remain stable, with no significant variation in the treatment group (Figure 2, e and f). The skeletonized structure evaluates the connectivity and morphological complexity of the mitochondrial network, quantifying these attributes by counting branches, branch junctions, and the length of branches within the skeleton. The results reveal significant increases in these parameters in treated groups with FAs compared with control groups, and no significant changes were observed in the vehicle group (Figure 2, g, h, and i).

Discussion

The polyunsaturated fatty acids, EPA (Omega-3) and LA (Omega-6), are distinguished by a single carbon-carbon double bond. EPA's carbon-carbon double bond is located at the ω-3 site, which is the third bond from the fatty acid's methyl end. The ω-6 situated, which is the sixth bond from the methyl end of FA, is where LA, on the other hand, displays the double bond (33). This study elucidates the effects of FAs, specifically LA and EPA, on mitochondrial biogenesis in mESCs. Mitochondria are capable of ATP production within cells via oxidative phosphorylation (34). This intricate mechanism involves the movement of electrons between complexes in the respiratory chain, which facilitates the translocation of protons across the mitochondrial membrane, ultimately driving ATP synthesis (35). In stem cells, during cardiomyocyte development, mitochondrial oxidative enzymes play a greater role, and the energy source for cardiomyocytes gradually shifts from glycolysis to β-oxidation of FAs (16,36).

Cardiomyocytes derived from human-induced pluripotent stem cells using FAs as the essential metabolic substrate have been demonstrated as follows (37), and another study found that FAs are necessary for the differentiation of blood cell progenitors (38). Moreover, ROS generated by electron leakage within mitochondria during cellular respiration primarily manifests as superoxide, which then undergoes dismutation to form hydrogen peroxide (H₂O₂) (39,40). Thus, ROS is involved in several physiological functions, such as cell proliferation, differentiation, and apoptosis, by acting as signaling molecules. Given that FAs induce ROS and mitochondrial remodeling, our findings suggest that EPA and LA might promote early differentiation in mESCs via mitochondrial reorganization (40). However, Mitochondria play an essential role in energy metabolism, signaling, and cellular differentiation. The observed variations in mitochondrial characteristics (27,41), particularly branching and junction formation, underscore the importance of FAs as modulators of mitochondrial behavior. Omega-3 supplementation has been found to enhance mitochondrial respiration sensitivity and increase mitochondrial ROS release capacity without altering the levels of oxidative products. This indicates that omega-3 FAs contribute to the reorganization of mitochondrial membrane composition (42). In previous studies, FAs have been shown to enhance mESC differentiation by producing ROS and nitric oxide (NO). FAs promote mESC differentiation through mechanisms involving biochemical components (ROS and NO) and alterations in intracellular calcium levels, which are associated with the energy detectors AMP-activated protein kinase (AMPK-α) and peroxisome proliferator-activated receptors (PPAR‐α) (8). However, Mitochondrial morphology is partially influenced by dynamics. The modulation of mitochondrial dynamics is evident in circumstances that require enhanced mitochondrial proliferation (43), is modified by hypoxia signaling mechanisms (44), as previously reported, and is closely linked to ROS production. Actually, extreme oxidative stress can lead to network fragmentation (45), and network modification may be regulated by elevated ROS level generation (46).

The findings of the present investigation demonstrate that mitochondrial counts differ notably between the treatment group and the untreated or vehicle groups. Some studies have also shown that high glucose concentrations enhance cell-specific morphological rearrangements of mitochondria and the mitochondrial network (47). In addition, this study's findings show significant alterations in mitochondrial branching and junctions, suggesting that LA and EPA may influence mitochondrial network dynamics. The study also explored form factor and aspect ratio, which are critical metrics for assessing mitochondrial shape (21). Significant changes were observed in these modifications following EPA treatment, indicating that while FAs may affect branching, they may also alter mitochondrial shape. The study also explored form factor and aspect ratio, which are critical metrics for assessing mitochondrial shape. Significant changes were observed in the form factor upon EPA treatment, while the aspect ratio remained unchanged. The observed aspect ratio suggests that mitochondria are not undergoing elongation, but rather branching, increasing in number through division, or undergoing morphological changes without altering their overall shape. This proposes a proportion between fusion and fission processes, or changes in mitochondrial shape without altering the length-to-width ratio (17).

The modulation of mitochondrial dynamics plays a pivotal role in stem cell differentiation (48). Implications of FAs on cellular processes. FAs are known to be integral to cellular signaling pathways that govern stem cell fate (49). A different study mentions that these long-chain FAs, such as palmitic acid and stearic acid, enhanced gastrointestinal permeability and energy deprivation, causing elevated proton leaks, mitochondrial transformation, and improved ROS generation in intestinal cells, in contrast to dodecanoic acid and tetradecanoic acid, which induced significant lipid storage and enhanced mitochondrial network fusion (6). Furthermore, research shows that a prolonged culture of cardiovascular myocytes in glucose media improves mitochondrial function and FA metabolism. Supplementation with palmitate and oleate aids mitochondrial remodeling, oxygen consumption, and ATP production. However, glucose-maintained cardiomyocytes show underdeveloped ultrastructural architecture and suboptimal development (50).

Mitochondrial biogenesis induction activates transcription factors and local protein translation in response to natural products such as 6-gingerol (ginger extracts) and ursolic acid. At the same time, a few synthetic drugs are recognized as such inducers (51-53). Natural extracts such as Kaempferia parviflora, tangeretin, salidroside, spice saffron, and polydatin have been shown to promote mitochondrial biogenesis by activating different pathways, including SIRT1/AMPK/PGC-1α/PPARδ, miR22/SIRT1, and resveratrol (54-56). Cells respond to energy demands by up-regulating transcription factors, stimulating or inhibiting mitochondrial biogenesis. Pathology-associated disturbances involve either impaired or abnormally elevated mitochondrial biogenesis (57). There is growing evidence that the regulation of mESCs' behaviours is actively mediated by mitochondria. Specific processes (biogenesis, fission, fusion, and mitophagy) occur in mitochondria during the self-renewal, proliferation, and differentiation of mESCs. Significant effects on mESCs' behavior result from changes in mitochondrial dynamics, which are regulated by stress signaling and mESCs' niche variables (20).

Finally, the analysis of the mitochondrial network indicated that all FA treatments, with either LA or EPA, altered it. This remodelling could manifest as either mitochondrial fusion, as evidenced by an increased average network branch length, or fission and fragmentation, as indicated by a decreased average network branch length (20).

Conclusion

The study shows that treatment with either 50 μM LA or EPA significantly alters mitochondrial metrics, including number, area, perimeter, and branching, while maintaining some morphological characteristics. This suggests potential effects on energy metabolism and mESC differentiation. These findings may have implications for the design of stem cell-based therapies or for the treatment of mitochondrial disorders.

Acknowledgment

I sincerely thank Professor Dr. Heinrich Sauer, affiliated with Justus Liebig University Giessen, Germany, for his collaboration and the invaluable assistance of the College of Veterinary Medicine at the University of Mosul.

Conflict of interest

The author declares that there are no conflicts of interest.