Skip to main content

Sodium selenite preserves rBM-MSCs’ stemness, differentiation potential, and immunophenotype and protects them against oxidative stress via activation of the Nrf2 signaling pathway



The physiological level of reactive oxygen species (ROS) is necessary for many cellular functions. However, during the in-vitro manipulations, cells face a high level of ROS, leading to reduced cell quality. Preventing this abnormal ROS level is a challenging task. Hence, here we evaluated the effect of sodium selenite supplementation on the antioxidant potential, stemness capacity, and differentiation of rat-derived Bone Marrow MSCs (rBM-MSCs) and planned to check our hypothesis on the molecular pathways and networks linked to sodium selenite’s antioxidant properties.


MTT assay was used to assess the rBM-MSCs cells’ viability following sodium selenite supplementation (concentrations of: 0.001, 0.01, 0.1, 1, 10 µM). The expression level of OCT-4, NANOG, and SIRT1 was explored using qPCR. The adipocyte differentiation capacity of MSCs was checked after Sodium Selenite treatment. The DCFH-DA assay was used to determine intracellular ROS levels. Sodium selenite-related expression of HIF-1α, GPX, SOD, TrxR, p-AKT, Nrf2, and p38 markers was determined using western blot. Significant findings were investigated by the String tool to picture the probable molecular network.


Media supplemented with 0.1 µM sodium selenite helped to preserve rBM-MSCs multipotency and keep their surface markers presentation; this also reduced the ROS level and improved the rBM-MSCs’ antioxidant and stemness capacity. We observed enhanced viability and reduced senescence for rBM-MSCs. Moreover, sodium selenite helped in rBM-MSCs cytoprotection by regulating the expression of HIF-1 of AKT, Nrf2, SOD, GPX, and TrxR markers.


We showed that sodium selenite could help protect MSCs during in-vitro manipulations, probably via the Nrf2 pathway.

Peer Review reports


Mesenchymal stem cells are an ideal source for fundamental studies in stem cell biology and many regenerative medicine applications [1, 2]. MSCs are available in adulthood, can be extracted from different tissues with no ethical concerns, have high genetic stability, rapid proliferation, and have multipotent differentiation capacity [3-5]. However, some challenges exist regarding the quality of MSCs used in clinical approaches and cell-based therapies, which can affect the outcomes of the applications of MSCs in clinical and basic research [6]. Among these challenges, a critical issue is a high level of oxidative stress (OS) in the culturing conditions, which is associated with an increased rate of apoptosis, cell inactivation, phenotypic alterations, reduced stemness and differentiation capability, and aging. A disproportion between the production of reactive oxygen species (ROS) and the antioxidant defense system of cells is defined as OS [7, 8]. OS can predispose senescence by upregulation of protein peroxides and increasing the rate of DNA and lipids oxidation, which leads to a wide range of molecular and cellular modifications [9-11].

Therefore, culture conditions for MSCs must be controlled to adjust the normal ROS level and maintain the efficacy of MSCs [12-14]. One approach is to supplement the culture media of MSCs with antioxidants [15]. Antioxidant agents are classified into several categories: enzymatic/non-enzymatic, hydrophilic/hydrophobic, endogenous/exogenous, and so forth. Enzymatic antioxidants primarily serve as intracellular protection against OS. Their function is to eliminate oxidant molecules and convert ROS into harmless entities. Superoxide dismutase (SOD), thioredoxin reductase (TrxR), and glutathione peroxidase (GPX) are the three known enzymes of this category. Non-enzymatic antioxidants encompass a great collection of molecules such as vitamins (A, C, E, and K), cofactors (Q10), minerals (Zn, Se, and others), uric acid, bilirubin, and albumin. Generally, these non-enzyme antioxidants do their function by deactivating free radicals, ROS, and other oxidating molecules [16-18]. The cellular antioxidant defense system functions through three main steps: (i). prevention (by agents such as GPX), (ii). interception (e.g., by TrxR), and (iii). repair [19].

Selenium (Se) is a trace nonmetal element essential for human nutrition [20]. According to the NIH fact sheet for health professionals ( the average daily selenium intake in Americans adults ranges from 134 to 93 mcg from foods for men and women respectively. These data raise to 151 t0 108 considering fool and supplements in nutrition regimen. Se is a fundamental component of seleno-proteins and some antioxidant enzymes, such as GPX and TrxR. Therefore, Selenium is an excellent component in the first and second levels of the antioxidant defense system [21-23]. Also, Selenium is an available and affordable agent with appropriate water solubility compared to other antioxidant agents. All these make Selenium an available and favorable choice for cell therapy and regenerative medicine applications [24].

Compared to other fields of medical and biological studies, the application of animal models in any type of stem cell-related research is inevitable. There are many debates and questions about the various aspects of stem cells’ applications, including choosing the best and most ethically acceptable cell type and answering many concerns about the high regenerative and developmental potential of stem cells that make them susceptible to hyperplasia and oncogenic transformation [25, 26]. In this context, consistency across different animal and human models and in-vitro, ex-vivo, and in-vivo experiments is another point that must be considered [27, 28]. In comparison to other animal model cells, rats are cheaper and their physiology, genetics, and cell processes are more close to humans and exhibited advantages in preclinical effectiveness studies resembling human stem cells [29]. Therefore, when they are used as model cells, the obtained data could be generalized to human cells.

Hence, in the following, we will mention some points regarding the application of Selenium to improve Bone Marrow MSCs (BM-MSCs) potency in animal and human models.

There are pieces of evidence indicating that selenium insufficiency is associated with diminished selenoproteins expression, increased inflammatory cytokines production, and oxidative stress in both animal and human models; on the other hand, selenium supplementation can help to decrease these inflammatory changes [30-32]. For example, thanks to the studies performed in animal models, at present, we know that due to its antioxidant properties, selenium alone or in combination with other antioxidants, such as vitamin E, can reduce OS-related damages in animal models of colitis [33, 34]. Moreover, we know that a low level of selenium is associated with several diseases in both animal and human models, such as cardiovascular diseases [35], type 2 diabetes [36], cancer [37], and neurological function [38]. All these observations have started an interest in selenium, and its derivatives, utilization as a defensive agent in various types of disorders, such as diabetes [39], nephropathy [40], and cancer [41]; also there are many kinds of research and trials on this issue [42, 43]. For example, in polycystic ovary syndrome (PCOS) rat models, sodium selenite has shown a significant effect on the restoration of GPX and has beneficial impacts on reproductive outcomes and oxidative stress markers [44-46].

However, as mentioned above, although several recent studies have described many beneficial effects of sodium selenite on MSCs derived from bone marrow [47, 48], adipose [49], and umbilical cord tissues [50], only sparse reports exist on the effects of sodium selenite on the stemness-associated marker genes, surface CD markers, and the selenium-linked OS signaling pathways. Hence, this study aimed to investigate the effects of Selenium supplementation on the viability of rBM-MSCs, stemness capacity (based on the expression level of OCT-4, NANOG, and SIRT1 genes [51]), adipocyte differentiation potential, CD markers profile, intracellular ROS, and expression of some relevant proteins that were chosen by reviewing the bulk of relevant previously-published papers and considering the results of in-silico network analysis.

Materials and methods

Chemicals and reagents

The entire reagents and chemical materials utilized in this work were of analytical grade. Propidium iodide (PI), Ribonuclease A, 3 (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Dimethyl sulfoxide(DMSO) were purchased from Sigma–Aldrich (St. Louis, MO). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), Penicillin Streptomycin solution, and trypsin were purchased from Gibco Invitrogen (Grand Island, NY). L-Glutamine, β-glycerophosphate (10 mM) (Sigma), Alizarin Red S (Sigma), and dexamethasone (Sigma). Sodium selenite (Sigma), DMEM medium (Bioidea, Iran), Oil Red (Sigma-Aldrich), Trizol reagent (Thermo Scientific), Syber Green Master Mix (Thermo Scientific), CD105-Percp-Cy5.5 (Biolegend), CD44-PE (eBioscience), CD45-FITC (BD Bioscience), and CD31-PE (eBioscience). Euthanasia chamber (Orchid Scientific).

Isolation, culture, and characterization of rBM-MSCs

All procedures related to rats in this research were approved by the Tabriz University of Medical Sciences Research Ethics Committee (No. IR.TBZMED.REC.1399.036). In this regard, we acted according to the well-accepted method of sacrificing and expression of some relevant proteins that were chosen sacrificing animal models guidelines of Tabriz University of Medical Sciences Animal models Ethics and related international guidelines ( The rats (No = 3), purchased from Animal house, Faculty of Veternary, Tabriz University. They were in aged three to five weeks, and about 250 g, were used for the isolation of bone marrow-derived mesenchymal stem cells. A euthanasia procedure was performed in a way that the rat rapidly lost consciousness without any pain or sensing any suffering. The rats were located in an anesthesia CO2 chamber. Then, anesthetized for about 6 min. Subsequently, the rats were picked up from the chamber at the time they stopped their motor activity and their blinking rate ended. Next, the rats were laid down on the operation bench and killed by cervical dislocation of rats. The femur and tibia bones were cut off the soft tissues around them were detached [52]. rBM-MSCs were isolated from the rats’ bone marrow in line with the earlier published protocol with minor changes [53]. The femur’s epiphysis was cut off from both ends in a biosafety cabinet, and the bone marrow was inserted with a syringe needle into one end of the bone. Then flushed out with serum-free DMEM/F12 medium (10 mL) mixed with 100 μg/ml streptomycin and 200 U/ml penicillin into a 60 mm culture dish. The cell suspension was achieved by pulling the marrow into syringes three times in a series employing decreasing-sized needles. The rBM-derived cell suspension was centrifuged at 1000xg. These cells were then incubated at 37 °C in a complete medium under the standard condition of 95% humidity and 5% CO2 to reach 70% cell confluency. According to the routine cell culture protocol, media were refreshed twice a week.

Immunophenotyping by flow cytometry was used to characterize the rBM-MSCs surface CD profile. For this purpose, the extracted rBM-MSCs were subcultured in 6 well format plates in routine cultured and harvested at 70% area confluence. Approximately 2 × 105 cells were transferred to FACS tubes and washed with phosphate-buffered saline (PBS). These cells were then incubated with solutions containing phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)- conjugated antibodies against rBM-MSC markers. Four fluorescent dye-conjugated Flow cytometry-specific antibodies, including anti-CD34-PE, anti-CD45-FITC, anti-CD90-FITC, and anti-CD73-PE, were used for cell staining in the dark. Finally, the presence of the rBM-MSCs specific surface antigens was assessed by a flow cytometer (BD FACS Caliber; BD Biosciences, San Jose, CA, USA), and Cell Quest software was used to analyze the obtained data (BD Biosciences).

Investigation of differentiation potential of rBM-MSCs

Adipogenesis and osteogenesis potential of extracted rBM-MSCs were investigated to confirm their multipotency. The confluent rBM-MSCs cells were harvested and counted, then a density of 5 × 104 cells per well was seeded in a 6-well plate vessel in an expansion medium with a base of DMEM enriched with 10% of fresh fetal bovine serum (FBS), Penstrp (Gibco), 1X ITS (Sigma), and dexamethasone 1 × 10–6 M at standard Co2 incubator. When cells fill 80% of the wells’ area, the medium mentioned above was replaced with either osteogenic or adipogenic differentiation induction cocktail media.

To evaluate the rBM-MSCs potential for adipocyte differentiation, the cells were cultured for 14 days in the differentiation medium. Adipogenesis induction medium consisted of 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 100 µM indomethacin, 10 µg/ml insulin, 1 µM dexamethasone. Considering the company’s recommended instructions. Differentiation Media were changed twice weekly, and special care was taken not to disturb the differentiation monolayer. By 14 days, cells were fixed and stained with Oil Red which stains neutral lipids. Meanwhile, to evaluate the differentiation potential of rBM-MSCs to osteocytes, the cells were cultured for 21 days in the differentiation medium, considering the company’s recommended instruction. Osteogenesis induction medium contained Alpha-Minimum Essential medium (α-MEM) enriched with 10 mM β-glycerol phosphate, 10% FBS, and 0.1 μM dexamethasone (Bioidea, Iran) Louis, MO, USA) and 10% FBS [54].

After 21 days, the differentiated cells were subjected to Alizarin/Red staining solution (Bioidea, Iran) to detect the mineralization within the calcium-containing osteocytes. The final images of both experiments were then observed and acquired by inverted microscopy (Hund, Wetzlar, Germany).

In previous works, the effect of Se on osteogenesis and chondrogenesis was checked [55] therefore in the present work, we checked the effect of sodium selenite on adipogenesis. In this way, rBM-MSCs (P5) were seeded in 6 well plates and treated with 0.1 µM sodium selenite supplemented in a routine medium for 72 h, then the medium was replaced with an adipogenesis induction cocktail. The oil droplets after adipogenesis was quantified using our previously deep learning-based method [56]. The microscopic images of oil droplets produced after adipogenic differentiation in control or sodium selenite treated cells were quantified. The captured microscopic photos were divided into square drawings, and the count of lipid droplets were studied, based on the deep learning methodology [56].

Evaluation of the optimal sodium selenite dosage and treatment course MTT assay and cell morphology observation

The tetrazolium-based MTT assay was employed to study the impact of different sodium selenite concentrations on the rBM-MSCs viability and proliferation. For this purpose, ~ 5000 rBM-MSCs were transferred to each well of a 96-well plate and incubated overnight. Cells were allowed to attach to the bottoms of microtiter wells and expand themselves in the expansion medium mentioned above. Then the medium was replaced with a series of low glucose DMEM media containing five different concentrations of sodium selenite (0.001, 0.01, 0.1, 1, and 10 µM) and further incubated for 24, 72 h. According to the previous report, the doubling time of the rBMSCs population is 49.9 ± 4.2 h [57] Therefore, in the present study, we performed a preliminary study by duration times less (24 h) and more than doubling time (72 h) and a series of concentrations. Following treatments, the media was aspirated from each well and discarded. 20 μl MTT solution in the fresh Selenium-free medium was added to each well and incubated in the dark for 4 h. After removing the supernatants, 200 μl of dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was added to each well and incubated for about 20 min to completely dissolve the produced formazan crystals. Subsequently, the absorbance of each well was measured at the wavelength of 570 nm in an ELISA plate reader. Moreover, morphological analysis of cells before and after treatment with sodium selenite was examined by inverted microscopy (OLYMPUS IX71).

Evaluation of stemness-related genes expression

The effect of sodium selenite treatment on the expression level of the three known stemness-related genes (i.e., OCT-4, NANOG, and SIRT1) was evaluated by qRT-PCR. For this purpose, the rBM-MSCs were cultured in a complete DMEM low glucose media supplemented with sodium selenite to the concentration of 0.1 µM. The control replicates were also included in which the rBM-MSCs were cultured in the same condition but without sodium selenite supplementation. After 72 h, cells from the treat and control groups were collected and used for total RNA extraction with Trizol reagent according to the manufacturer’s protocol. The RNA obtained from the Trizol extraction step was mixed with RNA-Free DNase to eliminate any DNA impurity. The concentration of the total extracted RNA for each replicate was then determined by the UV–Vis spectroscopy approach using NanoDrop (Thermo Scientific, Waltham, MA). The cDNA was synthesized using the Revert Aid cDNA synthesis kit from the Thermo Scientific company. The expression level of the three mentioned stemness-related genes was evaluated using cyber Green Master Mix and unique primers (Table 1) on a Rotor gene 6000 system (Corbett, Qiagen, Australia) at 40 cycles as follows: 95° C for 10 min, denaturation at 95° C for 10 min, annealing 60° C for the 30 s and extension step at 72° C for 30 s. The formula of fold chamg = 2–∆∆Ct was applied to analyze the acquired CTs, and the rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was referenced as the housekeeping gene. The control group expression (fold change) was normalized to 1 and the relative expression > 1 was considered as up-regulation and the 0 < relative expression < 1 as down-regulation for each gene.

Table 1 Primer sequences that were used for stemness gene expression in qPCR

Measurement of intracellular ROS level in live cells

The 2′,7′-dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay was used to measure the level of intracellular ROS accumulation after 72 h incubation in a cell culture medium with the concentration of 0.1 µM sodium selenite and also the control group.

The rBM-MSCs were first seeded in a 6-well plate and treated for 45 min with 30 µM DCFH-DA in the dark. The DCFH-DA treated rBM-MSCs were collected by trypsinization and washed twice in PBS. The DCF fluorescence was then measured using a FACS cytometer by FACS analyses (BD FACS Calibur (BD Biosciences, San Jose, CA, USA)) at an excitation wavelength of 488 nm and DCF band-passed emission filter of 530 nm. The histograms of flow cytometry analysis were obtained using the FlowJo software (Cell QuestTM software, BD Biosciences). Then PI dye was added to the tubes containing the rBM-MSCs, to the final concentration of 1 μg/ml. The whole PI staining process was performed on ice and in the dark, and the tubes immediately proceeded to the cytometry analysis. PI fluorescent data were collected in the FL-3 channel (620 nm) of FACS (BD FACS Calibur (BD Biosciences, San Jose, CA, USA)).

FACS characterization of the rBM-MSCs: effect of sodium selenite treatment

The rBM-MSCs treated with 0.1 µM of sodium selenite and controlled cells were characterized by FACSCalibur (Becton Dickinson). Antibodies and isotypes were used as follows: IgG1-FITC -Isotype control, IgG1-PE -Isotype control, and IgG1-PerCP. Cy5.5-Isotype control, CD105- (Biolegend), CD44-PE (eBioscience), CD45-FITC (BD Bioscience), and CD31-PE (eBioscience).

Western blot analysis

Western blot analysis was performed for AKT, P38, SIRT1, SOD, GPX, TrxR, HIF1-α, and Nrf2, which are among the key proteins involved in the regulation of oxidative stress. The analysis was carried out as previously described with some modifications. Briefly, the cell lysates were mixed with an equal volume of the 5X Laemmli sample buffer. The final protein concentrations were determined by the Bradford assay at OD595. The protein contents of the fifth passage of the rBM-MSCs (15 μg per lane) in the Laemmli sample buffer were boiled for 5 min to ensure complete solvation and then separated using an SDS-PAGE approach. The final SDS-PAGE pattern of these proteins was then electrotransferred onto a 0.2 μm immune-Blot™ polyvinylidene difluoride (PVDF) membrane (Cat No: 162–017,777; Bio-Rad Laboratories, CA, USA) in a transfer apparatus. At the next step, PVDF membranes were subjected to blocking in a 5% BSA solution for 120 min (Cat No: A-7888; Sigma Aldrich, MO, USA) in 0.1% Tween 20 for 1 h. Then, the membranes were incubated in the anti-HIF-1α (Cat No: ab51608, Abcam), anti-GPX (Cat No: ab96257, Abcam), anti-AKT (Cat No: 680302, Biolegend), anti-Nrf2 (Cat No: ab62352, Abcam), anti-p38 antibody (Cat No: ab170099, Abcam), anti-SOD (Cat No: ab83108, Abcam), anti-TrxR (Cat No: sc-28321, Santa Cruz) and anti-beta actin-loading control antibodies (Cat No: ab8227; Abcam) for 1 h at room temperature. After the first incubation in the solutions of the primary antibodies, for washing of the membranes TBST solution was rinsed three times and subsequently exposed to goat anti-rabbit IgG H&L HRP-conjugated (Cat No: ab6721; Abcam) and goat anti-mouse IgG (HRP) (Cat No: ab97240; Abcam) secondary antibodies. Afterward, the membranes were incubated with enhanced chemiluminescence (ECL)western blot reagents for 1–2 min and visualized using an enhanced chemiluminescence system. The expression of proteins was normalized to β-actin. The blots were then subjected to a densitometry analysis, and the relative concentration of the protein bands was measured using gel analyzer Version 2010 software (NIH, USA). To calculate the relative concentrations of each band, (Percentage area under the curve band/ the percentage area under the curve of actin band, based on the calculated values, the groups were compared as described previously [58].

Molecular function analysis by DAVID database

After analyzing our proteins with western blot, Entrez gene ID of AKT, P38, SIRT1, SOD, GPX, TrxR, HIF1-α, and Nrf2 were uploaded into the DAVID database (DAVID 6.8; to identify their molecular functions.

In-silico investigation of ROS stress

The protein–protein interaction (PPI) network analysis was performed using STRING (, to seek the interactions among AKT, P38, SIRT1, SOD, GPX, TrxR, HIF1-α, and Nrf2 in homo sapiens (Fig. 8B).

Statistical analysis

The experiments were designed in at least three replicates for all tests. The obtained data were analyzed using SPSS software (version 16), and the results were stated as mean ± SD. Differences between the means of each group were evaluated using Student’s t-test or ANOVA. A confident p-value range (p-Value < 0.05) was accepted as statistically significant.


rBM-MSCs isolation and validation

In the first step, the morphology of the isolated cells was studied under an inverted light microscope which was spindle-shaped after 48 h in vitro culture and reached 60% confluency after a week (Fig. 1A). Based on the FACS analysis, the isolated rBM-MSCs’ surface marker profile was in accordance with standard CD markers of MSCs [59]. In particular, the isolated rBM-MSCs were positively stained for CD90 (96.21%) and CD73 (97.09%), but almost the majority of them didn’t express CD45 or CD34 (CD45 (0.7%) and CD34 (3.67%)) (Fig. 1B).

Fig. 1
figure 1

Isolation, cultivation and characterization of rBM-MSC. A The rBM-MSCs morphology was spindle shape, and they established adherent culture within one week. B Most (97.63%) of rBM-MSCs showed positive surface expression for CD90 as a surface marker in MSCs. Also, 99.35% of them were positively stained for CD73. An ignorable percentage (less than 1%) of the rBM-MSCs showed expression of CD34 and CD45, which are markers of hematopoietic lineages. Ca Confirmation of adipogenesis potential was done by the presence of detection of intracellular lipid droplets. Cb Confirmation of osteogenesis potential was done by the presence of mineralization particles stained red. All experiments were done in triplicate

The rBM-MSCs were subjected to adipocyte differentiation to evaluate their multipotency capability. After 10 days of induction, lipid droplets were observed under a microscope, indicating the maintained adipogenesis potential of these cells. Subsequently, oil red staining was also performed and confirmed adipocyte differentiation (Fig. 1Ca). The osteoblast differentiation potential of rBM-MSCs was also assessed by exhibiting calcium deposits after the differentiation process. Accordingly, standard Alizarin red staining exhibited calcium accumulation detectable as red mineralization showing spots (Fig. 1Cb).

Cell proliferation study findings

Sodium selenite supplementation was performed in a range of 0.001 to 10 µM concentrations in the expansion medium of rBM-MSCs, and their proliferation was compared with the non-treated control group. The colorimetric proliferation MTT assay results demonstrated that the best concentration of sodium selenite for supporting the proliferation of the rBM-MSCs is 0.1 µM. Since the number of the cells increased significantly at concentrations up to 0.1 µM, while the impact of higher doses (0.001, 0.01, 0.1, 1, 10 µM) on proliferation potential was less when compared with 0.1 µM at different treatment intervals (24 h, 72 h) (Fig. 2). For the next experiments, 0.1 µM was used and other studies also reported the same dosage range: Yan et al. reported that 0.1 μM and 1 μM of Sodium selenite were able to promote the BMSCs proliferation and apoptosis, respectively. In addition, they found that using 0.1 μM of Sodium selenite significantly induce osteogenic and adipogenic differentiation via up-regulating associated factors such as LPL and PPRAG ( lipid factors) and RUNX2, COL1, and BGP ( as osteogenic factors) [47]. Ebert et al. showed that supplementation of selenite (0.1 μM) is an important countermeasure during oxidative stress in routine cell culture process due to increasing the anti-oxidative status of BMSCs by restoring basal GPx and TrxR, promoting mRNA expression of basal and ROS-stimulated SOD1, and finally decreasing the accumulation of ROS [48]. Che et al. concluded that in rats with Hashimoto’s thyroiditis (HT), the combination of Sodium selenite (0.5–20 µM) with adipose-derived mesenchymal stem cells (AMSCs) is able to accelerate the growth of AMSCs and their anti-oxidative potential by raising the glutathione level significantly. Moreover, Sodium selenite increased the level of inflammatory cytokines such as HGF (hepatocyte growth factor), TGF-β (transforming growth factor beta), and SCF (stem cell factor) [49].

Fig. 2
figure 2

Effects of Sodium selenite on the viability of rBM-MSCs, determined by MTT assay after 24 (A) and 72 (B), hours incubation with different concentrations (0.001, 0.01, 0.1, 1, 10 µM) concentration in comparison to the control group. In the rBM-MSCs optimum OD was obtained at 0.1 µM of Sodium selenite, which showed the best viability and proliferation.*p < 0.05. Number of samples = 3, Data: Mean ± SD

Gene expression

The qPCR results showed that the concentration of 0.1 µM sodium selenite significantly improved the expression of OCT-4, NANOG, and SIRT1 in the treated rBM-MSCs compared to the untreated control group (p-Value < 0.01) (Fig. 3).

Fig. 3
figure 3

Relative gene expression of Oct-4, NANOG, and SIRT1 at 0.1 µM Sodium Selenite and control groups. The Oct-4, NANOG, and SIRT1 were significantly upregulated. The error bars show mean ± SD.***(p-Value ≤ 0.001), **(p-Value ≤ 0.01). Number of samples = 3, Data: Mean ± SD

Effect of Sodium Selenite on intracellular reactive oxygen species and cell viability

In the presence of Sodium selenite as an antioxidant, the level of the oxidized form of DCFH was decreased in the rBM-MSC, and treatment with sodium selenite (0.1 µM) can decrease the level of cellular oxidative stress below the value of untreated cells (p-value:0.05) (Fig. 4A-D). Also, along with the ROS detection method, the living cell population was evaluated by PI staining, and although it cannot penetrate the cell membranes of living cells, it can penetrate those of dying or dead cells. Before Sodium selenite supplementation, the counted live cells were -5235 ± 261, but after treatment, they counted 6773 ± 135.46 (Fig. 4C). The results showed that the percentage of live cells was 69.1% ± 5.7 in the untreated group, but it was increased to 85.3% ± 3.6 in the Sodium selenite-treated group, which shows the viability enhanced by Sodium selenite treatment (Fig. 4C).

Fig. 4
figure 4

ROS generation and viability assessment by flow cytometry analysis. ROS generation in rBM-MSCs (A) cultured with Sodium Selenite (0.1 µM) (B). The ROS levels were determined by DCFH staining and flow cytometry. The viability of cells before and after Sodium selenite treatment was studied (C), which showed higher live cell populations after treatment. The ROS levels reduced after sodium selenite treatment (D). Number of samples = 3, Data: Mean ± SD

Morphological and FACS characterization of sodium selenite treated cells

Although in various studies for cell therapy applications using passage 3–5 of MSCs is recommended. A number of reports have proved that passage 5 of MSCs is at the border of initiating senescnce and reduction of stemness characteristics therefore could be examined as model cells to study the effects of antioxidants on stemness properties. Yang and colleagues documented that MSCs maintain their morphology and plasticity up to P5 but at later subcultures show irregular morphologies and sizes [51]. Even at lower passage number for instance in passage 3 MSCs showed reduced telomerase activity and chromosome anomalies may originating from in vitro expansion, which could impair stemness of them [60]. Also, study by Ridzuan et al., documented that the enzyme beta galctosidase as a senescence marker detected to become up regulated at passage 5. Besides G0/G1 arrested cell cycle because of cellular senescence at in vitro expansions were observed in passage 5 [61].

P5 passages of rBM-MSCs were cultured with 0.1 µM of sodium selenite for 72 h, and in comparison, with untreated cells, no significant morphological changes were observable. Both groups had spindle-shaped and elongated cells Fig. 5 a, b. A comparison of the adipogenesis potential of sodium selenite pre-treated cells and those of controls (Fig. 5c and d, respectively) exhibited a higher percentage of oil droplets in sodium selenite pre-treated cells (Fig. 5e). Such findings emphasized on positive effects of ROS regulation on the plasticity of the rBM-MSCs.

Fig. 5
figure 5

Morphological observation and adipogenic differentiation of control compared to Sodium selenite treated rBM-MSCs. Control rBM-MSCs (a) rBM-MSCs treated with sodium selenite exhibited spindle-shaped and elongated without significant morphological changes (b). rBM-MSCs subjected to adipogenesis (c), rBM-MSCs treated with sodium selenite showed increased lipid droplets after oil red o staining (d). The percentage of oil droplets were quantified and reported (e). Number of samples = 3, Data: Mean ± SD

The cell surface markers expression of treated and untreated rBM-MSCs were reported in Fig. 6 and Table 2. According to the findings, most of the rBM-MSCs exhibited CD105 and CD44 (Fig. 6 E–H). Also, Sodium selenite did not induce any spontaneous differentiation-related changes in markers when used as a supplementation in routine culture (Table 2).

Fig. 6
figure 6

FACS characterization of markers after Sodium Selenite treatment of the rBM-MSCs. Based on the FACS analysis of the rBM-MSCs, the number of cells for CD44 and CD105 markers increased by about 4.6% and 37%, respectively, after sodium selenite treatment. FACS characterization of negative markers for control (A,C, E,G) and Sodium Selenite treatment (B,D, F, H) of the rBM-MSCs. Based on the FACS analysis of the rBM-MSCs, the number of cells for CD31 and CD45 markers was comparable. Number of samples = 3, Data: Mean ± SD

Table 2 Frequencies of rBM-MSCs stained for CD markers quantified by FACS

Effect of sodium selenite on p38/MAPK/Nrf2 signaling pathway proteins

In the present study, the expression of the Nrf2 and its related enzymes was assessed by WB analysis. These proteins were selected by reviewing a bulk of relevant previously-published papers and considering our in-silico analysis results (3–7, 3–8 sections). Results of the WB analysis revealed a clear augmentation of the Nrf2 and other antioxidant enzymes, such as SOD, GPX, and TrxR, following sodium selenite exposure (p-Value < 0.05) (Fig. 7). To discover the effect of sodium selenite (0.1 µM) on ROS, the level of P38, the key protein in apoptosis activation, and the P38/MAPK signaling pathway and AKT (an essential protein in phosphorylation and deactivation of pro-apoptotic proteins) were also analyzed by WB. The results showed that after treatment with 0.1 µM sodium selenite, there were no significant differences between the control and treatment groups. The AKT level compared to the control group increased after 0.1 µM sodium selenite treatment(Fig. 7). The raw data for WB was included in the supplementary file (S1 and S2).

Fig. 7
figure 7

Western blot comparison of signaling molecules in response to Sodium Selenite (0.1 µM) in the rBM-MSCs. Expression of Nrf-2 and Selenium dependant antioxidant enzymes, such as SOD, GPX, and TrxR, increased compared with the control group. The level of AKT, P, increased after 72 h treatment with 0.1 µM sodium selenite. Data are represented as mean ± SD* p < 0.05, ** p < 0.01 significant level of treatment (T) vs. control (C) group. C1,2, and 3 are the three replicates of the control groups and T1,2, and 3 are three replicates of selenium (0.1 µM)treatment group. Full length blots are presented in the supplementary data. Number of samples = 3, Data: Mean ± SD

Bioinformatics analysis of oxidative stress signaling pathways and Nrf2 Oxidative stress activates the MAPK/p38 and other signaling pathways

Based on our literature review of the previously-published papers in this research area, we provide a list of proteins and signaling pathways affected by Selenium supplementation. A comprehensive search strategy was applied, and because there were not many published reports, data were entered without any cell type or case limitation (Table 3).

Table 3 Pathways affected by selenium

Among the signaling pathways affected by Selenium, we focused on mitogen‐activated protein kinase (MAPK or MAP kinase) pathways (ERK, JNK, and p38 kinase) activated in response to oxidative stress through ROS production [62, 63]. ROS are thought to activate MAPK pathways based on their oxidation potentials. ROS may achieve this by oxidizing MAPK signaling proteins and inactivating MKPs [75]. ERK regulates ROS levels and activation of Nrf2, which upregulates the main antioxidant enzymes (SOD, TrxR, GPX). Furthermore, by activating JNK, ROS can also cause cellular survival by upregulating FoxO expression, which enhances the levels of antioxidant production, and SIRT1, leading to the inhibition of p53-dependent transcription [76, 77].

In oxidative conditions, Keap1 is attached to Nrf2 and prevents its translocation to the nucleus; this leads to subsequent overexpression of the downstream genes [78]. In response to inducing ROS, Nrf2 is activated by oxidation of the cysteine residues in Keap1 and subsequently Nrf2 dissociation. OS leads to disulfide bridges, the Nrf2/Keap1 complex structure alteration, and the Nrf2 detachment from keap1. Alternatively, it has been documented that ROS levels can be affected by increasing translation levels of antioxidant proteins for instance Cu–Zn-SOD, Mn-SOD, GPX, GST-pi, MT3, and FHC through the NF- κB pathway [79]. Moreover, Recent studies have shown that oxidants produced from mitochondria are important mediators of molecular signaling. The ROS released by the electron chain in mitochondria is implicated in the mitochondria-dependent apoptotic pathway that involves proapoptotic (Bax, BAK) and antiapoptotic (Bcl-2) protein binding (14–3-3), the release of cytochrome c, and p53 signaling activation, leading to cellular death [80]. Oxidative stress activates the MAPK signaling pathway. ERK, JNK, and p38 kinase activation in response to oxidative stress through ROS can have both prosurvival and proapoptotic effects. ROS-activated PLC-gamma and Src phosphorylate Ras and Raf, and ERK. Activated ERK activates Nrf2, which upregulates antioxidants. ROS also increase the expression of JNK and p38. Like ERK, JNK has both prosurvival and proapoptotic roles in response to oxidative stress. Activated JNK can promote cell survival via the activation of FoxO, which upregulates antioxidant production, and SIRT1, which inhibits p53-dependent transcription. Conversely, activated JNK can promote apoptosis via activities both in the cytoplasm and in the nucleus. In the cytoplasm, JNK negatively regulates antiapoptotic proteins, such as Bcl-xL [81].

According to the DAVID database, the primary molecular function of these proteins is oxidoreductase. In the STRING database Nrf2, SOD, TrxR, GPX, AKT, P38, SIRT1, and P38 proteins were inserted in multiple proteins tab for homo sapiens and the signaling cascade has various topological characteristics, such as 28 nodes, 113 edges, 8.07 average node degrees, and 0.725 average local clustering coefficient. PPIs in this cascade have a confidence level of 0.400. The proteins are strongly connected with other proteins in the cascade. Therefore, in the present study, we investigated some of the most important proteins that are found in the Nrf2 signaling pathway and have an important role in oxidoreductase function (Nrf2, SOD, TrxR, GPX, AKT, P38, SIRT1, P38) found in defined signaling pathways and evaluated their role in Selenium treatment and ROS increment to determine the relationship between these pathways (Fig. 8). The number of the proteins involved in stress oxidative pathways were listed in Table 4. Overal results of this work were summarized in Fig. 9.

Fig. 8
figure 8

Schematic illustration of Nrf2 signaling pathway in oxidative stress (A) and protein network STRING (B). Nrf2-related signaling pathway plays a key role in directly regulating oxidative stress signaling pathway by overexpression of antioxidant enzymes (TrxR, SOD, GPX). Nrf2 is regulated by Keap1 in constitutive/oxidative conditions. Proteins coded light green were investigated in the present study, and those coded in dark green are bioinformatic predictions of the next layers in the network. The light green proteins was selected based theit ipmrtance in the siganling pathways, shown by the previously published reports (summerized in Table 3). The importance of these proteins were also confirmed by our by our bioinformatic analysis and pathway investigation by the String tool. A There are meaningful interactions among proteins (Nrf2, SOD, TrxR, GPX, SIRT1, AKT, P38, HIF1) based on the network and pathway analysis by STRING online tool. The different color lines show various interaction types among proteins on the system level. Dotted lines indicate inter-cluster and straight lines show cluster association (B)

Table 4 The biological process involved in ROS stress regulation, according to STRING analysis
Fig. 9
figure 9

Supplementation of rBM-MSCs culture with sodium selenite caused Nrf2 overexpression, reduced the ROS level, improved cytoprotection by regulating the expression of HIF-1 of AKT, SOD, GPX, and TrxR markers and enhanced the expression of stemness related OCT-4, Sox2, Nanog. Number of samples = 3, Data: Mean ± SD


In the basic research and clinical applications of stem cells, the cell sources must be plentiful and accessible and be efficiently delivered to the damaged sites. They also must be validated and optimized in the appropriate animal models, non-immunogenic and non-tumorigenic [82].

Among stem cell sources, MSCs have significant differentiation potential and could obtain easily from various tissues, including adipose tissue, bone marrow, and skeletal muscle [83]. Mainly, BM-MSCs are desirable cell sources due to their properties such as high proliferation, ease of access, harvesting simplicity, immune privilege, and differentiation potential into adipogenic, osteogenic, and chondrogenic lineages[84-86]. However, the stemness potential, differentiation capacity, and survival rate of cultured and transplanted stem cells to damaged sites could be limited by ROS-mediated oxidative stress imposed during isolation and in vitro expansion. This problem can lead to apoptosis and cell death in culture media and rejection of donor MSCs after transplantation [87, 88]. In addition, excessive ROS and oxidative stress could damage or modify cellular macromolecules, such as proteins, lipids, and DNA. Oxidative stress disrupts mitochondria, releases cytochrome C, and induces apoptosis by activating signal transduction pathways, such as P38 MAPK [12, 88]. The rat bone marrow-derived MSCs (rBM-MSCs) were employed for this work because they are available and user-friendly model cells in studies of basic stem cells and regenerative medicine [89]. The rBM-MSCs cells were isolated, cultured, and characterized and all results were in accordance with the International Society for Cell & Gene therapy (ISCT).

During the transplantation, the high mortality rate of BM-MSCs has been reported because oxidative stress-mediated apoptosis results in significantly decreased transplantation efficiency [90-92]. Mammalian cells have many anti-oxidative defense components to secure against oxidative stress. This incorporates the expression of specialized proteins such as catalase, superoxide dismutase, and a few peroxidases, as well as unspecific ROS-degrading antioxidants such as vitamin E, ascorbate, uric corrosive, carotenoids, or glutathione [93]. Therefore, using antioxidant supplements could increase the expression of exogenous antioxidant proteins and enzymes to improve the antioxidant ability of BM-MSCs, but their effect is limited [94]. Therefore, a valid strategy for protecting MSCs against in vitro acquired oxidative stress is necessary for entering these cells into clinical applications or getting real results in basic research. This study investigated sodium selenite as a ROS scavenging material to enhance the antioxidant ability of rBM-MSCs and reduce undesirable effects of oxidative stress [95]. Among methods coming to mind to protect MSCs, the cheapest and less challenging approach is the enrichment of media with available antioxidant elements. We selected Selenium since several reports of its positive impacts on MSCs were documented [66, 96]. However, few investigations performed on stemness, surface markers, differentiation potential, and probable signaling of Selenium mediated improvements of MSCs. In this work, the different doses of Sodium selenite were used to determine the optimum dose. The gene expression study using qRT-PCR showed that the transcription of NANOG and OCT-4 were significantly upregulated after Sodium selenite treatment.

Furthermore, supplementing rBM-MSCs culture with sodium selenite results in overexpression of Oct-4, NANOG, and SIRT1, which could improve the self-renewal and multipotency of rBM-MSCs. Previous studies have shown that regulation of p53 via Nrf2/p53/Sirt1 and OCT-4/Sirt1/p53 pathways leads to improved stemness ability of rBM-MSCs [97, 98]. Furthermore, p53 is a tumor suppressor and pro-apoptotic gene, which was recently identified to negatively mediates the stemness potential of stem cells [97, 99, 100].

SOX2, NANOG, and OCT-4 are stemness genes that maintain the multipotency and self-renewal of MSCs; it also is thought that Sirtuin 1 (Sirt1) has a role in the regulation of MSCs’ stemness ability. SIRT1 is a NAD-dependent protein deacetylase that controls several cellular mechanisms, including bone hemostasis, senescence, and metabolic pathways. In rBM-MSCs, Sirt1 can lead to long-term growth by reducing senescence during cell passages [97, 100]. Sirt1 activity can be suppressed by direct binding of P53 to its promoter, while Nrf2 can promote the degradation of p53 by regulating the expression of Mdm2, subsequently increasing the expression of Sirt1 and maintaining the stemness capacity of MSCs [97, 101].

Some studies indicated that Sirt1 regulates the expression of NANOG and OCT-4 by preventing p53 activation in human embryonic stem cells (hESC) and regulates SOX2 by post-translational modification in rBM-MSCs [98, 102]. In oxidative stress conditions, expression of Sirt1 prevents cytoplasmic translocation of p53 by its deacetylation, inhibits the p53-mediated repression of NANOG expression in stem cells, and maintains the stemness potential of stem cells [99, 100]. So increasing the level of Nrf2 protein after treatment with sodium selenite can maintain the undifferentiated state of rBM-MSCs via enhancing Sirt1 expression at the mRNA level. In addition, it was well known that OCT-4 preserves the stemness ability of stem cells by controlling NANOG and Sox2. OCT-4 maintains the stemness capacity of ESCs via inducing SIRT1, which inhibits p53 activity that induces ESCs differentiation through suppressing another stemness gene such as NANOG [98]. Aligned with these reports, our outcomes showed that sodium selenite could enhance the expression of stemness genes, including NANOG, OCT-4, and especially SIRT1 in mRNA level. The high level of SIRT1 is related to Nrf2- mediated p53 suppression and OCT-4-mediated inducing after treatment with sodium selenite in rBM-MSCs. Given the importance of these results in clinical applications, the role of sodium selenite in the maintenance of rBM-MSCs stemness potential is highlighted. On the one hand, overexpression of Nrf2 not only improves the antioxidant capacity [103] and survival rate of rBM-MSCs but could also regulate the stemness potential. Nevertheless, to date, no studies confirmed the association between Nrf2 and MSCs stemness potential or the interaction between Nrf2 and the expression of stemness genes [97]. Therefore, our study results were near to those of other mentioned studies and verified that sodium selenite could influence the rBM-MSCs in a positive manner.

Subsequently, the CD markers profile of rBM-MSCs were checked using flow cytometry. There are some reports regarding aged-associated lessening of CD markers profile [104, 105]. Moreover, some publications reported that MSCs surface markers decrease in higher passages [106] which subsequently, reduces the stemness and differentiation capacity and as a result, decreases the quality of MSCs. We selected 2 positive and 2 negative CD markers to check if Sodium selenite could preserve their decline during in-vitro culturing conditions. Interestingly, our results showed that Sodium selenite could improve CD markers expression profile of rBM-MSCs. Bouquest, et al. showed that CD31 negative MSCs up-regulate genes associated with stemness [107], thus we used this marker to check if could affect stemness potential. And, our results were in accordance to those of Bouquest. Also, Pham et al. investigated CD105 link with stemness and showed that the CD105 positive MSCs and CD105 negative MSCs have parallel stemness and differentiation potentials to three main lineages osteocyte, chondrocyte, and adipocytes [108, 109]. Previous report exhibited that CD44 expressed by MSCs has considered to play a role in stemness maintenance because of its involovment in contact between stem cells and progenitor cells within their niche [110].

Subsequently, the differentiation potential of rBM-MSCs was checked after Sodium selenite exposure, which exhibited enhanced adipogenesis in Sodium selenite pre-treated rBM-MSCs.

Our findings are in line with Wang et al., [111] who reported an increase in adipogenesis and fat deposition by Selenium pre-treatment. In line with our work previous reports documented that, sodium selenite improved chondrogenesis and osteogenesis of MSCs [56, 112].

The ROS levels of rBM-MSCs were investigated after Sodium selenite treatment. Sodium selenite supplementation reduced ROS levels in Cells.

In addition, previous studies confirmed that the overexpression of Nrf2 can improve MSCs’ resistance to oxidative stress and promote their survival. Nrf2 feels the level of intracellular ROS and can maintain the redox hemostasis under several conditions in MSCs. In response to oxidative stress, Nrf2 is activated and upregulates some downstream antioxidant and cytoprotective genes such as SOD and SIRT1 and some growth factors and cytokines like SDF-1 and VEGF, resulting in reduction or inhibition of apoptosis and senescence, and improvement of cell survival, migration, stemness maintenance, antioxidant capacity, and self-renewal of MSCs. [113]. Our results confirmed that supplementing sodium selenite into the culture medium could induce expression of Nrf2 protein in rBM-MSCs, followed by increasing the expression of SOD, GPX, and TrxR through Nrf2/ARE signaling pathway and ultimately decreased ROS level in rBM-MSCs.

Another investigated protein in cell survival and apoptosis is Akt. Akt, protein kinase B, is an essential component in the biological signaling pathway, such as the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, which prevents apoptosis, enhances cell survival, and improves angiogenesis and migration [114].

Kim et al. [115] reported that the AKT signaling pathway protected stem cells against oxidative stress damage by activating the Nrf2-ARE pathway. In addition, Martin et al. [116] indicated that Akt could increase the translocation of Nrf2 in the nucleus and bind to AREs by stabilizing of Nrf2 protein, which has a very short half-time. Also, they determined that activation of Akt increases stable protein levels, leading to enhanced Nrf2 protein levels. Besides, to improve the Nrf2 activity, Akt protein can decrease MSCs apoptosis and increase their survival rate through blocking the activity of pro-apoptotic protein Bax and enhancing anti-apoptotic protein Bcl-2.Based on our results, supplementation of sodium selenite could increase Akt protein level, improve antioxidant ability and stemness potential of rBM-MSCs by increasing Nrf2 stability and activity but also could decrease apoptosis rate of cells via regulation of Bax and BcL2.

Moreover, the MAPKs pathway was associated with apoptosis reduction of rBM-MSCs after treatment with sodium selenite. This pathway has critical functions in signal transduction and modulates stress responses and apoptosis, which their main subgroups include Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 [117].

It was indicated that p38 is a common regulator of mitochondrial and ER stress-induced apoptosis through caspase-3 and caspase-12 in MSCs. Therefore, activation of p38 is contributed to early apoptosis due to oxidative stress [117]. We found that supplementing rBM-MSCs culture media with sodium selenite as an antioxidant doesn’t change the P38 related to the MAPK signaling pathway in a significant way in this study, the Hypoxia-inducible factor 1α (HIF1α) after treatment with sodium selenite was decreased, while previously indicated that HIF1α expression is increased in hypoxia condition, which is necessary for pluripotency and survival of stem cells in hypoxic condition [118-120]. Additionally, the role of HIF1α in hypoxia conditions be considered that could affect various cellular physiology processes such as anti-apoptosis, proliferation, and migration of MSCs [119]. As a result, based on our findings, HIF1α had not a vital role in oxidative stress.

Previous studies showed that HIF-1a transcription is amplified in oxidative stress [121] (in agreement with previous reports, we observed that HIF-1a protein levels were high in cells and were considerably reduced after Selenium treatment. Also, we observed that Nrf2 shows up-regulation and inversely down-regulation of HIF-1a. This data is compatible with a previous study, which documented opposite responses to oxidative stress [122].


The final conclusion is that the ideal cytoprotection for rBM-MSCs during in-vitro experiments could be sodium selenite supplementation. Because, sodium selenite helps to maintain rBM-MSCs stemness capacity, differentiation potential, and expression of surface CD markers. Moreover, positively regulate antioxidant proteins expression via increased Nrf2 protein levels, leading to the suppression of oxidative stress. Our results verified the antioxidant and cytoprotective properties of sodium selenite on rBM-MSCs quality which may be mediated via the Nrf2 signaling pathway.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files are available from the corresponding author on reasonable request.



Bone marrow-derived mesenchymal stem cells


Mesenchymal stem cells


Reactive oxygen species


3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


2'-7'Dichlorofluorescin diacetate


Fluorescence-activated cell sorting


Hypoxia-inducible factors


Glutathione peroxidase


Superoxide dismutase


Thioredoxin reductase


Protein kinase B (PKB)


Nuclear factor erythroid 2-related factor 2

CD markers:

Cluster of differentiation


Octamer-binding transcription factor 4


Sirtunin 1


Nuclear factor erythroid 2-related factor 2


Oxidative stress


Mitogen-activated protein kinase




Phosphate-buffered saline




Fluorescein isothiocyanate


Fetal bovine serum


Dimethyl sulfoxide


Reverse transcription polymerase chain reaction


2′,7Dichloro-dihydro-fluorescein diacetate






Propidium iodide


Discounted cash flow


Sodium dodecyl sulphate–polyacrylamide gel electrophoresis


Polyvinylidene difluoride


Bovine serum albumin


Tris-buffered saline (TBS) plus Tween 20


Horseradish Peroxidase


Enhanced chemiluminescence


Extracellular signal-regulated kinas


Jun N-terminal kinase


Antioxidant response element


Kelch-like epichlorohydrin-associated protein 1


Nicotinamide adenine dinucleotide


Mouse double minute 2 homolog


Human embryonic stem cells


Hypoxia-inducible factor 1α


Spermatogonial stem cells


Neural progenitor cells


Cancer stem cells


Mouse Leydig cell line


Kashin–Beck disease


  1. Park J, Lee JH, Yoon BS, Jun EK, Lee G, Kim IY, You S. Additive effect of bFGF and selenium on expansion and paracrine action of human amniotic fluid-derived mesenchymal stem cells. Stem Cell Res Ther. 2018;9(1):293.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Pontikoglou C, Deschaseaux F, Sensebé L, Papadaki HA. Bone marrow mesenchymal stem cells: biological properties and their role in hematopoiesis and hematopoietic stem cell transplantation. Stem Cell Rev Rep. 2011;7(3):569–89.

    Article  PubMed  Google Scholar 

  3. Miao C, Lei M, Hu W, Han S, Wang Q. A brief review: the therapeutic potential of bone marrow mesenchymal stem cells in myocardial infarction. Stem Cell Res Ther. 2017;8(1):242.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Lin H, Sohn J, Shen H, Langhans MT, Tuan RS. Bone marrow mesenchymal stem cells: aging and tissue engineering applications to enhance bone healing. Biomaterials. 2019;203:96–110.

    Article  CAS  PubMed  Google Scholar 

  5. Khalili M, Zarebkohan A, Dianat-Moghadam H, Panahi M, Andre H, Alizadeh E. Corneal endothelial cell sheet bioengineering from neural crest cell-derived adipose stem cells on novel thermo-responsive elastin-mimetic dendrimers decorated with RGD. Chem Eng J. 2022;429:132523.

    Article  CAS  Google Scholar 

  6. Arzaghi H, Rahimi B, Adel B, Rahimi G, Taherian Z, Sanati AL, Dezfuli AS. Nanomaterials modulating stem cells behavior towards cardiovascular cell linage. Materials Adv. 2021;2(7):2231–62.

    Article  CAS  Google Scholar 

  7. Aruoma OI. Free radicals, oxidative stress, and antioxidants in human health and disease. J Am Oil Chem Soc. 1998;75(2):199–212.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Gu Y, Li T, Ding Y, Sun L, Tu T, Zhu W, Hu J, Sun X. Changes in mesenchymal stem cells following long-term culture in vitro. Mol Med Rep. 2016;13(6):5207–15.

    Article  CAS  PubMed  Google Scholar 

  9. Chen F, Liu Y, Wong N-K, Xiao J, So K-F. Oxidative stress in stem cell aging. Cell Transplant. 2017;26(9):1483–95.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Denu RA, Hematti P: Effects of oxidative stress on mesenchymal stem cell biology. Oxidative medicine and cellular longevity 2016;2016:1–9.

  11. Jeong S-G, Cho G-W. Endogenous ROS levels are increased in replicative senescence in human bone marrow mesenchymal stromal cells. Biochem Biophys Res Commun. 2015;460(4):971–6.

    Article  CAS  PubMed  Google Scholar 

  12. Wei H, Li Z, Hu S, Chen X, Cong X. Apoptosis of mesenchymal stem cells induced by hydrogen peroxide concerns both endoplasmic reticulum stress and mitochondrial death pathway through regulation of caspases, p38 and JNK. J Cell Biochem. 2010;111(4):967–78.

    Article  CAS  PubMed  Google Scholar 

  13. Shin TH, Lee S, Choi KR, Kim Y, Paik MJ, Seo C, Kang S, Jin MS, Yoo TH, Kang SH. Quality and freshness of human bone marrow-derived mesenchymal stem cells decrease over time after trypsinization and storage in phosphate-buffered saline. Sci Rep. 2017;7(1):1–8.

    Google Scholar 

  14. Rahimi B, Panahi M, Saraygord-Afshari N, Taheri N, Bilici M, Jafari D, Alizadeh E. The secretome of mesenchymal stem cells and oxidative stress: challenges and opportunities in cell-free regenerative medicine. Mol Biol Rep. 2021;48(7):5607–19.

    Article  CAS  PubMed  Google Scholar 

  15. Panahi M, Rahimi B, Rahimi G, Yew Low T, Saraygord-Afshari N, Alizadeh E. Cytoprotective effects of antioxidant supplementation on mesenchymal stem cell therapy. J Cell Physiol. 2020;235(10):6462–95.

    Article  CAS  PubMed  Google Scholar 

  16. Mehta SK, Gowder SJT. Members of antioxidant machinery and their functions. Basic Principles Clin Significance Oxidative Stress. 2015;11:59–85.

    Google Scholar 

  17. Somogyi A, Rosta K, Pusztai P, Tulassay Z, Nagy G. Antioxidant measurements. Physiol Meas. 2007;28(4):R41.

    Article  PubMed  Google Scholar 

  18. Mirończuk-Chodakowska I, Witkowska AM, Zujko ME. Endogenous non-enzymatic antioxidants in the human body. Adv Med Sci. 2018;63(1):68–78.

    Article  PubMed  Google Scholar 

  19. Zoidis E, Seremelis I, Kontopoulos N, Danezis G. Selenium-dependent antioxidant enzymes: actions and properties of selenoproteins. Antioxidants. 2018;7(5):66.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Refaay DA, Ahmed DM, Mowafy AM, Shaaban S: Evaluation of novel multifunctional organoselenium compounds as potential cholinesterase inhibitors against Alzheimer’s disease. Med Chem Res. 2022;31:894–904.

  21. Tapiero H, Townsend D, Tew K. The antioxidant role of selenium and seleno-compounds. Biomed Pharmacother. 2003;57(3–4):134–44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Harthi AA: Audit on medicine information centre queries and adverse drug reaction reporting. Journal of Pharmacovigilance 2017;05(04).

  23. Hawkes WC, Alkan Z. Regulation of redox signaling by selenoproteins. Biol Trace Elem Res. 2010;134:235–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Constantinescu-Aruxandei D, Frîncu RM, Capră L, Oancea F. Selenium analysis and speciation in dietary supplements based on next-generation selenium ingredients. Nutrients. 2018;10(10):1466.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Steindler DA. Stem cells, regenerative medicine, and animal models of disease. ILAR J. 2007;48(4):323–38.

    Article  CAS  PubMed  Google Scholar 

  26. Ribitsch I, Baptista PM, Lange-Consiglio A, Melotti L, Patruno M, Jenner F, Schnabl-Feichter E, Dutton LC, Connolly DJ, van Steenbeek FG. Large animal models in regenerative medicine and tissue engineering: to do or not to do. Front Bioengineering Biotechnol. 2020;8:972.

    Article  Google Scholar 

  27. Shi D, Mi G, Wang M, Webster TJ. In vitro and ex vivo systems at the forefront of infection modeling and drug discovery. Biomaterials. 2019;198:228–49.

    Article  CAS  PubMed  Google Scholar 

  28. Castelo-Branco DdSCM, Amando BR, Ocadaque CJ, Aguiar Ld, Paiva DDdQ, Diógenes EM, Guedes GMdM, Costa CL, Santos-Filho ASP, Andrade ARCd. Mini-review: from in vitro to ex vivo studies: an overview of alternative methods for the study of medical biofilms. Biofouling. 2020;36(9):1129–48.

    PubMed  Google Scholar 

  29. Yang X, Zhou J, He J, Liu J, Wang H, Liu Y, Jiang T, Zhang Q, Fu X, Xu Y. An immune system-modified rat model for human stem cell transplantation research. Stem Cell Rep. 2018;11(2):514–21.

    Article  CAS  Google Scholar 

  30. Ala M, Jafari RM, Nematian H, Shadboorestan A, Dehpour AR. Sodium selenite modulates IDO1/Kynurenine, TLR4, NF-κB and Bcl2/Bax pathway and mitigates acetic acid-induced colitis in rat. Cell Physiol Biochem. 2022;56(S1):24–35.

    Article  PubMed  Google Scholar 

  31. Goda K, Muta K, Yasui Y, Oshida S-I, Kitatani K, Takekoshi S. Selenium and glutathione-depleted rats as a sensitive animal model to predict drug-induced liver injury in humans. Int J Mol Sci. 2019;20(13):3141.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hogan C, Perkins AV. Selenoproteins in the human placenta: how essential is selenium to a healthy start to life? Nutrients. 2022;14(3):628.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Bitiren M, Karakilcik AZ, Zerin M, Ozardali I, Selek S, Nazligül Y, Ozgonul A, Musa D, Uzunkoy A. Protective effects of selenium and vitamin E combination on experimental colitis in blood plasma and colon of rats. Biol Trace Elem Res. 2010;136(1):87–95.

    Article  CAS  PubMed  Google Scholar 

  34. Pasha A, Calvani M, Favre C. β3-Adrenoreceptors as ROS Balancer in Hematopoietic Stem Cell Transplantation. Int J Mol Sci. 2021;22(6):2835.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Shimada BK, Alfulaij N, Seale LA. The impact of selenium deficiency on cardiovascular function. Int J Mol Sci. 2021;22(19):10713.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Ueno H, Shimizu R, Okuno T, Ogino H, Arakawa T, Murano K, Nakamuro K. Effect of seleno-L-methionine on oxidative stress in the pancreatic islets of a short-term induced diabetic mouse model in insufficient selenium status. Biol Pharm Bull. 2018;41(1):80–5.

    Article  CAS  PubMed  Google Scholar 

  37. Schwarzlin R, Pušenjak N, Makuc D, Križman M, Vovk I, Plavec J, Švajger U. Synergistic complex from plants Solanaceae exhibits cytotoxicity for the human hepatocellular carcinoma cell line HepG2. BMC Complement Altern Med. 2016;16(1):395.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Zhang Y, Cui J, Lu Y, Huang C, Liu H, Xu S. Selenium deficiency induces inflammation via the iNOS/NF-κB pathway in the brain of pigs. Biol Trace Elem Res. 2020;196(1):103–9.

    Article  CAS  PubMed  Google Scholar 

  39. Vural P, Kabaca G, Firat RD, Degirmencioglu S. Administration of selenium decreases lipid peroxidation and increases vascular endothelial growth factor in streptozotocin induced diabetes mellitus. Cell J. 2017;19(3):452–60.

    PubMed  PubMed Central  Google Scholar 

  40. Dominiak A, Wilkaniec A, Wroczyński P, Adamczyk A. Selenium in the therapy of neurological diseases. Where is it going? Curr Neuropharmacol. 2016;14(3):282–99.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lipinski B. Sodium selenite as an anticancer agent. Anticancer Agents Med Chem. 2017;17(5):658–61.

    Article  CAS  PubMed  Google Scholar 

  42. Xie D, Jiang L, Lin Y, Liu Z. Antioxidant activity of selenium-enriched Chrysomyia megacephala (Fabricius) larvae powder and its impact on intestinal microflora in D-galactose induced aging mice. BMC Complementary Medicine and Therapies. 2020;20(1):264.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Nafiu AB, Rahman MT. Selenium added unripe carica papaya pulp extracts enhance wound repair through TGF-β1 and VEGF-a signalling pathway. BMC Complement Altern Med. 2015;15(1):369.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Atef MM, Abd-Ellatif RN, Emam MN, Abo El gheit RE, Amer AI, Hafez YM. Therapeutic potential of sodium selenite in letrozole induced polycystic ovary syndrome rat model: targeting mitochondrial approach (selenium in PCOS). Arch Biochem Biophys. 2019;671:245–54.

    Article  CAS  PubMed  Google Scholar 

  45. El-Boshy ME, Risha EF, Abdelhamid FM, Mubarak MS, Hadda TB. Protective effects of selenium against cadmium induced hematological disturbances, immunosuppressive, oxidative stress and hepatorenal damage in rats. J Trace Elem Med Biol. 2015;29:104–10.

    Article  CAS  PubMed  Google Scholar 

  46. Zadeh Modarres S, Heidar Z, Foroozanfard F, Rahmati Z, Aghadavod E, Asemi Z. The effects of selenium supplementation on gene expression related to insulin and lipid in infertile polycystic ovary syndrome women candidate for in vitro fertilization: a randomized, double-blind Placebo-Controlled Trial. Biol Trace Element Res. 2018;183(2):218–25.

    Article  CAS  Google Scholar 

  47. Yan D, Tang B, Yan L, Zhang L, Miao M, Chen X, Sui G, Zhang Q, Liu D, Wang H. Sodium selenite improves the therapeutic effect of BMSCs via promoting the proliferation and differentiation, thereby promoting the hematopoietic factors. Onco Targets Ther. 2019;12:9685.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ebert R, Ulmer M, Zeck S, Meissner-Weigl J, Schneider D, Stopper H, Schupp N, Kassem M, Jakob F. Selenium supplementation restores the antioxidative capacity and prevents cell damage in bone marrow stromal cells in vitro. Stem cells. 2006;24(5):1226–35.

    Article  CAS  PubMed  Google Scholar 

  49. Che K, Liu X, Chi J, Li P, Gao J, Fu Z, Yan S, Xing X, Hu J. The effects of adipose-derived mesenchymal stem cells combined with sodium selenite on Hashimoto’s thyroiditis. Am J Transl Res. 2020;12(10):6422.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Hosseinzadeh Anvar L, Hosseini-Asl S, Mohammadzadeh-Vardin M, Sagha M. The telomerase activity of selenium-induced human umbilical cord mesenchymal stem cells is associated with different levels of c-Myc and p53 expression. DNA Cell Biol. 2017;36(1):34–41.

    Article  CAS  PubMed  Google Scholar 

  51. Yang Y-HK, Ogando CR, Wang See C, Chang T-Y, Barabino GA. Changes in phenotype and differentiation potential of human mesenchymal stem cells aging in vitro. Stem Cell Res Therapy. 2018;9:1–14.

    Article  Google Scholar 

  52. Zhang L, Chan C. Isolation and enrichment of rat mesenchymal stem cells (MSCs) and separation of single-colony derived MSCs. J Vis Exp. 2010;37:1852.

    Google Scholar 

  53. Lennon DP, Haynesworth SE, Young RG, Dennis JE, Caplan AI. A chemically defined medium supports in vitro proliferation and maintains the osteochondral potential of rat marrow-derived mesenchymal stem cells. Exp Cell Res. 1995;219(1):211–22.

    Article  CAS  PubMed  Google Scholar 

  54. Aval SF, Zarghami N, Alizadeh E, Mohammadi SA. The effect of ketorolac and triamcinolone acetonide on adipogenic and hepatogenic differentiation through miRNAs 16/15/195: possible clinical application in regenerative medicine. Biomed Pharmacother. 2018;97:675–83.

    Article  CAS  PubMed  Google Scholar 

  55. Lee SC, Lee NH, Patel KD, Jang TS, Knowles JC, Kim HW, Lee HH, Lee JH. The effect of selenium nanoparticles on the osteogenic differentiation of MC3T3-E1 cells. Nanomaterials (Basel). 2021;11(2):557.

    Article  PubMed  PubMed Central  Google Scholar 

  56. Yan J, Tian J, Zheng Y, Han Y, Lu S. Selenium promotes proliferation of chondrogenic cell ATDC5 by increment of intracellular ATP content under serum deprivation. Cell Biochem Funct. 2012;30(8):657–63.

    Article  CAS  PubMed  Google Scholar 

  57. Lotfy A, Salama M, Zahran F, Jones E, Badawy A, Sobh M. Characterization of mesenchymal stem cells derived from rat bone marrow and adipose tissue: a comparative study. Int J Stem Cells. 2014;7(2):135–42.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Asadian S, Alibabrdel M, Daei N, Cheraghi H, Maedeh Jafari S, Noshadirad E, Jabarpour M, Siavashi V, Nassiri SM. Improved angiogenic activity of endothelial progenitor cell in diabetic patients treated with insulin plus metformin. J Cell Biochem. 2019;120(5):7115–24.

    Article  CAS  PubMed  Google Scholar 

  59. Sarikhani M, Deylam M, Alizadeh E, Hejazy M, Alizadeh-Salteh S, Moeini H, Firouzamandi M. Anti-aging effects of peppermint (Mentha piperita L.) and Shirazi thyme (Zataria multiflora Boiss.) plant extracts. Food Bioscience. 2021;41:100930.

    Article  CAS  Google Scholar 

  60. Jiang T, Xu G, Wang Q, Yang L, Zheng L, Zhao J, Zhang X. In vitro expansion impaired the stemness of early passage mesenchymal stem cells for treatment of cartilage defects. Cell Death Dis. 2017;8(6):e2851–e2851.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Ridzuan N, Al Abbar A, Yip WK, Maqbool M, Ramasamy R. Characterization and expression of senescence marker in prolonged passages of rat bone marrow-derived mesenchymal stem cells. Stem Cells Int. 2016;2016:8487264.

    Article  PubMed  PubMed Central  Google Scholar 

  62. Fatima S, Alfrayh R, Alrashed M, Alsobaie S, Ahmad R, Mahmood A. Selenium nanoparticles by moderating oxidative stress promote differentiation of mesenchymal stem cells to osteoblasts. Int J Nanomed. 2021;16:331–43.

    Article  Google Scholar 

  63. Liu H, Bian W, Liu S, Huang K. Selenium protects bone marrow stromal cells against hydrogen peroxide-induced inhibition of osteoblastic differentiation by suppressing oxidative stress and ERK signaling pathway. Biol Trace Elem Res. 2012;150(1):441–50.

    Article  PubMed  Google Scholar 

  64. Pang K-L, Chin K-Y. Emerging anticancer potentials of selenium on osteosarcoma. Int J Mol Sci. 2019;20(21):5318.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Shi L, Duan Y, Yao X, Song R, Ren Y. Effects of selenium on the proliferation and apoptosis of sheep spermatogonial stem cells in vitro. Anim Reprod Sci. 2020;215: 106330.

    Article  CAS  PubMed  Google Scholar 

  66. Zheng C, Wang J, Liu Y, Yu Q, Liu Y, Deng N, Liu J. Functional selenium nanoparticles enhanced stem cell osteoblastic differentiation through BMP signaling pathways. Adv Func Mater. 2014;24(43):6872–83.

    Article  CAS  Google Scholar 

  67. Park S-H, Kim J-H, Nam S-W, Kim B-W, Kim G-Y, Kim W-J, Choi YH. Selenium improves stem cell potency by stimulating the proliferation and active migration of 3T3-L1 preadipocytes. Int J Oncol. 2014;44(1):336–42.

    Article  CAS  PubMed  Google Scholar 

  68. Yeo JE, Kang SK. Selenium effectively inhibits ROS-mediated apoptotic neural precursor cell death in vitro and in vivo in traumatic brain injury. Biochim Biophys Acta Mol Basis Dis. 2007;1772(11):1199–210.

    Article  CAS  Google Scholar 

  69. Murdolo G, Bartolini D, Tortoioli C, Piroddi M, Torquato P, Galli F: Chapter Nine - Selenium and Cancer Stem Cells. In: Advances in Cancer Research. Volume 136, edn. Edited by Tew KD, Galli F: Academic Press; 2017: 235–257.

  70. Kim JH, Lee MR, Kim JH, Jee MK, Kang SK. IFATS collection: selenium induces improvement of stem cell behaviors in human adipose-tissue stromal cells via SAPK/JNK and stemness acting signals. Stem cells (Dayton, Ohio). 2008;26(10):2724–34.

    Article  CAS  PubMed  Google Scholar 

  71. Ren X, Wang S, Zhang C, Hu X, Zhou L, Li Y, Xu L. Selenium ameliorates cadmium-induced mouse leydig TM3 cell apoptosis via inhibiting the ROS/JNK /c-jun signaling pathway. Ecotoxicol Environ Saf. 2020;192: 110266.

    Article  CAS  PubMed  Google Scholar 

  72. Bao R-k, Zheng S-f, Wang X-y. Selenium protects against cadmium-induced kidney apoptosis in chickens by activating the PI3K/AKT/Bcl-2 signaling pathway. Environ Sci Pollut Res. 2017;24(25):20342–53.

    Article  CAS  Google Scholar 

  73. Dai X, Song R, Xiong Y. The expression of ERK and JNK in patients with an endemic osteochondropathy Kashin-Beck disease. Exp Cell Res. 2017;359(2):337–41.

    Article  CAS  PubMed  Google Scholar 

  74. Kim SH, Johnson VJ, Shin T-Y, Sharma RP. Selenium attenuates lipopolysaccharide-induced oxidative stress responses through modulation of p38 MAPK and NF-κB signaling pathways. Exp Biol Med. 2004;229(2):203–13.

    Article  CAS  Google Scholar 

  75. Son Y, Cheong Y-K, Kim N-H, Chung H-T, Kang DG, Pae H-O. Mitogen-activated protein kinases and reactive oxygen species: how can ROS activate MAPK pathways? J Signal Transduction. 2011;2011:792639.

    Article  Google Scholar 

  76. Liu J, Ma L, Chen X, Wang J, Yu T, Gong Y, Ma A, Zheng L, Liang H. ERK inhibition sensitizes cancer cells to oleanolic acid-induced apoptosis through ERK/Nrf2/ROS pathway. Tumor Biol. 2016;37(6):8181–7.

    Article  CAS  Google Scholar 

  77. Shi Y, Nikulenkov F, Zawacka-Pankau J, Li H, Gabdoulline R, Xu J, Eriksson S, Hedström E, Issaeva N, Kel A, et al. ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis. Cell Death Differ. 2014;21(4):612–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Rahimi G, Heydari S, Rahimi B, Abedpoor N, Niktab I, Safaeinejad Z, Peymani M, Seyed Forootan F, Derakhshan Z, Esfahani MHN, et al. A combination of herbal compound (SPTC) along with exercise or metformin more efficiently alleviated diabetic complications through down-regulation of stress oxidative pathway upon activating Nrf2-Keap1 axis in AGE rich diet-induced type 2 diabetic mice. Nutr Metab. 2021;18(1):14.

    Article  CAS  Google Scholar 

  79. Zhang J, Wang X, Vikash V, Ye Q, Wu D, Liu Y, Dong W. ROS and ROS-Mediated Cellular Signaling. Oxid Med Cell Longev. 2016;2016:4350965.

    Article  PubMed  PubMed Central  Google Scholar 

  80. Nomura M, Shimizu S, Sugiyama T, Narita M, Ito T, Matsuda H, Tsujimoto Y. 14-3-3 Interacts directly with and negatively regulates pro-apoptotic Bax. J Biol Chem. 2003;278(3):2058–65.

    Article  CAS  PubMed  Google Scholar 

  81. Rezatabar S, Karimian A, Rameshknia V, Parsian H, Majidinia M, Kopi TA, Bishayee A, Sadeghinia A, Yousefi M, Monirialamdari M, et al. RAS/MAPK signaling functions in oxidative stress, DNA damage response and cancer progression. J Cell Physiol. 2019;234(9):14951–65.

    Article  CAS  PubMed  Google Scholar 

  82. Russo V, Young S, Hamilton A, Amsden BG, Flynn LE. Mesenchymal stem cell delivery strategies to promote cardiac regeneration following ischemic injury. Biomaterials. 2014;35(13):3956–74.

    Article  CAS  PubMed  Google Scholar 

  83. Kan I, Melamed E, Offen D. Integral therapeutic potential of bone marrow mesenchymal stem cells. Curr Drug Targets. 2005;6(1):31–41.

    Article  CAS  PubMed  Google Scholar 

  84. Hassanlou L, Meshgini S, Alizadeh E. Evaluating adipocyte differentiation of bone marrow-derived mesenchymal stem cells by a deep learning method for automatic lipid droplet counting. Comput Biol Med. 2019;112:103365.

    Article  CAS  PubMed  Google Scholar 

  85. Karamouzian S, Nematollahi-Mahani SN, Nakhaee N, Eskandary H. Clinical safety and primary efficacy of bone marrow mesenchymal cell transplantation in subacute spinal cord injured patients. Clin Neurol Neurosurg. 2012;114(7):935–9.

    Article  PubMed  Google Scholar 

  86. Hu M, Guo G, Huang Q, Cheng C, Xu R, Li A, Liu N, Liu S. The harsh microenvironment in infarcted heart accelerates transplanted bone marrow mesenchymal stem cells injury: the role of injured cardiomyocytes-derived exosomes. Cell Death Dis. 2018;9(3):1–14.

    Article  Google Scholar 

  87. Amiri F, Jahanian-Najafabadi A, Roudkenar MH. In vitro augmentation of mesenchymal stem cells viability in stressful microenvironments. Cell Stress Chaperones. 2015;20(2):237–51.

    Article  CAS  PubMed  Google Scholar 

  88. Cai B, Li X, Wang Y, Liu Y, Yang F, Chen H, Yin K, Tan X, Zhu J, Pan Z. Apoptosis of bone marrow mesenchymal stem cells caused by homocysteine via activating JNK signal. PLoS One. 2013;8(5):e63561.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Sarikhani M, Vaghefi Moghaddam S, Firouzamandi M, Hejazy M, Rahimi B, Moeini H, Alizadeh E. Harnessing rat derived model cells to assess the toxicity of TiO2 nanoparticles. J Mater Sci Mater Med. 2022;33(5):1–14.

    Article  Google Scholar 

  90. Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation. 2002;105(1):93–8.

    Article  PubMed  Google Scholar 

  91. Song C, Song C, Tong F. Autophagy induction is a survival response against oxidative stress in bone marrow–derived mesenchymal stromal cells. Cytotherapy. 2014;16(10):1361–70.

    Article  CAS  PubMed  Google Scholar 

  92. Lee KA, Shim W, Paik MJ, Lee SC, Shin JY, Ahn YH, Park K, Kim JH, Choi S, Lee G. Analysis of changes in the viability and gene expression profiles of human mesenchymal stromal cells over time. Taylor & Francis; 2009.

    Google Scholar 

  93. Sahlender B, Windolf J, Suschek CV. Superoxide dismutase and catalase significantly improve the osteogenic differentiation potential of osteogenetically compromised human adipose tissue-derived stromal cells in vitro. Stem Cell Res. 2022;60:102708.

    Article  CAS  PubMed  Google Scholar 

  94. Román F, Urra C, Porras O, Pino AM, Rosen CJ, Rodríguez JP. Real-time H2O2 measurements in bone marrow mesenchymal stem cells (MSCs) show increased antioxidant capacity in cells from osteoporotic women. J Cell Biochem. 2017;118(3):585–93.

    Article  PubMed  Google Scholar 

  95. Park J, Lee JH, Yoon BS, Jun EK, Lee G, Kim IY, You S. Additive effect of bFGF and selenium on expansion and paracrine action of human amniotic fluid-derived mesenchymal stem cells. Stem Cell Res Ther. 2018;9(1):1–17.

    Article  Google Scholar 

  96. Ahmed HH, Aglan HA, Mabrouk M, Abd-Rabou AA, Beherei HH. Enhanced mesenchymal stem cell proliferation through complexation of selenium/titanium nanocomposites. J Mater Sci Mater Med. 2019;30(2):1–14.

    Article  Google Scholar 

  97. Yoon D, Choi Y, Lee J. Cellular localization of NRF2 determines the self-renewal and osteogenic differentiation potential of human MSCs via the P53–SIRT1 axis. Cell Death Dis. 2016;7(2):e2093–e2093.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Zhang ZN, Chung SK, Xu Z, Xu Y. Oct4 maintains the pluripotency of human embryonic stem cells by inactivating p53 through Sirt1-mediated deacetylation. Stem Cells. 2014;32(1):157–65.

    Article  PubMed  Google Scholar 

  99. Han M-K, Song E-K, Guo Y, Ou X, Mantel C, Broxmeyer HE. SIRT1 regulates apoptosis and Nanog expression in mouse embryonic stem cells by controlling p53 subcellular localization. Cell Stem Cell. 2008;2(3):241–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Lin T, Chao C, Saito Si, Mazur SJ, Murphy ME, Appella E, Xu Y. p53 induces differentiation of mouse embryonic stem cells by suppressing Nanog expression. Nat Cell Biol. 2005;7(2):165–71.

    Article  CAS  PubMed  Google Scholar 

  101. Nemoto S, Fergusson MM, Finkel T. Nutrient availability regulates SIRT1 through a forkhead-dependent pathway. Science. 2004;306(5704):2105–8.

    Article  CAS  PubMed  Google Scholar 

  102. Yuan H-F, Zhai C, Yan X-L, Zhao D-D, Wang J-X, Zeng Q, Chen L, Nan X, He L-J, Li S-T. SIRT1 is required for long-term growth of human mesenchymal stem cells. J Mol Med. 2012;90(4):389–400.

    Article  CAS  PubMed  Google Scholar 

  103. Wang J, Liu C, Zhao Y, Wang J, Li J, Zheng M. Selenium regulates Nrf2 signaling to prevent hepatotoxicity induced by hexavalent chromium in broilers. Poult Sci. 2023;102(2):102335.

    Article  CAS  PubMed  Google Scholar 

  104. Duscher D, Rennert RC, Januszyk M, Anghel E, Maan ZN, Whittam AJ, Perez MG, Kosaraju R, Hu MS, Walmsley GG. Aging disrupts cell subpopulation dynamics and diminishes the function of mesenchymal stem cells. Sci Rep. 2014;4(1):1–9.

    Article  Google Scholar 

  105. Dam PT, Hoang VT, Bui HTH, Hang LM, Hoang DM, Nguyen HP, Lien HT, Tran HTT, Nguyen X-H, Nguyen Thanh L. Human adipose-derived mesenchymal stromal cells exhibit high HLA-DR levels and altered cellular characteristics under a xeno-free and serum-free condition. Stem Cell Rev Rep. 2021;17:2291–303.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Halfon S, Abramov N, Grinblat B, Ginis I. Markers distinguishing mesenchymal stem cells from fibroblasts are downregulated with passaging. Stem Cells Dev. 2011;20(1):53–66.

    Article  CAS  PubMed  Google Scholar 

  107. Boquest AC, Shahdadfar A, Frønsdal K, Sigurjonsson O, Tunheim SH, Collas P, Brinchmann JE. Isolation and transcription profiling of purified uncultured human stromal stem cells: alteration of gene expression after in vitro cell culture. Mol Biol Cell. 2005;16(3):1131–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Pham LH, Vu NB, Van Pham P. The subpopulation of CD105 negative mesenchymal stem cells show strong immunomodulation capacity compared to CD105 positive mesenchymal stem cells. Biomed Res Ther. 2019;6(4):3131–40.

    Article  Google Scholar 

  109. Aslan H, Zilberman Y, Kandel L, Liebergall M, Oskouian RJ, Gazit D, Gazit Z. Osteogenic differentiation of noncultured immunoisolated bone marrow-derived CD105+ cells. Stem Cells. 2006;24(7):1728–37.

    Article  PubMed  Google Scholar 

  110. Zhu H, Mitsuhashi N, Klein A, Barsky LW, Weinberg K, Barr ML, Demetriou A, Wu GD. The role of the hyaluronan receptor CD44 in mesenchymal stem cell migration in the extracellular matrix. Stem Cells. 2006;24(4):928–35.

    Article  CAS  PubMed  Google Scholar 

  111. Wang X, Wu H, Long Z, Sun Q, Liu J, Liu Y, Hai C. Differential effect of Se on insulin resistance: Regulation of adipogenesis and lipolysis. Mol Cell Biochem. 2016;415(1):89–102.

    Article  CAS  PubMed  Google Scholar 

  112. Khoshdel A, Fahmidehkar MA, Hajizadeh MR, Mahmoodi M, Mirzaei MR, Akyash F, Aflatoonian B. Vitamin E and selenium facilitate the osteogenesis and adipogenesis of the human adipose tissue-derived mesenchymal stem/stromal cells. Int J Med Lab. 2020;7(3):159–67.

    Google Scholar 

  113. Dai X, Yan X, Wintergerst KA, Cai L, Keller BB, Tan Y. Nrf2: redox and metabolic regulator of stem cell state and function. Trends Mol Med. 2020;26(2):185–200.

    Article  CAS  PubMed  Google Scholar 

  114. Liu W, Zhang L, Shi J, Liu Y, Zhou L, Hou K, Qu X, Teng Y. Clinical significance of pAkt and pErk1/2 expression in early-stage breast cancer patients treated with anthracycline-based adjuvant chemotherapy. Oncol Lett. 2015;9(4):1707–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Kim D, Kim H, Kim K, Roh S. The protective effect of indole-3-acetic acid (IAA) on H2O2-damaged human dental pulp stem cells is mediated by the AKT pathway and involves increased expression of the transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) and its downstream target heme oxygenase 1 (HO-1). Oxidative medicine and cellular longevity. 2017;2017:8639485.

    Article  PubMed  PubMed Central  Google Scholar 

  116. Martin D, Rojo AI, Salinas M, Diaz R, Gallardo G, Alam J, De Galarreta CMR, Cuadrado A. Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3-kinase/Akt pathway and the Nrf2 transcription factor in response to the antioxidant phytochemical carnosol. J Biol Chem. 2004;279(10):8919–29.

    Article  CAS  PubMed  Google Scholar 

  117. Chen T, Zhu H, Wang Y, Zhao P, Chen J, Sun J, Zhang X, Zhu G. Apoptosis of bone marrow mesenchymal stromal/stem cells via the MAPK and endoplasmic reticulum stress signaling pathways. Am J Transl Res. 2018;10(8):2555.

    CAS  PubMed  PubMed Central  Google Scholar 

  118. Kumar S, Vaidya MJM, biochemistry c: Hypoxia inhibits mesenchymal stem cell proliferation through HIF1α-dependent regulation of P27. Mol Cell Biochem. 2016;415(12):29–38.

  119. Lee HJ, Jung YH, Choi GE, Kim JS, Chae CW, Han HJJIjosc: Role of HIF1α regulatory factors in stem cells. Int J Stem Cells. 2019;12(1):8.

  120. Barzegari A, Omidi Y, Gueguen V, Meddahi‐Pellé A, Letourneur D, Pavon‐Djavid GJB: Nesting and fate of transplanted stem cells in hypoxic/ischemic injured tissues: The role of HIF1α/sirtuins and downstream molecular interactions. 2020.

  121. Li H-S, Zhou Y-N, Li L, Li S-F, Long D, Chen X-L, Zhang J-B, Feng L, Li Y-P. HIF-1α protects against oxidative stress by directly targeting mitochondria. Redox Biol. 2019;25:101109.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Wei H, Handa JT. Nrf2 and Hif1a have opposite responses to oxidative stress in ARPE-19 cells. Invest Ophthalmol Vis Sci. 2016;57(12):6049–6049.

    Google Scholar 

Download references


The authors would like to acknowledge the Deputy for Research and Technology, Tabriz University of Medical Sciences, for grant support (63680) with the ethics code: IR.TBZMED.REC.1399.


This work was funded by Deputy for Research and Technology, Tabriz University of Medical Sciences, for grant number: 63680.

Author information

Authors and Affiliations



Bahareh Rahimi and Mohammad Panahi:Conceptualization, Validation, Methodology, Formal analysis, Writing – original draft. HajieLotfi, Mostafa Khalili, and AstirehSalehi:Writing – review & editing. NedaSaraygord-Afshari, and EffatAlizadeh: Conceptualization, Writing- Reviewing and editingthe final draft. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Neda Saraygord-Afshari or Effat Alizadeh.

Ethics declarations

Ethics approval and consent to participate

All animal and cellular work was approved by Ethics committee located in Tabriz University of Medical Sciences, with an ethics code: IR.TBZMED.REC.1399.036, and performed in accordance with relevant guidelines/regulations. All experimental methods were reported in conformity with ARRIVE guidelines ( regarding animal tests.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: S1. 

The SDS-PAG gel before transfeering to membrane during western blot protocol.

Additional file 2: S2. 

The bands for western blot analysis after visualization by chemiluminscence.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rahimi, B., Panahi, M., Lotfi, H. et al. Sodium selenite preserves rBM-MSCs’ stemness, differentiation potential, and immunophenotype and protects them against oxidative stress via activation of the Nrf2 signaling pathway. BMC Complement Med Ther 23, 131 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


  • Mesenchymal stem cells
  • Reactive oxygen species (ROS)
  • Selenium selenite
  • Stemness
  • Nrf pathway