Mesenchymal Stromal Cell Mitochondrial Transfer as a Cell Rescue Strategy in Regenerative Medicine: A Review of Evidence in Preclinical Models.

Mesenchymal stromal cells (MSC) have excellent clinical potential and numerous properties that ease its clinical translation. Mitochondria play a crucial role in energy metabolism, essential for cellular activities, such as proliferation, differentiation, and migration. However, mitochondrial dysfunction can occur due to diseases and pathological conditions. Research on mitochondrial transfer from MSCs to recipient cells has gained prominence. Numerous studies have demonstrated that mitochondrial transfer led to increased adenosine triphosphate (ATP) production, recovered mitochondrial bioenergetics, and rescued injured cells from apoptosis. However, the complex mechanisms that lead to mitochondrial transfer from healthy MSCs to damaged cells remain under investigation, and the factors contributing to mitochondrial bioenergetics recovery in recipient cells remain largely ambiguous. Therefore, this review demonstrates an overview of recent findings in preclinical studies reporting MSC mitochondrial transfer, comprised of information on cell sources, recipient cells, dosage, route of administration, mechanism of transfer, pathological conditions, and therapeutic effects. Further to the above, this research discusses the potential challenges of this therapy in its clinical settings and suggestions to overcome its challenges.

Critical factors for lentivirus-mediated PRDX4 gene transfer in the HepG2 cell line.

Optimization of critical factors affects transduction efficiency and is able to reduce reagent consumption. The present study aimed to determine the optimum transduction conditions of small hairpin (sh)RNA against peroxiredoxin 4 (PRDX4) in the HepG2 cell line. Cell viability assays were conducted based on serum condition, incubation time, polybrene concentration and antibiotic dose selection. Non-targeting control shRNA was transduced into HepG2 cells in a 5-fold serial dilution, and colonies positive for green fluorescent protein were counted using ImageJ software. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to validate PRDX4 expression. The optimum cell density for transduction was 5.0×103 cells/well in 96-well plates to achieve 40 to 50% confluency the following day. The transduction media consisted of 10% fetal bovine serum (FBS) and 12 µg/ml polybrene, and was used to dilute lentiviral particles at a functional titer of 4.9×105 TU/ml for multiplicity of infection (MOI) of 20, 15 and 10, for 24 h of incubation. Selection with 7 µg/ml puromycin was performed in transduced cells. shRNA 3 was revealed to inhibit PRDX4 mRNA and protein expression. In conclusion, PRDX4 was successfully silenced in 5.0×103 HepG2 cells cultured with 10% FBS and 12 µg/ml polybrene, at a 4.9×105 TU/ml functional titer for MOI of 20, 15 and 10.

Tocotrienol-Rich Fraction Ameliorates Antioxidant Defense Mechanisms and Improves Replicative Senescence-Associated Oxidative Stress in Human Myoblasts.

During aging, oxidative stress affects the normal function of satellite cells, with consequent regeneration defects that lead to sarcopenia. This study aimed to evaluate tocotrienol-rich fraction (TRF) modulation in reestablishing the oxidative status of myoblasts during replicative senescence and to compare the effects of TRF with other antioxidants (α-tocopherol (ATF) and N-acetyl-cysteine (NAC)). Primary human myoblasts were cultured to young, presenescent, and senescent phases. The cells were treated with antioxidants for 24 h, followed by the assessment of free radical generation, lipid peroxidation, antioxidant enzyme mRNA expression and activities, and the ratio of reduced to oxidized glutathione. Our data showed that replicative senescence increased reactive oxygen species (ROS) generation and lipid peroxidation in myoblasts. Treatment with TRF significantly diminished ROS production and decreased lipid peroxidation in senescent myoblasts. Moreover, the gene expression of superoxide dismutase (SOD2), catalase (CAT), and glutathione peroxidase (GPX1) was modulated by TRF treatment, with increased activity of superoxide dismutase and catalase and reduced glutathione peroxidase in senescent myoblasts. In comparison to ATF and NAC, TRF was more efficient in heightening the antioxidant capacity and reducing free radical insults. These results suggested that TRF is able to ameliorate antioxidant defense mechanisms and improves replicative senescence-associated oxidative stress in myoblasts.

The Tocotrienol-Rich Fraction Is Superior to Tocopherol in Promoting Myogenic Differentiation in the Prevention of Replicative Senescence of Myoblasts.

Aging results in a loss of muscle mass and strength. Myoblasts play an important role in maintaining muscle mass through regenerative processes, which are impaired during aging. Vitamin E potentially ameliorates age-related phenotypes. Hence, this study aimed to determine the effects of the tocotrienol-rich fraction (TRF) and α-tocopherol (ATF) in protecting myoblasts from replicative senescence and promoting myogenic differentiation. Primary human myoblasts were cultured into young and senescent stages and were then treated with TRF or ATF for 24 h, followed by an analysis of cell proliferation, senescence biomarkers, cellular morphology and differentiation. Our data showed that replicative senescence impaired the normal regenerative processes of myoblasts, resulting in changes in cellular morphology, cell proliferation, senescence-associated ß-galactosidase (SA-ß-gal) expression, myogenic differentiation and myogenic regulatory factors (MRFs) expression. Treatment with both TRF and ATF was beneficial to senescent myoblasts in reclaiming the morphology of young cells, improved cell viability and decreased SA-ß-gal expression. However, only TRF treatment increased BrdU incorporation in senescent myoblasts, as well as promoted myogenic differentiation through the modulation of MRFs at the mRNA and protein levels. MYOD1 and MYOG gene expression and myogenin protein expression were modulated in the early phases of myogenic differentiation. In conclusion, the tocotrienol-rich fraction is superior to α-tocopherol in ameliorating replicative senescence-related aberration and promoting differentiation via modulation of MRFs expression, indicating vitamin E potential in modulating replicative senescence of myoblasts.

Vitamin E in sarcopenia: current evidences on its role in prevention and treatment.

Sarcopenia is a geriatric syndrome that is characterized by gradual loss of muscle mass and strength with increasing age. Although the underlying mechanism is still unknown, the contribution of increased oxidative stress in advanced age has been recognized as one of the risk factors of sarcopenia. Thus, eliminating reactive oxygen species (ROS) can be a strategy to combat sarcopenia. In this review, we discuss the potential role of vitamin E in the prevention and treatment of sarcopenia. Vitamin E is a lipid soluble vitamin, with potent antioxidant properties and current evidence suggesting a role in the modulation of signaling pathways. Previous studies have shown its possible beneficial effects on aging and age-related diseases. Although there are evidences suggesting an association between vitamin E and muscle health, they are still inconclusive compared to other more extensively studied chronic diseases such as neurodegenerative diseases and cardiovascular diseases. Therefore, we reviewed the role of vitamin E and its potential protective mechanisms on muscle health based on previous and current in vitro and in vivo studies.

Reversal of myoblast aging by tocotrienol rich fraction posttreatment.

Skeletal muscle satellite cells are heavily involved in the regeneration of skeletal muscle in response to the aging-related deterioration of the skeletal muscle mass, strength, and regenerative capacity, termed as sarcopenia. This study focused on the effect of tocotrienol rich fraction (TRF) on regenerative capacity of myoblasts in stress-induced premature senescence (SIPS). The myoblasts was grouped as young control, SIPS-induced, TRF control, TRF pretreatment, and TRF posttreatment. Optimum dose of TRF, morphological observation, activity of senescence-associated ß-galactosidase (SA-ß-galactosidase), and cell proliferation were determined. 50 µg/mL TRF treatment exhibited the highest cell proliferation capacity. SIPS-induced myoblasts exhibit large flattened cells and prominent intermediate filaments (senescent-like morphology). The activity of SA-ß-galactosidase was significantly increased, but the proliferation capacity was significantly reduced as compared to young control. The activity of SA-ß-galactosidase was significantly reduced and cell proliferation was significantly increased in the posttreatment group whereas there was no significant difference in SA-ß-galactosidase activity and proliferation capacity of pretreatment group as compared to SIPS-induced myoblasts. Based on the data, we hypothesized that TRF may reverse the myoblasts aging through replenishing the regenerative capacity of the cells. However, further investigation on the mechanism of TRF in reversing the myoblast aging is needed.

Senescence-related changes in gene expression of peripheral blood mononuclear cells from octo/nonagenarians compared to their offspring.

Mechanisms determining both functional rate of decline and the time of onset in aging remain elusive. Studies of the aging process especially those involving the comparison of long-lived individuals and young controls are fairly limited. Therefore, this research aims to determine the differential gene expression profile in related individuals from villages in Pahang, Malaysia. Genome-wide microarray analysis of 18 samples of peripheral blood mononuclear cells (PBMCs) from two groups: octo/nonagenarians (80-99 years old) and their offspring (50.2 ± 4.0 years old) revealed that 477 transcripts were age-induced and 335 transcripts were age-repressed with fold changes ≥1.2 in octo/nonagenarians compared to offspring. Interestingly, changes in gene expression were associated with increased capacity for apoptosis (BAK1), cell cycle regulation (CDKN1B), metabolic process (LRPAP1), insulin action (IGF2R), and increased immune and inflammatory response (IL27RA), whereas response to stress (HSPA8), damage stimulus (XRCC6), and chromatin remodelling (TINF2) pathways were downregulated in octo/nonagenarians. These results suggested that systemic telomere maintenance, metabolism, cell signalling, and redox regulation may be important for individuals to maintain their healthy state with advancing age and that these processes play an important role in the determination of the healthy life-span.