Skip to main content
NCBI home page
Scientific Reports logoLink to Scientific Reports
. 2026 Jan 26;16:6219. doi: 10.1038/s41598-026-37517-8

Validation of a novel genomic biomarker of mesenchymal stem cell scalability and implications of genotype status on cellular senescence phenotypes

I Kade Karisma Gita Ardana 1, Vitali V Maldonado 2, C Lowry Barnes 3, Rebekah M Samsonraj 1,2,3,4,✉
PMCID: PMC12905334  PMID: 41588136

Abstract

Ex vivo expansion impairs the regenerative potential of bone marrow-derived mesenchymal stem cells (BM-MSCs), primarily by inducing cellular senescence. Interestingly, populations of BM-MSCs that exhibit resistance to senescence even after prolonged expansion have been reported. However, a reliable strategy to identify these populations is still underway. Previously, the GSTT1 gene has been identified as a biomarker for BM-MSC scalability, but its effects on BM-MSC senescence have not yet been studied. Here, we investigate the role of GSTT1 genotype in BM-MSC senescence and proliferation. First, we categorized six BM-MSC groups into GSTT1 positive and GSTT1 negative groups based on their genotype. Then, we performed long-term in vitro culture and exposed cells to irradiation as senescence models. After that, their proliferative potential, SASP, and the expression of key genes were investigated. The results show that GSTT1 null BM-MSCs have a higher proliferative potential at low passages and exhibit fewer senescent cells in culture when compared to the other genotypes. Additionally, these cells exhibit a lower expression of p21 Waf1, p14ARF and IL-6, and a higher expression of ACTA2 throughout culture. A GSTT1 null genotype can serve as a potential biomarker to identify BM-MSC populations with higher resistance to senescence.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-37517-8.

Keywords: Mesenchymal stem cells, Senescence, Biomarker, GSTT1 polymorphism, Cell expansion, Cell therapies

Subject terms: Cell biology, Genetics, Molecular biology, Stem cells

Introduction

Although bone marrow-derived mesenchymal stem cells (BM-MSCs) have great potential for treating multiple diseases1–3, the lack of consistent and/or effective treatment outcomes poses a significant challenge for clinical translation4–7. Multiple factors play a role in hindering the therapeutic potential of BM-MSCs including the process of ex vivo expansion, which, although necessary for obtaining clinically relevant number of cells8, pushes cells towards senescence - a state or irreversible growth arrest - altering their phenotype and lowering their therapeutic potential9–12. Furthermore, it has been observed that donor-to-donor heterogeneity plays a critical role in dictating the potency and the onset of senescence in BM-MSCs13–15, suggesting the existence of intrinsic inter donor differences that provide resistance to senescence. However, to date, these donor-to-donor differences are poorly understood, limiting the possibility of utilizing BM-MSC screening methods to improve BM-MSC therapies.

The GSTT1 gene, a member of the glutathione S-transferase superfamily which is not present in about 14–30% of the population’s genome16, has been studied in the context of disease development and progression. For example, individuals with null GSTT1 gene seem to be at higher risk of developing cancer, Parkinson’s disease, and have a higher mortality rate due to COVID-1917–20. This increased vulnerability may be associated with the primary function of the enzyme coded by this gene: the detoxification of various endogenous and exogenous substances including oxidative stress products, major players in cellular damage and senescence21,22. Moreover, Sathiyanathan et al. reported that BM-MSCs from individuals with GSTT1 null genotypes grow faster and possess longer telomeres than those from individuals with the homozygous positive genotypes23 incentivizing more research on these somewhat contradicting but important results.

This study aims to investigate the effect of GSTT1 polymorphism on BM-MSC growth and senescence. First, the genomic status of six BM-MSC groups was determined. Then, cell senescence was induced through both in vitro irradiation and continuous cell passaging. Finally, BM-MSC proliferative capacity, telomere length, senescence-associated secretory phenotype (SASP), and gene expression profiles were assessed at different time points to determine whether GSTT1 genotype status has any relationship with proliferative capacity and the onset of cell senescence.

Results

Genotyping allowed BM-MSC grouping into two categories determined by GSTT1 status

BM-MSCs from multiple donors were genotyped to determine GSTT1 status using a multiplex PCR wherein the first primer pair amplified the GSTT1 gene which is approximately 0.97 kb long, whereas the second primer amplified the region lacking the GSTT1 gene, with an approximate size of 3.1 kb. A visual representation of how the forward and reverse primers used were able to detect the presence or absence of the GSTT1 gene is shown in Fig. 1.

Fig. 1.

Fig. 1

Open in a new tab

Location of the primers used for genotyping through multiplexing PCR. When present, the GSTT1 gene will be amplified using the GSTT1 F and GSTT1 R primers generating a 3.1 kb product. The GSTT1 deletion (0.97 kb) will only be amplified when the GSTT1 gene is missing since the product of Reverse Del and Forward Del is too big to be amplified when present (60 kb).

Genotyping allowed the grouping of BM-MSCs into two categories: GSTT1+ (presence of the GSTT1 gene) and GSTT1− (absence of the GSTT1 gene). BM-MSCs 310277,198, and 238 were identified to be GSTT1+ and BM-MSCs 164, 310264, and 00227 were determined to be GSTT1−. The genotyping results are shown in Fig. 2, Panel (A). Additionally, mRNA quantification showed that GSTT1− BM-MSCs showed no expression of GSTT1 mRNA whereas GSTT1+ groups showed positive expression of this gene as shown in Fig. 2, Panel (B) Nevertheless, it was noted that GSTT1 gene expression was independent of passage number, with some donors expressing higher GSTT1 at low passages and others at higher passages. Additionally, the GSTT1+ group showed varied expressions of the GSTT1 deletion amplicon, which made it challenging to distinguish between GSTT1 heterozygous and homozygous positive BM-MSCs. Therefore, BM-MSCs that expressed the GSTT1 gene were grouped into the GSTT1+ group, regardless of their heterozygous or homozygous positive genomic status.

Fig. 2.

Fig. 2

Open in a new tab

GSTT1 genotype of six bone marrow-derived mesenchymal stem cell (BM-MSC) groups derived from different donors. (A) Gel pictures of the amplified product obtained from multiplex PCR. BM-MSC groups 310277, 238, and 198 show a GSTT1 positive genotype and BM-MSC groups 164, 00227, and 310264 show a GSTT1 negative genotype. (B) Bar graphs depicting the average mRNA expression of the GSTT1 quantification using RT-qPCR. The relative gene expression is normalized to the respective RPLP0 expression.

GSTT1 null genotype is predictive of higher proliferative potential in BM-MSCs

The proliferative capacity of BM-MSCs was analyzed through a growth curve and a BrDU assay. From the 8-day growth curve, it was observed that BM-MSCs with a GSTT1 null genotype had a significantly higher proliferative potential when compared to GSTT1+ groups as shown in Fig. 3, Panel A. On day 8, the GSTT1 null groups BM-MSC 164 and BM-MSC 227 had significantly more cells than the GSTT1+ BM-MSCs as shown in Supplemental Fig. 1. However, when growth was assessed over multiple passages, there seemed to be no significant differences between the proliferative potential of different BM-MSC groups as shown in Supplemental Fig. 2 displaying a cumulative growth assay. Additionally, a BrdU assay was performed with one representative sample for each GSTT1 genotype at low passages (P6) and high passages (P12). Our results indicate that BM-MSCs with a GSTT1− (negative) genotype showed significantly higher proliferation capacity compared to GSTT1+ (positive) BM-MSCs at young passages as shown in Fig. 3, Panel B.

Fig. 3.

Fig. 3

Open in a new tab

Bone marrow-derived mesenchymal stem cell (BM-MSC) proliferative potential. (A) 8-day growth curve of six BM-MSC groups divided into GSTT1 genotypic groups. GSTT1 positive BM-MSCs are shown in gray and GSTT1 negative BM-MSCs are shown in orange. The average number of cells nine independent cell counts is shown in the graph. (B) Bar graphs depicting the average absorbance readings at 370 nm from the bromodeoxyuridine (BrdU) assay. Three readings for each group at low passages (P6) and high passages (P12) were performed and averaged. Data analysis was performed with one-way ANOVA. (C) 7-day growth curve depicting the number of cells each day of GSTT1 positive BM-MSC 238 without siRNA, with the addition of a negative control siRNA, and with the addition of a GSTT1 siRNA. 3 wells with 3 counts each (n = 9 per group each day) are depicted in the graph. (D) Bar graphs showing the average number of cells at day 7 of each BM-MSC treatment group. Significance was verified by one-way ANOVA. (E) GSTT1 expression after BM-MSC siRNA treatment. Significance was verified by one-way ANOVA.

To further investigate the effect of GSTT1 on the proliferation potential of BM-MSCs at low passages, GSTT1 silencing was performed in a GSTT1 positive BM-MSC group as shown in Fig. 3, Panel C, finding that the average number of cells at day 7 is increased after GSTT1 silencing. However, this difference was non-significant when compared to the cells treated with the negative control siRNA as shown in Fig. 3, Panels D-E.

BM-MSC telomere length attrition and hTERT expression are not related to GSTT1 genotype

In order to assess BM-MSC replicative capacity, their telomere length was assessed from passage 3 to passage 15. The results show that all BM-MSC groups experienced telomere shortening as shown in Fig. 4, Panel A. Even though there was fluctuation in the recorded telomere lengths from passage to passage, the overall slope was negative in all BM-MSC groups. This shortening process did not appear to be related to the GSTT1 genotype but to be donor-dependent.

Fig. 4.

Fig. 4

Open in a new tab

Telomere length and telomerase reverse transcriptase (hTERT) expression of bone marrow-derived mesenchymal stem cells (BM-MSCs) isolated from six different donors. (A) Relative telomere length at passages 3–15 from BM-MSCs. The slope of the best fit line (dotted line) is listed at the bottom of each graph. (B) Bar graphs depicting the average hTERT relative expression normalized to RPLP0 measured by RT-qPCR. The white bar on the left of each graph depicts the hTERT expression of induced pluripotent stem cells (iPSCs). The gray bars represent hTERT expression of BM-MSCs from passage 3/5–15.

Additionally, this study analyzed the expression of the hTERT gene. hTERT or human telomere reverse transcriptase is a gene associated with the synthesis of telomere lengthening. In general, normal diploid mature cells do not express the hTERT gene since this gene is usually expressed by pluripotent cells. The results showed that all BM-MSC groups had low hTERT gene expression with no significant differences between each other as shown in Fig. 4, Panel B. Nevertheless, hTERT expression is higher at lower passages for some BM-MSC groups and decreases as BM-MSCs are passaged. hTERT gene expression does not appear to be related to the GSTT1 genotype status.

GSTT1 genotype status affects expression of p21 Waf1 and p14ARF but not the expression of p16INK4A, γH2AX and TP53

To assess the effects of GSTT1 expression in senescence-related genes, RT-qPCR was used to quantify p14ARF, p16INK4A, p21 Waf1, TP53 and γH2AX in GSTT1+ and GSTT1− BM-MSCs as shown in Fig. 5. The results show that GSTT1+ BM-MSCs 310277, 238, and 198 showed a fluctuating but high expression of p14ARF when compared to most of the GSTT1 negative samples. Two of the three GSTT1 negative samples (164 and 00227) had low expression of p14ARF even at high passages. However, GSTT1 negative BM-MSC 310264 showed a fluctuating but relatively high expression of p14ARF. Furthermore, after grouping the GSTT1- and GSTT1+ BM-MSCs, GSTT1+ cells had a significantly higher expression of p14ARF at passages 5, 7, and 11 as shown in Supplemental Fig. 3. Moreover, p16INK4a only showed a significant difference between GSTT1 genotypes at p7, where GSTT1+ cells expressed this gene at higher levels. However, as the passage increases, no significant differences are observed between groups for p16INK4a.

Fig. 5.

Fig. 5

Open in a new tab

Gene expression of senescence-associated genes throughout long-term culture. p14ARF, p16INK4A, p21 Waf1, γH2AX, and TP53 were quantified at each passage in six different bone-marrow derived mesenchymal stem cell (BM-MSCs) groups isolated from different donors. BM-MSCs are grouped by their genotype as GSTT1 positive (GSTT1+) or GSTT1 negative (GSTT1−). Gene expressions are normalized to their respective RPLP0 expressions. Data analysis was performed using a one-way ANOVA, n = 3 for each group.

Our results showed that p21 Waf1 expression increased with passage and showed significant differences between genotypic groups. GSTT1+ BM-MSCs 238, 198, and 310277 showed the greatest p21 Waf1 expression at higher passages and a significantly higher p21 Waf1 expression when compared to GSTT1- BM-MSCs at p7, p9, p11, and p13 as shown in Supplemental Fig. 3. γH2AX expression showed high variation between passages with no specific pattern or clear relation to genotype. TP53 was highly expressed in GSTT1− BM-MSCs 00227 and 310264 throughout culture as well as in GSTT1+ donor 310277 at p9 and p11. Interestingly, GSTT1− BM-MSC 238 had the lowest TP53 expression throughout culture, making it difficult to associate TP53 expression to genotype status.

GSTT1 genotype does not influence TWIST1/2 expression

In addition to the senescence-related genes quantified in this study, genes related to cell proliferation were analyzed, namely TWIST1 and TWIST2 as shown in Fig. 6, Panel A. The results from this study showed that TWIST1 expression varied at each passage with mostly non-significant differences between GSTT1 genotypes as shown in Supplemental Fig. 4. Similarly, TWIST2 showed low expression and no significant differences between genotypes.

Fig. 6.

Fig. 6

Open in a new tab

TWIST1/2 and senescence-associated β galactosidase (SA-β-gal) expression in bone marrow-derived mesenchymal stem cells (BM-MSCs) during and after expansion. (A) TWIST1 and TWIST2 gene expression in BM-MSCs measured through RT-qPCR at multiple passages during long-term in vitro culture. BM-MSCs are grouped by their genotype as GSTT1 positive (GSTT1+) or GSTT1 negative (GSTT1−). (B) SA-β-gal staining from two selected BM-MSC groups, one from each genotype. (C) Bar graphs depicting the average number of SA-β-gal positive cells from each well divided by the total number of cells. Data analysis was performed using a one-way ANOVA, n = 9 for each group.

GSTT1 null BM-MSCs have the lowest percentage of senescent cells after long-term in vitro culture

To quantify the percentage of senescent cells, SA-β-gal staining was performed in cells after long-term in vitro culture (passage 15) in two representative BM-MSC groups (one from each genotype). SA-β-gal expression is known to be part of BM-MSC SASP and it is a reliable strategy to identify senescent cells. The results show that GSTT1 negative BM-MSCs have a significantly lower percentage of senescent cells in culture compared to GSTT1+ BM-MSCs as shown in Fig. 6, Panel B.

GSTT1 genotype has a significant effect on the expression of senescence-associated genes at higher passages

In addition to the genes directly involved in cell cycle arrest and DNA damage repair, other genes known to be part of the senescence-associated secretory phenotype (SASP) or key BM-MSC processes were quantified. Here, the expressions of IL-6, SOD1, EZH2, ACTA2, and PDXN were measured at multiple passages to study the effect of long-term culture and GSTT1 genotype on their expression as shown in Fig. 7, Panel A-B. IL-6 expression increases with increasing passage while EZH2 expression decreases with increasing passage in all GSTT1 genotypes. However, the expression of both IL-6 and EZH2 is significantly higher in GSTT1+ positive BM-MSCs compared to GSTT1 negative BM-MSCs when they reached higher passages. Furthermore, IL-6 seemed to be consistently lower in GSTT1 negative BM-MSCs throughout culture. It is to be noted that, at low passages, the GSTT1+ group had the lowest and highest expression of IL-6 and EZH2 respectively and experienced the greatest shifts in gene expression with long-term culture.

Fig. 7.

Fig. 7

Open in a new tab

Gene expression of IL-6, EZH2, ACTA2, SOD1, and PDXN throughout long-term in vitro culture of bone marrow-derived mesenchymal stem cells (BM-MSCs) measured through RT-qPCR. Gene expression is normalized to the respective RPLP0 expression. (A) Graphs depicting the average gene expression every two passages from passage 3/5 to passage 15 of two selected BM-MSC groups, one from each genotype. (B) Bar graphs comparing gene expression averages at low passages (P3/P5) and high passages (P13/P15) and between genotypic groups. Data analysis was performed using a two-way ANOVA, n = 3 for each group.

SOD1 expression increases with passage in all BM-MSC groups. However, it significantly increases only in the GSTT1+ group. Additionally, SOD1 expression at high passages in the GSTT1+ group is significantly higher than its expression in GSTT1− cells. The other two genes quantified in this study, namely ACTA2 and PDXN, do not show a clear trend of increasing or decreasing with passage, suggesting that the expression pattern is more heterogeneous across BM-MSC groups. Interestingly, ACTA2 expression throughout culture and at high passages was significantly higher in the GSTT1 negative group when compared to GSTT1+ BM-MSCs. Moreover, PDXN expression was significantly higher in GSTT1+ BM-MSCs when compared to the GSTT1 negative BM-MSCs at higher passages.

BM-MSCs with a GSTT1 null genotype are resistant to some of the phenotypic changes produced by irradiation

Besides long-term in vitro culture, BM-MSC irradiation was performed as an additional aging model to assess the impact of GSTT1 genotype on BM-MSC senescence. Then, the percentage of senescent cells was assessed through SA-β-gal staining and the expression of senescence-associated genes 53BP1, γH2AX, p14ARF, p16INK4A, p21 Waf1, TP53, TWIST1, and TWIST2 were quantified.

Our results show that, with irradiation, the percentage of senescent cells increases in all BM-MSC groups as shown in Fig. 8, Panel A-B. However, the percentage of senescent cells is significantly lower in the GSTT1 null group compared to the GSTT1+ group.

Fig. 8.

Fig. 8

Open in a new tab

Senescence-associated β galactosidase (SA-β-gal) staining and senescence-associated gene expression after irradiation of bone marrow-derived mesenchymal stem cells (BM-MSCs). (A) SA-β-gal expression of BM-MSCs without irradiation (CTRL) and after 5 and 10 GY of irradiation from two selected BM-MSC groups, one from each genotype. (B) Bar graphs depicting the average number of SA-β-gal positive cells over the total number of cells. (C) Bar graphs depicting the average gene expression of 53BP1, γH2AX, p14ARF, p16INK4A, p21Waf1, TP53, TWIST1, and TWIST2 without irradiation (CTRL) and after 10 GY of irradiation exposure. Gene expression was normalized to their respective RPLP0 expressions. Data analysis was performed using a one-way ANOVA, n = 3 for each group.

Additionally, gene expression results after irradiation show some interesting patterns as shown in Fig. 8, Panel C. The expressions of 53BP1 and p21 Waf1 significantly increase in the GSTT1 positive group after irradiation. However, no significant changes for these genes were observed in the GSTT1 negative group, resulting in a significantly lower expression post-irradiation. Nevertheless, p16INK4A experienced a similar pattern but resulted in a significantly lower expression post-irradiation in the GSTT1 null group when compared to the GSTT1+ group. Moreover, γH2AX, p14ARF, and TP53 show similar expression patterns with irradiation no matter the genotypic group. Interestingly, although TWIST1 and TWIST2 expressions are initially higher in the GSTT1 negative group, they significantly decrease with irradiation. This phenomenon is only observed in the GSTT1 negative group as no significant changes are observed in the GSTT1+ group.

BM-MSC differentiation potential is not affected by GSTT1 genotype

An important phenotypic change that BM-MSCs undergo as they are continuously passaged is their decrease in differentiation potential. Thus, to asses how their multipotency differs between GSTT1 genotypes, we subjected BM-MSCs to osteogenic and adipogenic differentiation. After quantifying the stain intensity produced by Alizarin Red and Oil-Red-O staining respectively, we detected no differences in differentiation potential between GSTT1 genotypes as shown in Supplemental Fig. 5.

Discussion

In vitro expansion of BM-MSCs is necessary to obtain the sufficient number of cells for clinical use8. However, extended culture often leads to cellular senescence, or a state of irreversible growth arrest while cells remain metabolically active24,25. This phenomenon negatively affects BM-MSC potency, leading to a diminished proliferative potential, decreased multipotentiality, and a modified secretome as part of their senescence-associated secretory phenotype (SASP)9,10,26. Notably, different groups have reported BM-MSCs populations with high proliferative potential and sustained phenotype despite prolonged culture11,27–29. This can be partially attributed to the well-acknowledged heterogeneity that BM-MSCs intrinsically possess. However, this phenomenon is not well understood, leading to a lack of reliable strategies to identify BM-MSC populations that can undergo in vitro expansion without compromising their regenerative potential.

Previously, the GSTT1 gene, present in 14–30% of the population16, has been studied in the context of BM-MSC proliferative potential. Sathiyanathan et al. showed that BM-MSCs lacking the GSTT1 gene can proliferate faster and possess longer telomeres than their GSTT1 positive counterparts23. Building upon these findings, we further assess how GSTT1 genotype affects BM-MSC phenotype during long-term in vitro culture and irradiation, discovering that a GSTT1 null genotype provides resistance to some of the changes associated with cell senescence. Consistent with earlier reports, we observed that cells with a GSTT1 null genotype proliferate faster than BM-MSCs with GSTT1 positive genotypes23, something that was also true after GSTT1 silencing. However, this finding was only true when comparing the cell number at low passages, something that was diminished as the cells were cultured over multiple passages. Furthermore, SA-β-gal expression, a well acknowledged marker of senescence24, increased after subjecting BM-MSCs to both long-term culture and irradiation. Nevertheless, this increase was significantly attenuated in the GSTT1 null group when compared to the GSTT1 positive group, implying a partial resistance to senescence.

Given the established link between telomere shortening and senescence24,26, we investigated BM-MSC telomere length and shortening rates. Our findings show no correlation between GSTT1 status and telomere length or rate of telomere shortening. Although Sathiyanathan et al. reported longer telomeres in GSTT1 null BM-MSCs, they similarly observed that the rate of telomere shortening was the same between the GSTT1 null and GSTT1 positive groups23, which partially aligns with our results. Similarly, hTERT expression did not show any correlation to GSTT1 genotype, suggesting that neither the telomere length nor hTERT are responsible for the increased proliferative abilities and resistance to senescence in GSTT1 null BM-MSCs.

We then investigated whether GSTT1 status affects the expression of several genes related to senescence and cell cycle arrest in BM-MSCs, namely p14ARF, p16INK4A, p21 Waf1, γH2AX, 53BP1, and TP53. Both p16INK4A and p21 Waf1 have been studied in the context of senescence and have been shown to be elevated after long-term culture and following irradiation26,30–33. A similar trend was observed in most of the BM-MSC groups during this study. However, GSTT1 genotype had an important effect on the expression of these cyclin-dependent kinase inhibitors, indicating that a GSTT1 null genotype leads to a diminished increase in p16INK4A expression post-irradiation and p21 Waf1 expression during long-term culture and following irradiation. These results suggest that a GSTT1 null genotype can lower the percentage of cells undergoing cell cycle arrest, thus lowering the percentage of senescent cells in culture.

Similarly, p14ARF expression was generally higher in the GSTT1 positive BM-MSC groups even at earlier passages during expansion while its expression did not significantly differ between GSTT1 genotypes after irradiation. Even through p14ARF is not as thoroughly studied as p16INK4A and p21 Waf1 in the context of cell senescence, this cyclin-dependent kinase inhibitor has important p53-dependent and p53-independent functions, inhibiting p53 suppression and interacting with multiple proteins involved in chromosome stability and transcription34,35. Interestingly, this study observed a decrease in p14ARF expression with passaging in some BM-MSC groups, a trend that has been recorded previously9 which might suggest other regulatory pathways for p14ARF expression. Although p16INK4A, p21 Waf1, and p14ARF are involved in the activation of p53 (involved in the conservation of the DNA integrity)36, TP53, the gene responsible for coding this protein, did not seem to be related to GSTT1 genotype during long-term culture or irradiation.

To assess DNA damage repair responses, γH2AX and 53BP1 expressions were evaluated since these two proteins are involved specifically in double-strand breaks, a DNA lesion that can trigger permanent growth arrest and senescence37. γH2AX and 53BP1 are typically upregulated when BM-MSCs are exposed to irradiation37–39. As expected, γH2AX increased in all BM-MSC groups after irradiation with no genotype-specific differences, indicating that all BM-MSC genotypes can activate the γH2AX repair mechanism when DNA double-strand breaks are present, preserving the genomic integrity when cells are exposed irradiation. However, when γH2AX was quantified during prolonged expansion, a link between passage number or BM-MSC genotype and γH2AX expression was not found, consistent with previous reports showing impairment of the DNA damage response in BM-MSCs as they are extensively passaged40,41. On the other hand, when 53BP1 was quantified after irradiation, its expression increased in all BM-MSC groups except for the GSTT1 null group. This is unexpected since 53BP1 is usually co-expressed with γH2AX post-irradiation39,42. Nonetheless, this could mean that GSTT1 null BM-MSCs prefer the homologous recombination double-strand break repair mechanism, which do not necessarily require the 53BP1 protein, in contrast to the non-homozygous end joining pathway37.

We further quantified the expression of TWIST1 and TWIST2, genes involved in maintaining BM-MSC stemness43. Our results indicate that, at low passages, TWIST1 expression was the highest in the GSTT1 null groups and decreased with long-term culture in all BM-MSC groups. Previously, TWIST1 expression in BM-MSCs has been correlated with a higher proliferative capacity and resistance to senescence27,44–46, agreeing with our results as shown in the growth curve and TWIST1 quantification. Interestingly, after irradiation, the GSTT1 null group was the only one that experienced a significant decrease in TWIST1 and TWIST2 expression, which might be due to them having the highest TWIST1 and TWIST2 expression pre-irradiation in the first place. Moreover, this decrease in TWIST1 and TWIST2 expression could suggest that GSTT1 null BM-MSCs are not able to conserve an advantage in their proliferative capacities post-irradiation compared to the other BM-MSC genotypes, but more research on the topic is encouraged.

Genes usually altered with senescence, IL-6, SOD1, EZH2, ACTA2, and PDXN, were quantified to evaluate how SASP changes depending on the genotype of BM-MSCs. With long-term culture, there were changes in the secretome of BM-MSCs from both genotypes. However, the GSTT1 null group expressed lower levels of IL-6, a pro-inflammatory cytokine that is part of the BM-MSC SASP10,31,47 throughout culture expansion and PDXN, whose elevated levels might contribute to oxidative stress and apoptosis48 at higher passages, indicating that GSTT1 null BM-MSCs have a less inflammatory and pro-apoptotic secretome when compared to the other BM-MSC groups. Additionally, the GSTT1 null group showed signs of higher multipotentiality as shown by the elevated levels of ACTA2, a myogenic differentiation marker49, throughout culture. Finally, EZH2 levels decreased in all BM-MSC groups but were the lowest in the GSTT1 null group. Previously, EZH2 expression has been shown to diminish senescence and SASP in BM-MSCs50,51, therefore its decrease in expression with long-term passage is predicted. However, the particularly low expression of EZH2 in the GSTT1 null group is unexpected and needs to be further studied since it does not seem to increase the number of senescent cells as shown by the SA-β-gal activity.

Although the findings from this study are promising for the field of BM-MSC biomanufacturing, it has some important limitations to consider. First, the sample size was small, with only three representative samples for each GSTT1 genotype. Additionally, for some experiments, only one representative sample from each group was used. This indicates that, although some results were significant, they need to be considered carefully. Moreover, for this study, media with 10% FBS was used, something that is non-compliant with GMP standards. If these findings were to be translated to BM-MSC biomanufacturing, a different media choice such as xeno- and serum-free would be used. This is important because media composition can significantly impact BM-MSC properties52,53, possibly skewing the data obtained in this study. Finally, even though BM-MSCs were isolated from donors aged between 20 and 30 years old, there were still differences in donor ages between BM-MSCs, something known to affect cell phenotype54,55 which might play a role in some of the differences we observed between BM-MSC groups. The donor ages from each BM-MSC group are included in Supplemental Table 1. Therefore, a bigger sample size, a narrower donor age gap, and the use of xeno- and serum-free media are encouraged in further investigations to obtain more significant data that can be used in BM-MSC biomanufacturing.

Overall, the absence of the GSTT1 gene in BM-MSC grants these cells with partial resistance to some of the phenotypic changes that are originated with senescence, increasing their proliferative potential at low passages and decreasing some components of MSC SASP. Even though a GSTT1 null genotype has been shown to be deleterious in the context of diseases like cancer18,20, it can have important advantages in regenerative medicine applications. This specific genotype can be used as a biomarker to screen for BM-MSC populations that might be less susceptible to the deleterious changes caused by long-term culture. Consequently, this will enable cell manufacturing companies to produce high numbers of BM-MSCs through expansion without compromising their quality and potency, generating more consistent and effective results for clinical applications.

Conclusions

In conclusion, this study demonstrates that GSTT1 null BM-MSCs exhibit an enhanced proliferative capacity at low passages and partial resistance to senescence. Additionally, these cells have reduced SASP and expression of important senescence-associated genes, namely p21 Waf1, p14ARF, 53BP1, IL-6, and PDXN. Additionally, BM-MSCs with this genotype express TWIST1 and ACTA2 at higher levels, something that might contribute to their stemness and proliferation potential. While further investigations are needed, a GSTT1 null genotype can serve as a potential biomarker to identify BM-MSCs populations that can be expanded in vitro without compromising their quality too much.

Materials and methods

Genotyping

For genotyping, BM-MSC genomic DNA was isolated from 80% confluent T75 flasks using DNeasy Blood & Tissue Kit (Qiagen, cat.no. 69504) following manufacturer’s instructions. Briefly, cells were lysed using the lysis buffer with proteinase K. Then, lysates were transferred into a binding membrane and washed several times using guanidine/ethanol buffer. Finally, DNA was eluted using 200 µL of elution buffer and quantified using a NanoPhotometer® N60 (IMPLEN). Subsequently, PCR was performed by multiplexing using Platinum Multiplex PCR Master Mix (Applied Biosystems, cat.no. 446429) consisting of 2 different sets of primers shown in Supplemental Table 2 as described in Buchard et al.56. The first set of primers detects the deletion of GSTT1. When the gene is present, the DNA fragment between the forward and reverse primers will be greater than 60 kb, which is too long for amplification; however, if deletion is present (i.e. absence of GSTT1), the DNA fragment will be around 3.1 kb which corresponds to the pseudo GSTT1 gene and results in amplification. The second set of primers detects the presence of the GSTT1 gene, yielding a fragment of around 0.97 kb that will undergo amplification if the gene is present and resulting in no amplification if the gene is absent. The PCR profile used for this study includes heating a thermal cycler CFX96 (Bio-Rad) at 95 °C for 5 min followed by a cycle of 95 °C for 2 min, 60 °C for 30 s, and 72 °C for 1 min for the last cycle, followed by an additional final extension at 72o c for 10 min. The amplicons obtained were run on a 0.8% agarose/TBE electrophoresis gel at 50 V for 60 min, followed by analyses using Chemidoc (Bio-Rad) gel documentation system. During this study, a total of 11 BM-MSCs derived from different donors were genotyped to achieve a 1:1:1 genotypic ratio.

Culture and expansion of mesenchymal stem cells

BM-MSCs purchased from RoosterBio from healthy individual donors between the age of 20–30 were cultured in T75 flasks using low-glucose DMEM media supplemented with 1% antibiotic (penicillin-streptomycin), 1% of L-Glutamine, and 10% fetal bovine serum. The cells were cultured under standard conditions of 5% CO2, and 37 °C in sterile incubators and continuously expanded from passage 4–5 until passage 15. For expansion, BM-MSCs were harvested using TrypLE™ (Thermo Fisher Scientific, cat.no. 12605010) once they reached 80% confluency. Then, they were reseeded at a density of 5,000 cells/cm2 into new T75 flasks. At each passage, a fraction of cells was collected for DNA and RNA analysis.

Irradiation of BM-MSCs

BM-MSCs from each genotype were seeded at a density of 5,000 cells/cm2 in 6-well plates and allowed to reach 75% confluency. Prior to irradiation, cells were treated with fresh maintenance media. BM-MSCs were irradiated using an X-ray source for 5 min every day for three days to provide a total of 10 Gy dose strength. Post-irradiation, the cells were returned to normal culture conditions with no media change for the next 3 days. At 72 h post-irradiation, cells were harvested for mRNA analysis to assay gene expression of senescence-related markers or fixed for assessment of senescence-associated beta-galactosidase (SA-β-gal) activity.

Growth curve and cumulative growth assay

To perform the 8-day growth curve, BM-MSCs from GSTT1 + and GSTT1 – groups were seeded in 12-well plates at a density of 5000 cells/cm2. Then, every two days, cells from three wells were resuspended using TripLE™, neutralized with low-glucose DMEM, and counted using an automated cell counter (Countess 3 FL, Invitrogen). Each well was counted 3 times using the same sample volume and the total number of cells per well was recorded at days 2,4,6, and 8. A similar growth curve was performed to assess growth after GSTT1 silencing, but for this experiment, BM-MSCs were seeded at a 3000 cells/cm2 density and counted on days 3,5,and 7. For the cumulative growth assay, BM-MSCs were seeded in 5 cm dishes at a density of 3000 cells/cm2. When the dishes reached 70–80% confluence, cells were detached using TripLE™ and seeded in a new 5 cm dish at the same density from passage 6 to passage 15. During the re-seeding, the cell number in each dish was recorded by counting the cells 3 times with an automatic cell counter (Countess 3 FL, Invitrogen). Then, the number of cells per well was recorded and the cumulative cell number was graphed.

Cell proliferation assay by Bromodeoxyuridine incorporation

Two representative samples, one from each genotype group (BM-MSC 164 GSTT1− and BM-MSC 310277 GSTT1+) were chosen to perform a bromodeoxyuridine (BrDU) assay. BM-MSCs at low passages (< P6) or high passages (P12< ) were seeded at a density of 2000cells/well into 96-well plates with 6 replicates per sample. BrDU was performed using a BrDU ELISA Colorimetric kit (Roche, cat.no. 11647229001) following manufacturer’s instructions. Briefly, cells were incubated at 37 °C with 5% CO2 for 24 h. Then, BM-MSCs were checked for confluency and treated with 10 µM of BrDu labeling solution followed by an additional incubation for 24 h. Plates were then heated for 1 h at 60o C using a hot plate. After that, 200 µL of FixDenat solution was added to each well followed by a 30-minute incubation at ambient temperature. Then, the FixDenat solution was removed, and a secondary antibody was added. Next, cells were incubated for 90 min at ambient temperature. After removing the secondary antibody solution, cells were washed three times with 200 µL of 1X PBS. Finally, chromogenic substrate was added and incubated at room temperature for 25 min followed by measurement of the absorbance at 370 nm for 25, 30, and 35 min of incubation corresponding to BrDU activity.

Real-time quantitative polymerase chain reaction

Total RNA from individual cell cultures were isolated using RNeasy Miniprep kit (Qiagen, cat.no. 74104) following manufacturer’s protocol recommendations. Briefly, BM-MSCs were lysed using a lysis buffer. Then, lysates were transferred into silica binding membrane and washed several times using wash buffers provided in the kit. RNA was then eluted using DNase/RNase free water followed by quantification and purity assessment using a NanoPhotometer® N60 (IMPLEN). cDNA synthesis was performed using 500 ng mRNA using SuperScript™ VILO™ cDNA Synthesis Kit (Invitrogen, cat.no. 11754250) following manufacturer’s protocol. The cDNA was diluted to 50 ng/µL and approximately 5ng of cDNA was used as a template for all downstream real-time quantitative polymerase chain reaction (RT-qPCR) assays using SYBR Green. Primer sequences for all genes assayed in the study are listed in Supplemental Table 3. RT-qPCR was performed in a CFX96 (Bio-Rad) thermal cycler using standard reaction set up that comprised an activation at 95o C for 2 min, 40 cycles of 95 °C for 1 min and 60 °C for 30s of annealing and extension followed by a melt curve. All Ct values obtained were normalized to a housekeeping gene (RPLP0) and presented as a relative expression (2–∆∆Ct) as described previously57.

Telomere length analysis

The relative telomere lengths of BM-MSCs from multiple donors were quantified RT-qPCR using previously established protocols57. Genomic DNA of BM-MSCs at multiple passages was isolated using the DNeasy Blood & Tissue Kit (Qiagen, cat.no. 69504) following manufacturer’s instructions and used in a SYBR green-based RT-qPCR reaction with Tel 1 and Tel 2 primers and single copy genes of 36B4u and 36B4d. The primer sequences are shown in Supplemental Table 4. The thermal cycler CFX96 (BioRad) set up involved activation at 95 °C for 1 min, followed by 40 cycles at 95 °C (1 min), 60 °C (30 s), and 2 min at 54 °C. Then, a melt curve analysis was performed. A modification in the reaction set up was applied for the single copy gene analyses which involve 40 cycles of the program: 1 min at 95 °C, 30 s at 60 °C, and 1 min at 58 °C. Standard curves for both telomere and single copy genes were first obtained from iPSC gDNA for the standard curve at the following dilutions: 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 ng/ml. Telomere length RT-qPCR using Tel primers and 36B4 primers was performed using 12.5 ng of gDNA (for each sample at specified passage in three replicates) and the data was analyzed using 2–∆∆Ct as described previously57.

hTERT gene expression analysis

The expression of telomerase reverse transcriptase (hTERT), the gene responsible for telomere repeats, was assessed in all BM-MSC groups using RT-qPCR. As an internal control, mRNA from iPSCs was used since these cells have a robust expression of hTERT. The primer sequences for the hTERT gene are provided in Supplemental Table 4. RNA from BM-MSCs and iPSCs was isolated using RNeasy mini kit (Qiagen, cat.no. 74104) followed by quantification and reverse transcription as described in previous sections. RT-qPCR was performed using our established standard protocol (95 °C for 2 min, 40 cycles of 95 °C for 1 min and 60 °C for 30 s) in an CFX96 (BioRad) thermal cycler, followed by melt curve analysis. Data was analyzed using 2–∆∆Ct as described previously57 using RPLP0 as the housekeeping gene.

Senescence-associated beta-galactosidase (SA-β-gal) staining

Representative BM-MSCs from each GSTT1 genotype (BM-MSC 164− and BM-MSC 310277+) were cultured according to standard conditions and sampled at a specific passage/timepoint during expansion. Cells were seeded in glass bottomed dishes (MatTek, cat.no. P35GC-1.5-14-C) in maintenance media. At 75% confluency, cells were either irradiated for a total dose of 10 Gy or were left non-irradiated in preparation for assessing SA-β-gal activity using the senescence-associated β-galactosidase staining kit (Cell Signaling, cat.no. 9860) following manufacturer’s instructions. Briefly, cells were fixed in 1X fixative solution for 15 min at room temperature followed by washes with 1X PBS. Fixed cells were stained with β-galactosidase Staining Solution. Dishes were sealed in parafilm and incubated at 37 °C overnight in a dry incubator (no CO2). Post-incubation, cells were evaluated under the microscope for development of blue color which indicates SA-β-gal activity. In addition to the irradiation studies, SA-β-gal activity was assessed in continuously expanded cells until passage 15 to assess in vitro replicative senescence. The experiment was performed in triplicates (three wells for each BM-MSC group) and three fields of view were analyzed in each well. Stained images were examined under bright field microscopy. Cells stained positively were counted to estimate the percentage of SA-β-gal+ cells.

GSTT1 Silencing

A representative sample from the GSTT1 positive group was chosen to undergo silencing (BM-MSC 238). For this, BM-MSCs were seeded in a 12-well plate at a density of 3000 cells/cm2. The next day, 15 pmol of either GSTT1 SiRNA (Thermo Fisher Scientific, cat.no. 4392420) or negative control SiRNA (Thermo Fisher Scientific, cat.no. 4390843) were combined with the manufacturer’s recommended amount of OPTI-MEM (Gibco, cat.no. 31985-062) and lipofectamine RNAiMAX (Thermo Fisher Scientific, cat.no. 13778-150). After letting the mixture rest for 5 min, it was added to complete DMEM and subsequently to the BM-MSCs. 48 h after the SiRNA treatment RNA was isolated form the samples to quantify GSTT1 expression. The rest of the wells were used to generate a 7-day growth curve as described previously.

Osteogenic and adipogenic differentiation

BM-MSCs differentiation was performed as described previously9. Briefly, cells from each group were seeded at a 5000 cells/cm2 density in a 6-well plate. Once they reached 70–80% confluency, wells undergoing differentiation were treated with either osteogenic media (complete DMEM with 10 mM β-Glycerolphosphate, 10 mM of Dexamethasone, and 25 µg/mL of L-Ascorbic Acid) or adipogenic media (complete DMEM made with high glucose basal media, 1µM of Dexamethasone, 10 µg/mL of Insulin, 11.6 µg/mL of 3-isobutyl-1-methylxanthine, and 100 µM of Indomethacin). The control wells were treated with either complete DMEM or complete DMEM made with high glucose basal media respectively. After 21 days of osteogenic differentiation and 28 days of adipogenic differentiation, the osteogenic plates were stained with Alizarin Red and the adipogenic plates were stained with Oil-Red-O. Finally, pictures of the wells were taken in a microscope using the 4x lens.

Statistical analysis

To compare phenotypic measurements and gene expression between both genotypes, one-way ANOVA, two-way ANOVA, and t-test analyses where applicable were performed. An α value of 0.05 was used to determine significance in the compact letter display. For the bar graphs, * was used for a p-value ≤ 0.05, ** for a p-value ≤ 0.01, *** for a p-value ≤ 0.001, and **** for a p-value ≤ 0.0001.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (916.9KB, pdf)

Author contributions

I.K.K.A performed experiments. V.V.M and I.K.K.A. drafted the main manuscript. R.M.S. conceived the idea for the study, generated financial support, provided input, and edited the manuscript. C.L.B provided financial support and edited the manuscript. All authors reviewed and approved the final version of the manuscript.

Funding

We acknowledge funding support to Dr. Rebekah Samsonraj from the National Institute of Health COBRE funding (P20GM125503), MTF Biologics Foundation Junior Investigator Grant, National Science Foundation (2134494), University of Arkansas Women’s Giving Circle, University of Arkansas College of Engineering, and the Arkansas Biosciences Institute. We also thank the Fulbright Scholarship awarded to I Kade Karisma Gita Ardana.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

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

References

  • 1.Maldonado, V. V. et al. Clinical utility of mesenchymal stem/stromal cells in regenerative medicine and cellular therapy. J. Biol. Eng.17 (1). 10.1186/s13036-023-00361-9 (2023). [DOI] [PMC free article] [PubMed]
  • 2.Samsonraj, R. M. et al. Concise Review: Multifaceted Characterization of Human Mesenchymal Stem Cells for Use in Regenerative Medicine, Stem Cells Transl. Med., vol. 6, no. 12, pp. 2173–2185, 10.1002/sctm.17-0129 (2017). [DOI] [PMC free article] [PubMed]
  • 3.Williams, T. et al. Versatility of mesenchymal stem cell-derived extracellular vesicles in tissue repair and regenerative applications. Biochimie207, 33–48. 10.1016/j.biochi.2022.11.011 (2023). [DOI] [PubMed] [Google Scholar]
  • 4.Kabat, M., Bobkov, I., Kumar, S. & Grumet, M. Trends in mesenchymal stem cell clinical trials 2004-2018: is efficacy optimal in a narrow dose range? Stem. Cells Transl. Med.9 (1), 17–27. 10.1002/sctm.19-0202 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Lozano-Rivas, N. et al. AB1011 Clinical trial of intravenous infusion of fucosylated bone marrow mesenchymal stem cells in patients with osteoporosis, Annals Rheumatic Dis., vol. 77, no. Suppl 2, p. 1625, 10.1136/annrheumdis-2018-eular.4728 (2018).
  • 6.Theodosaki, A. M. et al. Bone regeneration with mesenchymal stem cells in scaffolds: systematic review of human clinical trials. Stem. cell. Rev. Rep.20 (4), 938–966. 10.1007/s12015-024-10696-5 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Li, Y. et al. Current status of clinical trials assessing mesenchymal stem cell therapy for graft versus host disease: a systematic review. Stem Cell Res. Ther.13 (1), 93–93. 10.1186/s13287-022-02751-0 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Binato, R. et al. Stability of human mesenchymal stem cells during in vitro culture: considerations for cell therapy. Cell Prolif.46 (1), 10–22. 10.1111/cpr.12002 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Maldonado, V. V., Jensen, H., Barnes, C. L. & Samsonraj, R. M. Phenotypic changes associated with continuous long term in vitro expansion of bone marrow-derived mesenchymal stem cells, Biochimie, vol. 234, pp. 62–75 10.1016/j.biochi.2025.04.002, (2025). [DOI] [PMC free article] [PubMed]
  • 10.Samsonraj, R. M., Law, S. F., Chandra, A. & Pignolo, R. J. An unbiased proteomics approach to identify the senescence-associated secretory phenotype of human bone marrow-derived mesenchymal stem cells. Bone Rep.18, 101674–101674. 10.1016/j.bonr.2023.101674 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Heinrichs, D. P., Maldonado, V. V., Ardana, I. K. K., Porter, R. M. & Samsonraj, R. M. Assessing the effects of dasatinib on mesenchymal Stem/Stromal cells: assessing the effects of dasatinib, Cellular and molecular bioengineering, 17, 6, pp. 609–618,10.1007/s12195-024-00830-1 (2024). [DOI] [PMC free article] [PubMed]
  • 12.Needy, T. et al. Examining the effects of Quercetin on phenotypic characteristics of human mesenchymal stem cells. Cell. Mol. Bioeng.10.1007/s12195-025-00849-y (2025). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Wang, T., Zhang, J., Liao, J., Zhang, F. & Zhou, G. Donor genetic backgrounds contribute to the functional heterogeneity of stem cells and clinical outcomes. Stem Cells Transl. Med.9 (12), 1495–1499. 10.1002/sctm.20-0155 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Maged, G., Abdelsamed, M. A., Wang, H. & Lotfy, A. The potency of mesenchymal stem/stromal cells: does donor sex matter? Stem. Cell Res. Ther.15 (1), 112–112. 10.1186/s13287-024-03722-3 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Turlo, A. J. et al. Mesenchymal Stromal Cell Secretome Is Affected by Tissue Source and Donor Age, Stem cells (Dayton, Ohio), vol. 41, no. 11, pp. 1047–1059, 10.1093/stmcls/sxad060 (2023). [DOI] [PMC free article] [PubMed]
  • 16.Nakanishi, G. et al. GSTM1 and GSTT1 polymorphisms in healthy volunteers - a worldwide systematic review. Drug Metab. Rev.54 (1), 37–45. 10.1080/03602532.2022.2036996 (2022). [DOI] [PubMed] [Google Scholar]
  • 17.Guedi, A. B. et al. Glutathione S-transferase polymorphisms (GSTM1/GSTT1) outcomes in clinical profile and treatment responsiveness among Tunisian cohort of parkinson’s disease. J. Neural Transm.132 (1), 117–127. 10.1007/s00702-024-02815-w (2025). [DOI] [PubMed] [Google Scholar]
  • 18.Ye, Z. & Song, H. Glutathione s-transferase polymorphisms (GSTM1, GSTP1 and GSTT1) and the risk of acute leukaemia: A systematic review and meta-analysis, Eur. J. Cancer), 10.1016/j.ejca.2005.01.014 (2005). [DOI] [PubMed]
  • 19.Abbas, M. et al. Association of GSTM1 and GSTT1 gene polymorphisms with COVID-19 susceptibility and its outcome. J. Med. Virol.93 (9), 5446–5451. 10.1002/jmv.27076 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Zhang, W. P. et al. Individual and combined effects of GSTM1, GSTT1, and GSTP1 polymorphisms on lung cancer risk: A meta-analysis and re-analysis of systematic meta-analyses. Med. (Baltim).100 (26), e26104–e. 10.1097/MD.0000000000026104 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Dasari, S., Ganjayi, M. S. & Meriga, B. Glutathione S-transferase is a good biomarker in acrylamide induced neurotoxicity and genotoxicity. Interdisciplinary Toxicol.11 (2), 115–121. 10.2478/intox-2018-0007 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Kudlova, N., De Sanctis, J. B., Hajduch, M., Senescence, C. & Molecular Targets, Biomarkers, and senolytic drugs. Int. J. Mol. Sci.23 (8), 4168. 10.3390/ijms23084168 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Sathiyanathan, P. et al. A genomic biomarker that identifies human bone marrow-derived mesenchymal stem cells with high scalability. Stem Cells. 38 (9), 1124–1136. 10.1002/stem.3203 (2020). [DOI] [PubMed] [Google Scholar]
  • 24.Huang, W., Hickson, L. J., Eirin, A., Kirkland, J. L. & Lerman, L. O. Cellular senescence: the good, the bad and the unknown. Nat. Rev. Nephrol.18 (10), 611–627. 10.1038/s41581-022-00601-z (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Wagner, K. D. & Wagner, N. The Senescence Markers p16INK4A, p14ARF/p19ARF, and p21 in Organ Development and Homeostasis, Cells (Basel, Switzerland), vol. 11, no. 12, p. 2022,10.3390/cells11121966 (1966). [DOI] [PMC free article] [PubMed]
  • 26.Liu, J., Ding, Y., Liu, Z. & Liang, X. Senescence in mesenchymal stem cells: functional Alterations, molecular Mechanisms, and rejuvenation strategies. Front. cell. Dev. Biology. 8, 258–258. 10.3389/fcell.2020.00258 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Samsonraj, R. M. et al. Establishing Criteria for Human Mesenchymal Stem Cell Potency, Stem cells (Dayton, Ohio), vol. 33, no. 6, pp. 1878–1891, 10.1002/stem.1982 (2015). [DOI] [PMC free article] [PubMed]
  • 28.DiGirolamo, C. M. et al. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br. J. Haematol.107 (2), 275–281. 10.1046/j.1365-2141.1999.01715.x (1999). [DOI] [PubMed] [Google Scholar]
  • 29.Muraglia, A., Cancedda, R. & Quarto, R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J. Cell Sci.113 (7), 1161–1166. 10.1242/jcs.113.7.1161 (2000). [DOI] [PubMed] [Google Scholar]
  • 30.Ho, J. H. et al. Cell contact accelerates replicative senescence of human mesenchymal stem cells independent of telomere shortening and p53 activation: roles of Ras and oxidative stress, Cell Transpl., 20, 8, pp. 1209–1220, 10.3727/096368910X546562 (2011). [DOI] [PubMed]
  • 31.Marote, A. & Comparison of bone marrow derived and induced mesenchymal stem cells and their secretome over long term culture. 202 - Mesenchymal Stem/Stromal Cells: Cellular aging secretes: A, Cytotherapy (Oxford, England), vol. 25, no. 6, pp. S86-S87, 10.1016/S1465-3249(23)00286-4 (2023). [DOI] [PubMed]
  • 32.Bai, J. et al. Irradiation-induced senescence of bone marrow mesenchymal stem cells aggravates osteogenic differentiation dysfunction via paracrine signaling. Am. J. Physiology: Cell. Physiol.318 (5, pp. C1005-C1017, ). 10.1152/ajpcell.00520.2019 (2020). [DOI] [PubMed]
  • 33.Fekete, N. et al. Effect of High-Dose Irradiation on Human Bone-Marrow-Derived Mesenchymal Stromal Cells, Tissue engineering. Part C, Methods, vol. 21, no. 2, pp. 112–122, 10.1089/ten.tec.2013.0766 (2015). [DOI] [PMC free article] [PubMed]
  • 34.Cilluffo, D., Barra, V. & Di Leonardo, A. P14ARF: The Absence that Makes the Difference, Genes, vol. 11, no. 7, p. 824 10.3390/genes11070824, (2020). [DOI] [PMC free article] [PubMed]
  • 35.Sherr, C. J. Divorcing ARF and p53: an unsettled case. Nat. Rev. Cancer. 6 (9), 663–673. 10.1038/nrc1954 (2006). [DOI] [PubMed] [Google Scholar]
  • 36.Borrero, L. J. H. & El-Deiry, W. S. Tumor suppressor p53: Biology, signaling pathways, and therapeutic targeting, Biochimica et biophysica acta. Reviews on cancer, vol. 1876, no. 1, pp. 188556–188556, 10.1016/j.bbcan.2021.188556 (2021). [DOI] [PMC free article] [PubMed]
  • 37.Panier, S. & Boulton, S. J. Double-strand break repair: 53BP1 comes into focus. Nat. Rev. Mol. Cell Biol.15 (1), 7–18. 10.1038/nrm3719 (2014). [DOI] [PubMed] [Google Scholar]
  • 38.Dickey, J. S. et al. H2AX: functional roles and potential applications, Chromosoma, vol. 118, no. 6, pp. 683–692, 10.1007/s00412-009-0234-4 (2009). [DOI] [PMC free article] [PubMed]
  • 39.Tsvetkova, A. et al. γH2AX, 53BP1 and Rad51 protein foci changes in mesenchymal stem cells during prolonged X-ray irradiation, Oncotarget, vol. 8, no. 38, pp. 64317–64329, 10.18632/oncotarget.19203, (2017). [DOI] [PMC free article] [PubMed]
  • 40.Bao, X. et al. Extended in vitro culture of primary human mesenchymal stem cells downregulates Brca1-related genes and impairs DNA double‐strand break recognition. FEBS open. Bio.. 10 (7), 1238–1250. 10.1002/2211-5463.12867 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Hladik, D. et al. Long-term culture of mesenchymal stem cells impairs ATM-dependent recognition of DNA breaks and increases genetic instability. Stem Cell Res. Ther.10 (1), 218–218. 10.1186/s13287-019-1334-6 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Marková, E., Schultz, N. & Belyaev, I. Y. Kinetics and dose-response of residual 53BP1 γ-H2AX foci: Co-localization, relationship with DSB repair and clonogenic survival. Int. J. Radiat. Biol.83 (5), 319–329. 10.1080/09553000601170469 (2007). [DOI] [PubMed] [Google Scholar]
  • 43.Franco, H. L., Casasnovas, J., Rodríguez-Medina, J. R. & Cadilla, C. L. Redundant or separate entities?--roles of Twist1 and Twist2 as molecular switches during gene transcription. Nucleic Acids Res.39 (4), 1177–1186. 10.1093/nar/gkq890 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Toriumi, K. et al. LRRC15 expression indicates high level of stemness regulated by TWIST1 in mesenchymal stem cells, iScience, vol. 26, no. 7, pp. 106946 10.1016/j.isci.2023.106946 (2023). [DOI] [PMC free article] [PubMed]
  • 45.Voskamp, C. et al. Eur. Cells Mater., 42, 401–414, 10.22203/eCM.v042a25. (2021). [DOI] [PubMed] [Google Scholar]
  • 46.Voskamp, C., Anderson, L. A., Koevoet, W. J. & Narcisi, R. G. J. van Osch, and Twist1 regulates cellular senescence in mesenchymal stem cells, Osteoarthritis Cartilage, vol. 28, pp. S509-S510, 10.1016/j.joca.2020.02.800 (2020).
  • 47.Zheng, Y., Wu, S., Ke, H., Peng, S. & Hu, C. Secretion of IL-6 and IL-8 in the senescence of bone marrow mesenchymal stem cells is regulated by autophagy via FoxO3a. Exp. Gerontol.172, 112062–112062. 10.1016/j.exger.2022.112062 (2023). [DOI] [PubMed] [Google Scholar]
  • 48.Cheng, G. & Shi, R. Mammalian peroxidasin (PXDN): From physiology to pathology, Free radical biology & medicine, vol. 182, pp. 100–107, 10.1016/j.freeradbiomed.2022.02.026 (2022). [DOI] [PMC free article] [PubMed]
  • 49.Mistriotis, P. et al. Reverses the myogenic differentiation potential of senescent stem cells by restoring ACTIN filamentous organization and SRF-Dependent gene expression. Stem Cells (Dayton Ohio). 35 (1), 207–221. 10.1002/stem.2452 (2017). [DOI] [PubMed] [Google Scholar]
  • 50.Yang, R. et al. 1,25-Dihydroxyvitamin D protects against age‐related osteoporosis by a novel VDR‐Ezh2‐p16 signal axis, Aging cell, vol. 19, no. 2, 10.1111/acel.13095 (2020). [DOI] [PMC free article] [PubMed]
  • 51.Wang, T., Chen, J., Qu, B., Zhou, D. & Hong, Z. Scutellarin Alleviates Bone Marrow Mesenchymal Stromal Cellular Senescence via the Ezh2-Nrf2 Signalling Axis in Diabetes‐Induced Bone Loss, Cell proliferation, 10.1111/cpr.13790 (2025). [DOI] [PMC free article] [PubMed]
  • 52.Lee, J. Y. et al. Comparative analysis of mesenchymal stem cells cultivated in serum free media. Sci. Rep.12 (1), 8620–8615. 10.1038/s41598-022-12467-z (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Kang, M. et al. Comparative analysis of serum and serum-Free medium cultured mesenchymal stromal cells for cartilage repair. Int. J. Mol. Sci.25 (19), 10627. 10.3390/ijms251910627 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Zaim, M., Karaman, S., Cetin, G. & Isik, S. Donor age and long-term culture affect differentiation and proliferation of human bone marrow mesenchymal stem cells. Ann. Hematol.91 (8), 1175–1186. 10.1007/s00277-012-1438-x (2012). [DOI] [PubMed] [Google Scholar]
  • 55.Siegel, G. et al. Phenotype, donor age and gender affect function of human bone marrow-derived mesenchymal stromal cells, (in English), BMC Med., vol. 11, no. 1, pp. 146–146,10.1186/1741-7015-11-146 (2013). [DOI] [PMC free article] [PubMed]
  • 56.Buchard, A., Sanchez, J. J., Dalhoff, K., Morling, N., Multiplex, P. C. R. & Detection of GSTM1, GSTT1, and GSTP1 gene variants: simultaneously detecting GSTM1 and GSTT1 gene copy number and the allelic status of the GSTP1 Ile105Val genetic variant. J. Mol. Diagnostics: JMD. 9 (5), 612–617. 10.2353/jmoldx.2007.070030 (2007). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Samsonraj, R. M. et al. Telomere length analysis of human mesenchymal stem cells by quantitative PCR, Gene, vol. 519, no. 2, pp. 348–355 10.1016/j.gene.2013.01.039, (2013). [DOI] [PubMed]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (916.9KB, pdf)

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Articles from Scientific Reports are provided here courtesy of Nature Publishing Group

RESOURCES