Induced pluripotent stem cells (iPSCs) generated from human neonatal foreskin fibroblasts.
In cells, infected with reprogramming factors (retroviruses encoding human OCT4, SOX2, KLF4, and c-MYC), supplementation of the culture media with NAC significantly increased iPSCs survival and reduced ROS generation and the number of DNA double-stranded breaks in the reprogrammed cells.
Antioxidants significantly reduced ROS generation and the number of copy-number variations (CNVs: an indication of genomic aberrations) in treated iPSCs, compared to the untreated control group (). Treatment with NAC had no effect on transgene expression, silencing, and viral transduction efficiency.
Homemade antioxidant cocktail [ascorbate, glutathione, and α-tocopherol at 20 mM, 4 mM, and 1 mM, resp.].
Two human cell lines of iPSCs (201B7 and 253G1).
(i) Measurement of cellular ROS levels showed diminished ROS levels in cells, cultured with antioxidants, compared to the untreated group.
(ii) Moreover, the addition of homemade antioxidant cocktail reduced the number of genomic aberrations in treated cells.
(i) The components of homemade cocktail exerted a free-radical scavenging activity to neutralize ROS in treated cells.
(ii) For two months of culturing with low doses of antioxidants, iPSCs maintained the expression of stemness-related genes (Oct3/4, Nanog, SSEA-4, and ALP).
Roselle (Hibiscus sabdariffa L.) at 125, 500, or 1000 ng/mL.
Bone marrow-derived hematopoietic stem cells (HSCs) from murine bone marrow.
(i) Adding roselle (at 500 and 1000 ng/mL) significantly increased the survival of HSCs and protected them against H2O2-induced DNA damage.
(ii) Roselle supplementation was genoprotective, as evidenced by the nonremarkable difference on the percentage of tail DNA, compared to the control group (untreated BMSCs).
Compared to the control group, roselle enhanced the activity of SOD in HSCs (at 125, 500, and 1000 ng/mL) with a significant increase in GSH level (). However, there was no difference in ROS levels between roselle-treated and control groups.
Hematopoietic stem cells (HSCs) from mice fetal liver cells.
Ikeda et al. designed a biocompatible cell culture surface that can be used during ex vivo culturing and expansion of HSCs. This new surface has several advantages, compared to the currently used one including low molecular weight and antioxidant supplementation. It decreased ROS production, inhibited apoptosis, and increased the purity of separated cells.
The antioxidant culture surface (PEG-b-PMNT) scavenged nitric oxide radicals and reduced oxidative membrane damage without changing the mitochondrial membrane potential because it is not internalized within the cell as the conventional LMW systems.
LSK cells (Lin− Sca-1+ c-Kit+, a population enriched with HSCs).
(i) Aneuploidy/chromosomal instability during in vitro expansion of HSCs is ROS-mediated and can be minimized by maintaining a hypoxic condition (3% O2) during cell culturing.
(ii) Similarly, NAC administration significantly reduced the percentage of aneuploidy in HSCs, cultured under normoxic conditions, only at low dosages (0.1 μM).
(i) At optimum concentrations, NAC significantly reduced oxidative damage due to its ROS-scavenging activity.
(ii) Moreover, hypoxic conditions and NAC reduced the percentage of aneuploidy in both young and old aged stem cells.
Lipocalin-2 (Lcn2), a natural cytoprotective factor, generated within the cell upon exposure to stressful conditions.
Bone marrow-derived stem cells (BMSCs) from rat bone marrow (4–6 weeks old).
(i) Lcn2-expressing BMSCs showed a more potent defense against H2O2, hypoxia, and serum deprivation stresses, compared to control MSCs
(ii) Moreover, Lcn2 expression in MSCs increased cell proliferation and adhesion to culture media by 45% on H2O2 exposure, compared to control MSCs.
(iii) Lcn2-expressing BMSCs showed normal multipotency into different cell lineage with mild potentiation of adipogenic lineage compared to the control group.
(i) The antioxidant effect of Lcn2 expression is due to ROS scavenging activity, associated with upregulation of antioxidant enzymes’ genes, such as SOD.
(ii) This antioxidant effect is associated with an antiapoptotic property as indicated by the diminished number of apoptotic cells on oxidative stress exposure, compared to the control group.
Alpha-phenyl-t-butyl nitrone (PBN) at 800 μM and NAC at 5 mM.
Mesenchymal stem cells (MSCs) from mice embryos.
(i) Cultured MSCs with extracellular matrix from mouse embryonic fibroblast (MEF-ECM) under hypoxic conditions (2% O2) showed greater proliferation, lower generation of ROS, and increased chromosomal stability compared to the control group, cultured on plastic plates under normoxic conditions.
(ii) To further diminish chromosomal instability and based on the increased DNA damage on H2O2 exposure, antioxidant supplementation to the culture media significantly reduced the number of DNA micronuclei and karyotyping abnormalities. The use of both (antioxidants and MEF-ECM) in the initial isolation of cells from the marrow increased purity and normal cell karyotyping.
Although authors did not investigate the underlying mechanisms for antioxidants’ effects, they suggested that their findings can be attributed to the ability of both PBN and NAC to trap free radicals. Moreover, NAC serves as a precursor for glutathione, an intracellular antioxidant molecule.
Mesenchymal stem cells (MSCs) from rat bone marrow.
In general, 2-vinyl-8-hydroxyquinoline derivatives had a positive effect on MSCs proliferation in a dose-dependent manner.
2-Vinyl-8-hydroxyquinoline derivatives are phenol compounds that perform their antioxidant activity through reaction of their hydroxyl group with free radicals.
Ascorbic acid-2-phosphate (AAP) at 0, 5, 50 250, 500 mM.
Mesenchymal stem cells (MSCs) from adult human bone marrow [39] and human umbilical cord blood-derived stem cells (hUCB-SCs) from umbilical vein [79].
(i) Ascorbic acid significantly increased the proliferation of MSCs/hUCB-SCs, compared to the control group (with the highest proliferation rate at 250 mM). It had no effect on cellular antigenic expression and differentiation.
(ii) Moreover, AAP significantly increased osteogenic differentiation at 50 mM (highest calcium deposition at this concentration). In contrast, a significant induction of adipogenic differentiation with oil droplets formation was noted at 250 mM and higher.
(i) AAP improved the amount of collagen production per cell and increased the amount of calcium (at 50 mM) and oil deposition (at ≥250 mM), enhancing MSCs/hUCB-SCs differentiation.
(ii) The positive effect of AAP on cellular proliferation is dose-dependent (highest at 250 mM and decreases with higher doses due to inhibitory effect on glycosaminoglycan formation).
Human umbilical cord blood-derived stem cells (hUCB-SCs) from umbilical vein.
(i) Exposure to genotoxic stress (H2O2) in culture media caused a more significant reduction in cellular proliferation and DNA synthesis in hUCB-SCs, compared to control cells (cancer cells and human primary fibroblasts).
(ii) Moreover, hUCB-SCs showed low resistance to oxidative stress with cellular senescence and apoptosis at H2O2 levels much lower than those of control groups.
Measuring the cellular antioxidant capacity showed that hUCB-SCs had a lower antioxidant capacity than control cells. To confirm that, antioxidant supplementation increased this capacity and diminished cellular damage upon exposure to oxidative stress.
Human umbilical cord blood-derived stem cells (hUCB-SCs) from umbilical vein.
(i) Unlike the pro-oxidant (diethyl maleate), edaravone significantly reduced lipopolysaccharide (LPS)/H2O2-induced damage and increased stem cell viability ().
(ii) In diabetic mice with severe combined immunodeficiency, only three mice died in the group, injected with (Gal/LPS and hUCB-SCs), compared to 50% loss in the group, injected with Gal/LPS only.
(iii) Pretreatment with edaravone rescued all mice with potentiation of the hepatic cell regenerative power. Furthermore, it diminished the levels of cellular injury and proinflammatory markers in treated mice, compared to the control group.
(i) LPS/H2O2 challenge induced apoptosis by augmenting oxidative stress and increasing Bax/Bcl2 ratio. However, pretreatment with edaravone abolished these changes.
(ii) Moreover, edaravone increased the expression of endogenous antioxidant enzymes (superoxide dismutase, catalase).
Tempol (SOD mimetic) at 0 to 10 mm/L concentration.
Rat cardiomyoblasts, transfected by a bioluminescence reporter gene for in vivo detection and transplanted into the myocardium, guided by high-resolution ultrasound.
(i) Cells, exposed to hypoxic/oxidative stress conditions during in vitro culturing, showed decreased cell viability with increased ROS production and NADPH-oxidase-1 expression, compared to the control group. These effects were significantly reduced after adding antioxidants in a dose-dependent manner.
(ii) After transplantation into rat myocardium, antioxidant-treated cells showed significantly higher cell viability within the first three days of transplantation, compared to untreated cells.
Hypoxia induces oxidative stress by increasing the expression of NAD(P)H oxidase enzyme. Interestingly, adding antioxidant did not reduce NAD(P)H expression, suggesting that tempol reduces oxidative stress by neutralizing free radicals rather than decreasing their production.
Homemade antioxidant cocktail consisting of 100 ML-ascorbate, L-glutathione, and α-tocopherol acetate.
Cardiac stem cells (CSCs) from the endomyocardial tissue of a patient undergoing a cardiac procedure.
(i) Cells, cultured under hypoxic conditions (5% O2) had a lower number of chromosomal abnormalities, compared to cells, cultured under normoxic conditions (20% O2).
(ii) Unexpectedly, increased cytogenic abnormalities in number and severity were recorded in cells, cultured with antioxidant supplementation.
(iii) Antioxidant supplementation, if excessive, may hinder the physiological roles of ROS in stem cells’ proliferation and differentiation, which raises a new concept [reductive stress].
(i) Measuring c-H2AX foci (a marker of DNA breaks) showed a biphasic relationship between ROS levels and frequency of DNA breaks, that is, increased DNA damage occurs at low antioxidant/high ROS levels, while excessive suppression of ROS levels increases DNA damage.
(ii) There is an optimal level of ROS in stem cells, above which genomic instability occurs due to ROS-induced DNA damage and below which DNA repair enzymes are not activated to maintain the DNA stability.
(i) Ascorbic acid significantly increased ESCs differentiation into cardiac myocytes in a dose-dependent manner, as evidenced by increased expression of the cardiac specific gene (myosin heavy chain (MHC)).
(ii) Ascorbic acid, independent of its antioxidative property, induced ESCs differentiation into cardiac myocytes. This was evidenced by the absence of this effect with other antioxidants, such as NAC, Tiron, and vitamin E.
Ascorbic acid increased the expression of cardiac muscle genes, such as GATA4, Nkx2.5, α-MHC, β-MHC, and atrial natriuretic factor (ANF), with subsequent cardiac-specific protein production.
Human embryonic stem cell- (ESC-) derived vascular progenitors.
(i) Following vascular differentiation of ESCs into vascular progenitor cells (CD34+ cells), a quiescent state of cellular proliferation developed with 41% of the cells in the G0 phase, upregulation of the G1 checkpoint inhibitor (p21) protein and downregulation of mitosis-related genes.
(ii) Selenium increased cellular proliferation, reduced p21 expression, and decreased the number of cells in G0 phase of cell cycle. Moreover, it promoted the vascular differentiation of ESCs with no similar effect on endodermal or ectodermal potentiality.
(iii) Using NAC totally inhibited the vascular differentiation of ESCs.
(i) Physiologically, ROS are produced during vascular differentiation mainly by NADPH oxidase and is responsible for this quiescent state.
(ii) Selenium through increasing glutathione and thioredoxin activity moderately diminished ROS levels.
(iii) However, NAC, through complete scavenging of ROS, abolished their physiological role in vascular differentiation.
GV1001 [derived from human telomerase reverse transcriptase from 0 to 100 μM.
Neural stem cells (NSCs) from mice embryonic brain (cortical tissue).
GV1001 significantly reduced H2O2 effects on NSCs including diminished cellular proliferation, migration and increased apoptosis. Interestingly, GV1001 itself had no effect on normal untreated cells.
(i) GV1001 has an ROS-scavenging activity, preventing lipid peroxidation and DNA damage.
(ii) At the molecular level, GV1001 induced the expression of survival-related proteins and diminished the expression of apoptosis-related proteins.
Cyclosporine A (CsA), brain-derived neurotrophic factor (BDNF), and thyrotropin-releasing hormone (TRH).
Neural stem cells (NSCs) from the spinal cord of transgenic adult female rats (spinal cord injury model).
(i) Pretreatment with BDNF for 48 hours (before H2O2 exposure) significantly increased NSCs viability and decreased intracellular ROS accumulation, compared to the control group. However, CsA and TRH-treated cells showed no significant changes from the control group.
(ii) Interestingly, BDNF-treated cells showed no changes in cellular proliferation and differentiation compared to the control group.
The neuroprotective effect of BDNF is exerted through its ROS-scavenging activity and induction of antioxidant enzymes, such as GR and SOD. Moreover, significant reductions in apoptotic features were noted in BDNF-treated cells, compared to the control group.
Neural stem cells (NSCs) from mice embryonic cortical tissue.
(i) Melatonin significantly reduced LPS-induced toxicity and apoptosis of NSCs through reducing nitric oxide (NO) production and inducing antioxidant enzymes.
(ii) Furthermore, it maintained the neurosphere size in NSCs, treated with LPS.
(iii) Melatonin increased cell survival by activating PI3K/Akt pathway. This was confirmed by the addition of wortmannin (a PI3K inhibitor), which inhibited Nrf2 expression and subsequent antioxidant activities.
(i) Melatonin increased the expression of multiple transcriptional factors, involved in NSCs proliferation, self-renewal, and differentiation, such as orphan nuclear receptor TLX and fibroblast growth factor receptor-2.
(ii) Melatonin upregulated the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2), responsible for downstream activation of antioxidant enzymes’ genes.
N-Acetyl-L cysteine (NAC) at 2 mM and ascorbic acid-2-phosphate (AAP) at 0.2 mM in comparison to the effect of hypoxia.
Adipose-derived stem cells (ADSCs) from human adipose tissue.
ADSCs, grown in media, supplemented by antioxidants or under hypoxic conditions (5% po2), showed a more significant increase in cell proliferation and a decrease in doubling time than the control group, supplemented by fibroblast growth factor-2. Moreover, cytometric analysis showed that cells, cultured in antioxidant-supplemented and hypoxic media, had a greater proportion of cells in S1 phase of the cell cycle with diminished G0/G1 phase cells, compared to the control group.
In antioxidant-supplemented media, PCR showed diminished levels of cyclin-dependent kinase inhibitors (CDK: important cell cycle regulators that control entering S1 phase), with enhanced expression of stemness-related genes, compared to the control group.
Tempol (1-2 mM), NAC (5–20 mM), and resveratrol (20–40 μM).
Endometrial stem cells, isolated from desquamated endometrium of menstrual blood and ADSCs from adipose tissue.
(i) Reactive oxygen species are important regulators of stem cell self-renewal and proliferation upon exit of the quiescent stage.
(ii) Using a synchronized cell in G0 phase, there was a transient increase in ROS levels upon stimulation of cell proliferation and during initial stages of DNA synthesis.
(iii) Adding antioxidants to the medium after proliferation induction and before initiation of S1 phase blocked S1 transition. Antioxidant did not have the same effect when added after S1 initiation.
(i) Cells, treated with antioxidants, showed expression of the proliferative marker (Ki-67), which is absent in the nucleus of quiescent cells, indicating that the cell left the quiescent state and was arrested in G1 phase.
(ii) Antioxidants, through dose-dependent reduction of ROS levels, can be used to control cellular proliferation during in vitro culturing.
Fullerol (a polyhydroxylated fullerene) at 0.1, 0.3, 1, 3, and 10 μM.
Human adipose-derived stem cells (ADSCs).
(i) Fullerol enhanced the osteogenic differentiation of ADSCs, as indicated by increased expression of osteogenic markers (Runx2, OCN, and alkaline phosphatase) and mineralization.
(ii) Moreover, fullerol (at all concentrations) reduced ROS levels in the osteogenic culture media.
Fullerol exerted an antioxidant effect on ADSCs through potentiating the expression of the transcription factor FoxO1 and its downstream genes (Runx2 and SOD2), which promote ROS scavenging and osteoblastic differentiation.
Adipose-derived stem cells (ADSCs) from the subcutaneous adipose tissue from a female patient, undergoing abdominoplasty.
(i) Ascorbic acid significantly increased ADSCs proliferation, preserved cellular stemness and increased the potentiality for adipogenic, hepatic, neural and osteogenic differentiation.
(ii) In AAP-induced cell sheet, there was a significant increase in the genetic expression and secretion of collagen, laminin, and fibronectin proteins.
(iii) The AAP-induced cell sheet improved wound healing in a murine wound model, which indicates the possibility of ADSCs differentiation in a non-mesenchymal lineage.
(i) Adding AAP to ADSCs increased the expression of stemness-related proteins.
(ii) Using other antioxidants, such as NAC did not show an increase of stemness markers. In contrast, adding a collagen synthesis inhibitor abolished AAP-induced overexpression of stemness proteins, indicating that the involved mechanism in AAP action is collagen synthesis, not ROS scavenging.
NAC and AAP at 3 mM and 0.2 mM, respectively (for 20 hours).
Human ADSCs from 10 different human patients.
(i) Pretreated MSCs with antioxidants showed less apoptosis and lower caspase-3 levels upon exposure to advanced glycosylation end-products (AGE), compared to the control group.
(ii) The effects of NAC and AAP were significantly amplified after the addition of miRNA-223 mimetics and were significantly abolished by miRNA-223 inhibitors.
Antioxidants reduced ROS generation and apoptosis, induced by AGE. This can be explained by the effect of both on miR-223 (a regulator of intracellular apoptotic singling through modulation of fibroblast-like growth factor receptor-2 protein levels.
N-Acetyl-L cysteine at 10 mM in comparison to the pro-oxidant (diethyl maleate) at 50 μM.
Muscle-derived stem cells (MDSCs) from the skeletal muscle of 3-week-old female mice.
(i) In comparison to control cells, NAC-treated cells showed increased survival and differentiation into myotubes upon exposure to oxidative (H2O2) or inflammatory stress (tumor necrosis factor).
(ii) In failed mice hearts, injection with NAC-treated cells significantly improved systolic and diastolic function on echocardiographic assessment, compared to injection with untreated and DEM-treated cells. Moreover, scar tissue formation was significantly lower in NAC-treated cells than untreated cells.
(i) Increased cell survival after NAC treatment is probably related to stimulation of mitogen-activated protein kinases (MAPK) and extracellular signal-regulated kinase (ERK), kinase families involved in cellular survival and proliferation.
(ii) Interestingly, a similar increase in CD31+ endothelial cells in NAC-treated and untreated MDSCs was observed.
Spermatogonial stem cells (SSCs) from neonatal male mice testis.
Antioxidant supplementation of cryopreserved SSCs reduced oxidative damage to membranes and organelles and increased cell survival in a dose-dependent manner.
Catalase and α-tocopherol reduced ROS generation in treated cells, compared to control cells. Moreover, antioxidant-treated cells showed an increased expression of the anti-apoptotic BcL-2 gene with decreased expression of the pro-apoptotic BAX gene, compared to the control group.
