Validation of a Novel Genomic Biomarker of Mesenchymal Stem Cell Scalability and Implications of Genotype Status on Cellular Senescence Phenotypes

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Lowry Barnes, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7115326/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version 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. First, we identified the GSTT1 genotype (either homozygous positive, heterozygous, or homozygous negative) of nine BM-MSC groups. Then, we performed long-term in vitro culture and exposed cells to irradiation as senescence models. After that, their proliferative potential, their SASP, and the expression of key genes were investigated. The results show that GSTT1 null BM-MSCs have a higher proliferative potential and exhibit fewer senescent cells in culture when compared to the other genotypes. Additionally, these cells exhibit a lower expression of p21 Waf1, p14ARF, IL-6, PDXN, and 53BP1 and a higher expression of TWIST1 and ACTA2 genes, especially at low passages. A GSTT1 null genotype can serve as a potential biomarker to identify BM-MSC populations with higher resistance to senesce. Biological sciences/Cell biology Biological sciences/Genetics Biological sciences/Molecular biology Biological sciences/Stem cells Mesenchymal stem cells Senescence Biomarker GSTT1 polymorphism Cell expansion Cell therapies Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Although bone marrow-derived mesenchymal stem cells (BM-MSCs) have great potential for treating multiple diseases [ 1 – 3 ], the lack of consistent and/or effective treatment outcomes poses a significant challenge for their clinical translation [ 4 – 7 ]. Multiple factors can play a role in hindering the therapeutic potential of BM-MSCs including the process of ex vivo expansion, which, although necessary for obtaining a clinically relevant number of cells [ 8 ], pushes cells towards senescence - a state or irreversible growth arrest - altering their phenotype and lowering their therapeutic potential [ 9 – 12 ]. Furthermore, it has been observed that donor-donor heterogeneity plays a critical role in dictating the potency and the onset of senescence in BM-MSCs [ 13 – 15 ], suggesting the existence of intrinsic inter donor differences that provide resistance to senescence. However, to date, these donor-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 genome [ 16 ], 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-19 [ 17 – 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 senescence [ 21 , 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 genotypes [ 23 ] incentivizing more research on these somewhat contradicting but important results. This study aims to investigate the effect of the GSTT1 polymorphism (homozygous positive, heterozygous, or homozygous negative) on BM-MSC growth and senescence. First, the genomic status of nine 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 the onset of cell senescence. RESULTS 1. Genotyping allowed BM-MSCs grouping into three categories determined by their 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 1.5 kb long, whereas the second primer amplified the region flanking the GSTT1 gene, with an approximate size of 3.1 kb. The location in the genome of the forward and reverse primers used are shown in Fig. 1 , Panels A-B. Genotyping allowed the grouping of BM-MSCs into three categories: GSTT1 +/+ (homozygous positive), GSTT1 +/− (heterozygous) and GSTT1 −/− (homozygous null). BM-MSCs 310277,198, and 238 were identified to be GSTT1 +/+ ; BM-MSCs 310280, 257, and 172 were observed to have a GSTT1 +/− genotype, 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 the homozygous null BM-MSCs showed no expression of GSTT1 mRNA whereas the heterozygous and homozygous positive 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. While some BM-MSC groups showed higher expression in later passages (BM-MSC 172, BM-MSC 280, BM-MSC 238 and BM-MSC 310277), other donors showed higher expression in earlier passages (BM-MSC 257 and BM-MSC 198). No significant differences in GSTT1 gene expression were observed between the homozygous positive and heterozygous samples. 2. 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 the other two genotypes as shown in Fig. 3 , Panel A . At day 8, the GSTT1 null groups had significantly more cells than BM-MSCs from the other two groups. This difference was observed mainly in BM-MSCs 164 and 00227. The compact letter display for significant differences is shown in Supplemental Table 1. 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 −/− genotype showed significantly higher proliferation capacity compared to GSTT1 +/+ and GSTT1 +/− BM-MSCs at both young passages (p-value < 0.0001) as shown in Fig. 3 , Panel B . 3. 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. From the tested groups, BM-MSC 172 showed the slowest shortening rate, with a slope of -0.00038 while BM-MSC 164 showed the fastest shortening, with a slope of -0.065. Additionally, this study analyzed the expression of the hTERT gene. hTERT or human Telomere Retro 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. 4. GSTT1 genotype status affects the 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 homozygous positive, heterozygous, and homozygous negative BM-MSC groups as shown in Fig. 5 . The results show that GSTT1 homozygous positive BM-MSCs 310277, 238, and 198 showed a fluctuating but high expression of p14ARF when compared to most of the heterozygous or homozygous negative samples. Two of the three GSTT1 heterozygous samples (310280 and 172) and homozygous negative samples (164 and 00227) had low expression of p14ARF even at high passages. However, GSTT1 heterozygous BM-MSC 257 and GSTT1 homozygous negative BM-MSC 310264 showed a fluctuating but relatively high expression of p14ARF at young passages. Furthermore, p16INK4A showed a mostly uniform and low expression at young passages regardless of the GSTT1 genotype. It increased with passages in most BM-MSC groups, seeing mostly non-significant increases at passages 13–15 except for the homozygous positive 238 group, in which p16INK4A expression increased significantly from P13-P15 (p-value < 0.0001). Our results showed that p21 Waf1 expression increased with passage and showed the greatest but mostly non-significant differences between genotypic groups. Homozygous positive BM-MSC groups 238 and 198 showed the greatest p21 Waf1 expression at higher passages followed by homozygous positive 310277 and heterozygous 257 groups. Nevertheless, heterozygous 172 had the lowest p21 Waf1 expression even at high passages followed by homozygous negative 164. Then, γH2AX expression showed high variation between passages with no specific pattern or clear relation to genotype. TP53 showed the highest expression in heterozygous 310280 followed by homozygous negative 00227. Heterozygous 172 showed the lowest expression of TP53 followed by homozygous positive 238. However, a clear relationship between genotype and TP53 was not detected. The compact letter display that indicates significant differences between groups at each passage is shown in Supplemental Table 2 . 5. 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. The results from this study showed that TWIST1 expression varied at each passage with mostly non-significant differences between GSTT1 genotypes. Interestingly, homozygous negative 164 had the highest expression of TWIST1, particularly at passage 3 and 11. On the other hand, TWIST2 showed low expression in most samples except for the GSTT1 heterozygous BM-MSC 310280 which had significantly higher expression compared to the other samples as shown in Fig. 6 , Panel A . The compact letter display that indicates significant differences between groups at each passage is shown in Supplemental Table 3 . 6. 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 three 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 homozygous positive (p-value < 0.0001) and heterozygous (p-value < 0.0001) BM-MSCs as shown in Fig. 6 , Panel B . Moreover, no significant differences in SA-β-gal expression between the homozygous positive and heterozygous groups were observed. 7. 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. IL-6 expression increases with increasing passage while EZH2 expression decreases with increasing passage in all three GSTT1 genotypes. However, the expression of both IL-6 and EZH2 is significantly higher in the homozygous positive BM-MSCs compared to the homozygous negative BM-MSCs when they reached higher passages. It is to be noted that, at low passages, the GSTT1 homozygous positive 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. Then, SOD1 expression increases with passage in all BM-MSC groups. However, it significantly increases only in the GSTT1 homozygous positive group. Additionally, SOD1 expression at high passages in the GSTT1 homozygous positive group is significantly higher than its expression in the other two BM-MSC groups. 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 at high passages was significantly higher in the GSTT1 negative group when compared to the other two BM-MSC groups. Moreover, PDXN expression was significantly higher in GSTT1 homozygous positive BM-MSCs when compared to the other BM-MSC groups at high passages. 8. 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 was quantified. Our results show that, with irradiation, the percentage of senescent cells increases in all BM-MSC groups as shown in Fig. 7 , Panel A . However, the percentage of senescent cells is significantly lower in the GSTT1 null group compared to the other two groups. Additionally, gene expression results after irradiation show some interesting patterns as shown in Fig. 7 , Panel B. The expressions of 53BP1 and p21 Waf1 significantly increase in the heterozygous and homozygous positive groups 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 only when compared to the GSTT1 homozygous positive 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 homozygous negative group as no significant changes are observed in the other two genotypic groups. DISCUSSION In vitro expansion of BM-MSCs is necessary to obtain the necessary number of cells for clinical use [ 8 ]. However, extended culture often leads to cellular senescence, or a state of irreversible growth arrest while cells remain metabolically active [ 24 , 25 ]. This phenomenon negatively affects BM-MSCs 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 culture [ 11 , 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 population[ 16 ], 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 counterparts [ 23 ]. 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 senescence and some of the changes associated with it. Consistent with earlier reports, we observed that cells with a GSTT1 null genotype proliferate faster than BM-MSCs with GSTT1 positive genotypes [ 23 ]. Furthermore, SA-β-gal expression, a well acknowledged marker of senescence [ 24 ], 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 other two groups, implying a partial resistance to senescence. Given the established link between telomere shortening and senescence [ 24 , 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 groups [ 23 ], 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 irradiation [ 26 , 30 – 33 ]. A similar trend was observed in most of the BM-MSC groups during this study. However, the 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 that 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 homozygous positive BM-MSC groups even at earlier passages during expansion, except for the GSTT1 null 310264 and GSTT1 heterozygous 257, indicating that other variables beyond GSTT1 genotype may influence its regulation. Notably, p14ARF 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 transcription [ 35 , 36 ]. Interestingly, this study observed a decrease in p14ARF expression with passage in some BM-MSC groups, a trend that has been recorded previously [ 9 ] 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)[ 37 ], 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 senescence [ 38 ]. γH2AX and 53BP1 are typically upregulated when BM-MSCs are exposed to irradiation [ 38 – 40 ]. 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 passaged [ 41 , 42 ]. 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-irradiation [ 40 , 43 ]. 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 pathway [ 38 ]. We further quantified the expression of TWIST1 and TWIST2, genes involved in maintaining BM-MSC stemness [ 44 ]. 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 senescence [ 27 , 45 – 47 ], 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 a 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. After that, 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 all genotypes. However, the GSTT1 null group expressed lower levels of IL-6, a pro-inflammatory cytokine and part of BM-MSC SASP[ 10 , 31 , 48 ] and PDXN, whose elevated levels might contribute to oxidative stress and apoptosis[ 49 ], 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 marker [ 50 ]. 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-MSCs [ 51 , 52 ], 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 secretion. Overall, the absence of the GSTT1 gene in BM-MSC grants these cells with partial resistance to senescence, increasing their proliferative potential and decreasing some of their SASP. Even though a GSTT1 null genotype has been shown to be deleterious in the context of diseases like cancer [ 18 , 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 will conserve their potency even after long-term in vitro 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 when applied in a clinical setting. Further research on GSTT1 genotypes and their implications in BM-MSC phenotype is encouraged. CONCLUSIONS In conclusion, this study demonstrates that GSTT1 null BM-MSCs exhibit an enhanced proliferative capacity 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 4 . This strategy was used to detect homozygous and heterozygous BM-MSC populations as described in Buchard et al. [ 53 ]. 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 1.5 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 o C for 5 min followed by a cycle of 95 o C for 2 min, 60 o C for 30 sec, and 72 o C for 1 min for the last cycle, followed by an additional final extension at 72 o c for 10 min. The amplicons obtained were run on a 0.8% agarose/TBE electrophoresis gel at 50V for 60 min, followed by analyses using Chemidoc (Bio-Rad) gel documentation system. Culture and expansion of mesenchymal stem cells BM-MSCs from healthy male 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% CO 2, 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/cm 2 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/cm 2 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 minutes 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 hours 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. Cell proliferation assay by bromodeoxyuridine incorporation Three representative samples, one from each genotype group (BM-MSC 164 GSTT1 −/− , BM-MSC 310277 GSTT1 +/+ , and BM-MSC 257 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 o C with 5% CO 2 for 24 hours. Then, BM-MSCs were checked for confluency and treated with S-phase DNA-specific primary antibody followed by an additional incubation for 24 hours. Plates were then heated for 1 hour at 60 o 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 minutes followed by measurement of the absorbance at 370 nm for 25, 30, and 35 minutes 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 5 . RT-qPCR was performed in a CFX96 (Bio-Rad) thermal cycler using standard reaction set up that comprised an activation at 95 o C for 2 min, 40 cycles of 95 o C for 1 min and 60 o 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 previously [ 54 ]. Telomere length analysis The relative telomere lengths of BM-MSCs from multiple donors were quantified RT-qPCR using previously established protocols [ 54 ]. 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 6 . The thermal cycler CFX96 (BioRad) set up involved activation at 95 o C for 1 min, followed by 40 cycles at 95 o C (1min), 60 o C (30 s), and 2 min at 54 o 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 o C, 30 sec at 60 o C, and 1 min at 58 o 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 previously [ 54 ]. 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 6 . 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 o C for 2 min, 40 cycles of 95 o C for 1 min and 60 o C for 30 sec) in an CFX96 (BioRad) thermal cycler, followed by melt curve analysis. Data was analyzed using 2 –∆∆Ct as described previously [ 54 ] using RPLP0 as the housekeeping gene. Senescence-associated Beta-galactosidase (SA-b-gal) Staining Representative BM-MSCs from each GSTT1 genotype (BM-MSC 164 −/− , BM-MSC 310277 +/+ , and BM-MSC 257 +/− ) 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 10Gy or were left non-irradiated in preparation for assessing SA-β-gal activity using the senescence-associated b-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 CO 2 ). 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-b-gal activity was assessed in continuously expanded cells until passage 15 to assess in vitro replicative senescence. Stained images were examined under bright field microscopy. Cells stained positively were counted to estimate the percentage of SA-b-gal + cells. Statistical analysis To compare phenotypic measurements and gene expression between the three 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. Declarations DATA AVAILABILITY The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request FUNDING SUPPORT 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, 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. 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. 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Supplementary Files Supplementaltables.pdf Cite Share Download PDF Status: Published Journal Publication published 26 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 19 Sep, 2025 Reviews received at journal 16 Sep, 2025 Reviews received at journal 08 Sep, 2025 Reviewers agreed at journal 22 Aug, 2025 Reviewers agreed at journal 20 Aug, 2025 Reviewers agreed at journal 18 Aug, 2025 Reviews received at journal 04 Aug, 2025 Reviewers agreed at journal 30 Jul, 2025 Reviewers invited by journal 29 Jul, 2025 Editor assigned by journal 29 Jul, 2025 Editor invited by journal 25 Jul, 2025 Submission checks completed at journal 23 Jul, 2025 First submitted to journal 23 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Lowry Barnes","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"C.","middleName":"Lowry","lastName":"Barnes","suffix":""},{"id":493117648,"identity":"c42539be-3e48-47df-9d38-a2603cdb462c","order_by":3,"name":"Rebekah Margaret Samsonraj","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYDADfiBmbIBgIoFkA8laDA4Qq8V82vGHn278sckzvpH87OMMBhvZDQcIaJG5nWMsnduWVmx2I8145gaGNGOCWiSkcxikcxsOJ267kWDM+IDhcCIRWtIf/875czhx84z0z0At/4nRkmAmncMGNFwix5hxA8MBYrTkmFkD/ZI448ybYsYZBsnGM4lx2O2cPzaJ/e3pmxl7Kuxk+whpQQMGpCkfBaNgFIyCUYADAAD8k0Uuj0dr6QAAAABJRU5ErkJggg==","orcid":"","institution":"University of Arkansas","correspondingAuthor":true,"prefix":"","firstName":"Rebekah","middleName":"Margaret","lastName":"Samsonraj","suffix":""}],"badges":[],"createdAt":"2025-07-13 20:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7115326/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7115326/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-37517-8","type":"published","date":"2026-01-26T15:59:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88047722,"identity":"e0a3b4a2-ec87-4956-9fc5-3fd4950128ae","added_by":"auto","created_at":"2025-07-31 18:48:14","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":164786,"visible":true,"origin":"","legend":"\u003cp\u003eLocation of the primers used for genotyping through multiplexing PCR. \u003cstrong\u003eA) \u003c/strong\u003eWhen present, the GSTT1 gene will be amplified using the GSTT1 F and GSTT1 R primers generating a 3.1kb product. The GSTT1 deletion 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 (60kb). \u003cstrong\u003eB)\u003c/strong\u003e Genome location of the GSTT1 gene as well as the primer amplification site for the GSTT1 gene and the deletion of the GSTT1 gene.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7115326/v1/4bacd6b2c554bef635f4b54b.jpeg"},{"id":88047724,"identity":"4645bbaf-c3ad-4d51-8797-fb0e10d5339a","added_by":"auto","created_at":"2025-07-31 18:48:14","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":379153,"visible":true,"origin":"","legend":"\u003cp\u003eGSTT1 genotype of nine bone marrow-derived mesenchymal stem cell (BM-MSC) groups derived from different donors. \u003cstrong\u003eA) \u003c/strong\u003eGel pictures of the amplified product obtained from multiplex PCR. BM-MSC groups 310277, 238, and 198 show a GSTT1 homozygous positive genotype, BM-MSC groups 257, 310280, and 172 show a GSTT1 heterozygous genotype, and BM-MSC groups 164, 00227, and 310264 show a GSTT1 homozygous negative genotype. \u003cstrong\u003eB)\u003c/strong\u003e 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.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7115326/v1/40cf44f4bf59b882f9d16b44.jpeg"},{"id":88048242,"identity":"a9c02aa5-298e-4ec4-9dbc-a7d9c6ac9db0","added_by":"auto","created_at":"2025-07-31 18:56:14","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":312293,"visible":true,"origin":"","legend":"\u003cp\u003eBone marrow-derived mesenchymal stem cell (BM-MSC) proliferative potential. \u003cstrong\u003eA) \u003c/strong\u003e8-day growth curve of nine BM-MSC groups divided into their GSTT1 genotypic groups. GSTT1 homozygous positive BM-MSCs are shown in gray dots, GSTT1 heterozygous BM-MSCs are shown in green triangles, and GSTT1 homozygous negative BM-MSCs are shown in orange squares. The average number of cells after nine cell counts is shown in the graph. Significance was achieved through an unpaired two-tail t-test. \u003cstrong\u003eB) \u003c/strong\u003eBar graphs depicting the average absorbance readings at 370nm 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.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7115326/v1/2374a1d18939df56c65cf304.jpeg"},{"id":88048362,"identity":"cc06f98d-512e-40ce-9ca9-3e7654f3455b","added_by":"auto","created_at":"2025-07-31 19:04:14","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":694680,"visible":true,"origin":"","legend":"\u003cp\u003eTelomere length and telomerase reverse transcriptase (hTERT) expression of bone marrow-derived mesenchymal stem cells (BM-MSCs) isolated from nine different donors. \u003cstrong\u003eA) \u003c/strong\u003eRelative 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. \u003cstrong\u003eB)\u003c/strong\u003e 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.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7115326/v1/3377d2591b4fd2b754ae400d.jpeg"},{"id":88047727,"identity":"f6cacae6-7282-407c-aaad-2128cdd292bb","added_by":"auto","created_at":"2025-07-31 18:48:14","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":476937,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression of senescence-associated genes throughout long-term culture. p14ARF, p16INK4A, p21 Waf1, γH2AX, and TP53 were quantified at each passage in nine different bone-marrow derived mesenchymal stem cell (BM-MSCs) groups isolated from different donors. BM-MSCs are grouped by their genotype as GSTT1 homozygous positive (GSTT1 +/+), GSTT1 heterozygous (GSTT1 +/-), and GSTT1 homozygous 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.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7115326/v1/c7c165e2f8d26fbdfbb082ed.jpeg"},{"id":88048243,"identity":"a1fe2c3e-a8bc-4a43-b0e0-b13a025f18f9","added_by":"auto","created_at":"2025-07-31 18:56:14","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":474673,"visible":true,"origin":"","legend":"\u003cp\u003eTWIST1/2 and\u003cstrong\u003e \u003c/strong\u003esenescence-associated β galactosidase (SA-β-gal) expression in\u003cstrong\u003e \u003c/strong\u003ebone marrow-derived mesenchymal stem cells (BM-MSCs) during and after expansion. \u003cstrong\u003eA) \u003c/strong\u003eTWIST1 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 homozygous positive (GSTT1 +/+), GSTT1 heterozygous (GSTT1 +/-), and GSTT1 homozygous negative (GSTT1 -/-). \u003cstrong\u003eB) \u003c/strong\u003eSA-β-gal staining from three selected BM-MSC groups, one from each genotype. \u003cstrong\u003eC) \u003c/strong\u003eBar 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=3 for each group.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7115326/v1/78b0541e528279f84bd30849.jpeg"},{"id":88047736,"identity":"3de39291-4469-4d4b-bc28-3f90c779ceb8","added_by":"auto","created_at":"2025-07-31 18:48:15","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":490789,"visible":true,"origin":"","legend":"\u003cp\u003eGene 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. \u003cstrong\u003eA) \u003c/strong\u003eGraphs depicting the average gene expression every two passages from passage 3/5 to passage 15 of three selected BM-MSC groups, one from each genotype. \u003cstrong\u003eB) \u003c/strong\u003eBar 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.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7115326/v1/97b9eef7167259fe28c8b235.jpeg"},{"id":88047730,"identity":"f7ecb99e-26ae-4fc6-87e8-9d6d0f69dab1","added_by":"auto","created_at":"2025-07-31 18:48:14","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":615885,"visible":true,"origin":"","legend":"\u003cp\u003eSenescence-associated β galactosidase (SA-β-gal) staining and senescence-associated gene expression after irradiation of bone marrow-derived mesenchymal stem cells (BM-MSCs).\u003cstrong\u003eA) \u003c/strong\u003eSA-β-gal expression of BM-MSCs without irradiation (CTRL) and after experiencing 5 and 10 GY of irradiation from three selected BM-MSC groups, one from each genotype. \u003cstrong\u003eB) \u003c/strong\u003eBar graphs depicting the average number of SA-β-gal positive cells over the total number of cells. \u003cstrong\u003eC) \u003c/strong\u003eBar graphs depicting the average gene expression of 53BP1, γH2AX, p14ARF, p16INK4A, p21 Waf1, TP53, TWIST1, and TWIST2 without irradiation (CTRL) and after 10 GY of irradiation exposure. Gene expressions were normalized to their respective RPLP0 expressions. Data analysis was performed using a one-way ANOVA, n=3 for each group.\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7115326/v1/ffcc17524fb30ac1974ae1e1.jpeg"},{"id":101690574,"identity":"5912bacd-2570-485e-a6ef-935147018f85","added_by":"auto","created_at":"2026-02-02 16:05:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4714454,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7115326/v1/35eca829-167d-40cb-a288-70fc11e3dab1.pdf"},{"id":88048239,"identity":"c3bd72c8-4ce5-44c2-b710-45d2fe53003f","added_by":"auto","created_at":"2025-07-31 18:56:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":210098,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaltables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7115326/v1/ec4d8ddc6aab475974d98579.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Validation of a Novel Genomic Biomarker of Mesenchymal Stem Cell Scalability and Implications of Genotype Status on Cellular Senescence Phenotypes","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAlthough bone marrow-derived mesenchymal stem cells (BM-MSCs) have great potential for treating multiple diseases [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], the lack of consistent and/or effective treatment outcomes poses a significant challenge for their clinical translation [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Multiple factors can play a role in hindering the therapeutic potential of BM-MSCs including the process of ex vivo expansion, which, although necessary for obtaining a clinically relevant number of cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], pushes cells towards senescence - a state or irreversible growth arrest - altering their phenotype and lowering their therapeutic potential [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Furthermore, it has been observed that donor-donor heterogeneity plays a critical role in dictating the potency and the onset of senescence in BM-MSCs [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], suggesting the existence of intrinsic inter donor differences that provide resistance to senescence. However, to date, these donor-donor differences are poorly understood, limiting the possibility of utilizing BM-MSC screening methods to improve BM-MSC therapies.\u003c/p\u003e\u003cp\u003eThe GSTT1 gene, a member of the glutathione S-transferase superfamily which is not present in about 14\u0026ndash;30% of the population\u0026rsquo;s genome [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], 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\u0026rsquo;s disease, and have a higher mortality rate due to COVID-19 [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. 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 senescence [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. 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 genotypes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] incentivizing more research on these somewhat contradicting but important results.\u003c/p\u003e\u003cp\u003eThis study aims to investigate the effect of the GSTT1 polymorphism (homozygous positive, heterozygous, or homozygous negative) on BM-MSC growth and senescence. First, the genomic status of nine 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 the onset of cell senescence.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003e1.\u003c/b\u003e \u003cb\u003eGenotyping allowed BM-MSCs grouping into three categories determined by their GSTT1 status\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBM-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 1.5 kb long, whereas the second primer amplified the region flanking the GSTT1 gene, with an approximate size of 3.1 kb. The location in the genome of the forward and reverse primers used are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003ePanels A-B.\u003c/b\u003e Genotyping allowed the grouping of BM-MSCs into three categories: GSTT1\u003csup\u003e+/+\u003c/sup\u003e (homozygous positive), GSTT1\u003csup\u003e+/\u0026minus;\u003c/sup\u003e (heterozygous) and GSTT1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (homozygous null). BM-MSCs 310277,198, and 238 were identified to be GSTT1\u003csup\u003e+/+\u003c/sup\u003e; BM-MSCs 310280, 257, and 172 were observed to have a GSTT1\u003csup\u003e+/\u0026minus;\u003c/sup\u003e genotype, and BM-MSCs 164, 310264, and 00227 were determined to be GSTT1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e. The genotyping results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cb\u003ePanel A\u003c/b\u003e. Additionally, mRNA quantification showed that the homozygous null BM-MSCs showed no expression of GSTT1 mRNA whereas the heterozygous and homozygous positive groups showed positive expression of this gene as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cb\u003ePanel B.\u003c/b\u003e Nevertheless, it was noted that GSTT1 gene expression was independent of passage number. While some BM-MSC groups showed higher expression in later passages (BM-MSC 172, BM-MSC 280, BM-MSC 238 and BM-MSC 310277), other donors showed higher expression in earlier passages (BM-MSC 257 and BM-MSC 198). No significant differences in GSTT1 gene expression were observed between the homozygous positive and heterozygous samples.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.\u003c/b\u003e \u003cb\u003eGSTT1 null genotype is predictive of higher proliferative potential in BM-MSCs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe 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 the other two genotypes as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003ePanel A\u003c/b\u003e. At day 8, the GSTT1 null groups had significantly more cells than BM-MSCs from the other two groups. This difference was observed mainly in BM-MSCs 164 and 00227. The compact letter display for significant differences is shown in \u003cb\u003eSupplemental Table\u0026nbsp;1.\u003c/b\u003e 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\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e genotype showed significantly higher proliferation capacity compared to GSTT1\u003csup\u003e+/+\u003c/sup\u003e and GSTT1\u003csup\u003e+/\u0026minus;\u003c/sup\u003e BM-MSCs at both young passages (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003ePanel B\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.\u003c/b\u003e \u003cb\u003eBM-MSC telomere length attrition and hTERT expression are not related to GSTT1 genotype\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cb\u003ePanel A\u003c/b\u003e. 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. From the tested groups, BM-MSC 172 showed the slowest shortening rate, with a slope of -0.00038 while BM-MSC 164 showed the fastest shortening, with a slope of -0.065.\u003c/p\u003e\u003cp\u003eAdditionally, this study analyzed the expression of the hTERT gene. hTERT or human Telomere Retro 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cb\u003ePanel B\u003c/b\u003e. 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.\u003c/p\u003e\u003cp\u003e\u003cb\u003e4. GSTT1 genotype status affects the expression of p21 Waf1 and p14ARF but not the expression of p16INK4A, γH2AX and TP53\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo 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 homozygous positive, heterozygous, and homozygous negative BM-MSC groups as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The results show that GSTT1 homozygous positive BM-MSCs 310277, 238, and 198 showed a fluctuating but high expression of p14ARF when compared to most of the heterozygous or homozygous negative samples. Two of the three GSTT1 heterozygous samples (310280 and 172) and homozygous negative samples (164 and 00227) had low expression of p14ARF even at high passages. However, GSTT1 heterozygous BM-MSC 257 and GSTT1 homozygous negative BM-MSC 310264 showed a fluctuating but relatively high expression of p14ARF at young passages. Furthermore, p16INK4A showed a mostly uniform and low expression at young passages regardless of the GSTT1 genotype. It increased with passages in most BM-MSC groups, seeing mostly non-significant increases at passages 13\u0026ndash;15 except for the homozygous positive 238 group, in which p16INK4A expression increased significantly from P13-P15 (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOur results showed that p21 Waf1 expression increased with passage and showed the greatest but mostly non-significant differences between genotypic groups. Homozygous positive BM-MSC groups 238 and 198 showed the greatest p21 Waf1 expression at higher passages followed by homozygous positive 310277 and heterozygous 257 groups. Nevertheless, heterozygous 172 had the lowest p21 Waf1 expression even at high passages followed by homozygous negative 164. Then, γH2AX expression showed high variation between passages with no specific pattern or clear relation to genotype. TP53 showed the highest expression in heterozygous 310280 followed by homozygous negative 00227. Heterozygous 172 showed the lowest expression of TP53 followed by homozygous positive 238. However, a clear relationship between genotype and TP53 was not detected. The compact letter display that indicates significant differences between groups at each passage is shown in \u003cb\u003eSupplemental Table\u0026nbsp;2\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003e5. GSTT1 genotype does not influence TWIST1/2 expression\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn addition to the senescence-related genes quantified in this study, genes related to cell proliferation were analyzed, namely TWIST1 and TWIST2. The results from this study showed that TWIST1 expression varied at each passage with mostly non-significant differences between GSTT1 genotypes. Interestingly, homozygous negative 164 had the highest expression of TWIST1, particularly at passage 3 and 11. On the other hand, TWIST2 showed low expression in most samples except for the GSTT1 heterozygous BM-MSC 310280 which had significantly higher expression compared to the other samples as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cb\u003ePanel A\u003c/b\u003e. The compact letter display that indicates significant differences between groups at each passage is shown in \u003cb\u003eSupplemental Table\u0026nbsp;3\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e6. GSTT1 null BM-MSCs have the lowest percentage of senescent cells after long-term in vitro culture\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eTo quantify the percentage of senescent cells, SA-β-gal staining was performed in cells after long-term in vitro culture (passage 15) in three 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 homozygous positive (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and heterozygous (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) BM-MSCs as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cb\u003ePanel B\u003c/b\u003e. Moreover, no significant differences in SA-β-gal expression between the homozygous positive and heterozygous groups were observed.\u003c/p\u003e\u003cp\u003e\u003cb\u003e7. GSTT1 genotype has a significant effect on the expression of senescence-associated genes at higher passages\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn 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. IL-6 expression increases with increasing passage while EZH2 expression decreases with increasing passage in all three GSTT1 genotypes. However, the expression of both IL-6 and EZH2 is significantly higher in the homozygous positive BM-MSCs compared to the homozygous negative BM-MSCs when they reached higher passages. It is to be noted that, at low passages, the GSTT1 homozygous positive 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.\u003c/p\u003e\u003cp\u003eThen, SOD1 expression increases with passage in all BM-MSC groups. However, it significantly increases only in the GSTT1 homozygous positive group. Additionally, SOD1 expression at high passages in the GSTT1 homozygous positive group is significantly higher than its expression in the other two BM-MSC groups. 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 at high passages was significantly higher in the GSTT1 negative group when compared to the other two BM-MSC groups. Moreover, PDXN expression was significantly higher in GSTT1 homozygous positive BM-MSCs when compared to the other BM-MSC groups at high passages.\u003c/p\u003e\u003cp\u003e\u003cb\u003e8. BM-MSCs with a GSTT1 null genotype are resistant to some of the phenotypic changes produced by irradiation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBesides 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 was quantified.\u003c/p\u003e\u003cp\u003eOur results show that, with irradiation, the percentage of senescent cells increases in all BM-MSC groups as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, \u003cb\u003ePanel A\u003c/b\u003e. However, the percentage of senescent cells is significantly lower in the GSTT1 null group compared to the other two groups.\u003c/p\u003e\u003cp\u003eAdditionally, gene expression results after irradiation show some interesting patterns as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, \u003cb\u003ePanel B.\u003c/b\u003e The expressions of 53BP1 and p21 Waf1 significantly increase in the heterozygous and homozygous positive groups 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 only when compared to the GSTT1 homozygous positive 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 homozygous negative group as no significant changes are observed in the other two genotypic groups.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn vitro expansion of BM-MSCs is necessary to obtain the necessary number of cells for clinical use [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, extended culture often leads to cellular senescence, or a state of irreversible growth arrest while cells remain metabolically active [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This phenomenon negatively affects BM-MSCs potency, leading to a diminished proliferative potential, decreased multipotentiality, and a modified secretome as part of their senescence-associated secretory phenotype (SASP)[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Notably, different groups have reported BM-MSCs populations with high proliferative potential and sustained phenotype despite prolonged culture [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. 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.\u003c/p\u003e\u003cp\u003ePreviously, the GSTT1 gene, present in 14\u0026ndash;30% of the population[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], 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 counterparts [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. 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 senescence and some of the changes associated with it. Consistent with earlier reports, we observed that cells with a GSTT1 null genotype proliferate faster than BM-MSCs with GSTT1 positive genotypes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Furthermore, SA-β-gal expression, a well acknowledged marker of senescence [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], 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 other two groups, implying a partial resistance to senescence.\u003c/p\u003e\u003cp\u003eGiven the established link between telomere shortening and senescence [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], 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 groups [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], 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.\u003c/p\u003e\u003cp\u003eWe 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 irradiation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. A similar trend was observed in most of the BM-MSC groups during this study. However, the 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 that percentage of cells undergoing cell cycle arrest, thus lowering the percentage of senescent cells in culture.\u003c/p\u003e\u003cp\u003eSimilarly, p14ARF expression was generally higher in the GSTT1 homozygous positive BM-MSC groups even at earlier passages during expansion, except for the GSTT1 null 310264 and GSTT1 heterozygous 257, indicating that other variables beyond GSTT1 genotype may influence its regulation. Notably, p14ARF 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 transcription [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Interestingly, this study observed a decrease in p14ARF expression with passage in some BM-MSC groups, a trend that has been recorded previously [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] 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)[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], TP53, the gene responsible for coding this protein, did not seem to be related to GSTT1 genotype during long-term culture or irradiation.\u003c/p\u003e\u003cp\u003eTo 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 senescence [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. γH2AX and 53BP1 are typically upregulated when BM-MSCs are exposed to irradiation [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. 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 passaged [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. 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-irradiation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. 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 pathway [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe further quantified the expression of TWIST1 and TWIST2, genes involved in maintaining BM-MSC stemness [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. 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 senescence [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], 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 a 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.\u003c/p\u003e\u003cp\u003eAfter that, 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 all genotypes. However, the GSTT1 null group expressed lower levels of IL-6, a pro-inflammatory cytokine and part of BM-MSC SASP[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and PDXN, whose elevated levels might contribute to oxidative stress and apoptosis[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], 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 marker [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. 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-MSCs [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], 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 secretion.\u003c/p\u003e\u003cp\u003eOverall, the absence of the GSTT1 gene in BM-MSC grants these cells with partial resistance to senescence, increasing their proliferative potential and decreasing some of their SASP. Even though a GSTT1 null genotype has been shown to be deleterious in the context of diseases like cancer [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], 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 will conserve their potency even after long-term in vitro 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 when applied in a clinical setting. Further research on GSTT1 genotypes and their implications in BM-MSC phenotype is encouraged.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eIn conclusion, this study demonstrates that GSTT1 null BM-MSCs exhibit an enhanced proliferative capacity 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.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cb\u003eGenotyping\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor genotyping, BM-MSC genomic DNA was isolated from 80% confluent T75 flasks using DNeasy Blood \u0026amp; Tissue Kit (Qiagen, cat.no. 69504) following manufacturer\u0026rsquo;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 \u0026micro;L of elution buffer and quantified using a NanoPhotometer\u0026reg; 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 \u003cb\u003eSupplemental Table\u0026nbsp;4\u003c/b\u003e. This strategy was used to detect homozygous and heterozygous BM-MSC populations as described in Buchard \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. 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 1.5 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\u003csup\u003eo\u003c/sup\u003eC for 5 min followed by a cycle of 95\u003csup\u003eo\u003c/sup\u003eC for 2 min, 60\u003csup\u003eo\u003c/sup\u003eC for 30 sec, and 72\u003csup\u003eo\u003c/sup\u003eC for 1 min for the last cycle, followed by an additional final extension at 72\u003csup\u003eo\u003c/sup\u003e c for 10 min. The amplicons obtained were run on a 0.8% agarose/TBE electrophoresis gel at 50V for 60 min, followed by analyses using Chemidoc (Bio-Rad) gel documentation system.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCulture and expansion of mesenchymal stem cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBM-MSCs from healthy male individual donors between the age of 20\u0026ndash;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% CO\u003csub\u003e2,\u003c/sub\u003e and 37\u0026deg;C in sterile incubators and continuously expanded from passage 4\u0026ndash;5 until passage 15. For expansion, BM-MSCs were harvested using TrypLE\u0026trade; (Thermo Fisher Scientific, cat.no. 12605010) once they reached 80% confluency. Then, they were reseeded at a density of 5,000 cells/cm\u003csup\u003e2\u003c/sup\u003e into new T75 flasks. At each passage, a fraction of cells was collected for DNA and RNA analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIrradiation of BM-MSCs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBM-MSCs from each genotype were seeded at a density of 5,000 cells/cm\u003csup\u003e2\u003c/sup\u003e 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 minutes 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 hours 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.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell proliferation assay by bromodeoxyuridine incorporation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThree representative samples, one from each genotype group (BM-MSC 164 GSTT1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, BM-MSC 310277 GSTT1\u003csup\u003e+/+\u003c/sup\u003e, and BM-MSC 257 GSTT1\u003csup\u003e+/\u0026minus;\u003c/sup\u003e) were chosen to perform a bromodeoxyuridine (BrDU) assay. BM-MSCs at low passages (\u0026lt;\u0026thinsp;P6) or high passages (P12\u0026lt;) 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\u0026rsquo;s instructions. Briefly, cells were incubated at 37\u003csup\u003eo\u003c/sup\u003eC with 5% CO\u003csup\u003e2\u003c/sup\u003e for 24 hours. Then, BM-MSCs were checked for confluency and treated with S-phase DNA-specific primary antibody followed by an additional incubation for 24 hours. Plates were then heated for 1 hour at 60\u003csup\u003eo\u003c/sup\u003e C using a hot plate. After that, 200 \u0026micro;L 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 \u0026micro;L of 1X PBS. Finally, chromogenic substrate was added and incubated at room temperature for 25 minutes followed by measurement of the absorbance at 370 nm for 25, 30, and 35 minutes of incubation corresponding to BrDU activity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eReal-time quantitative polymerase chain reaction\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA from individual cell cultures were isolated using RNeasy Miniprep kit (Qiagen, cat.no. 74104) following manufacturer\u0026rsquo;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\u0026reg; N60 (IMPLEN). cDNA synthesis was performed using 500 ng mRNA using SuperScript\u0026trade; VILO\u0026trade; cDNA Synthesis Kit (Invitrogen, cat.no. 11754250) following manufacturer\u0026rsquo;s protocol. The cDNA was diluted to 50 ng/\u0026micro;L 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 \u003cb\u003eSupplemental Table\u0026nbsp;5\u003c/b\u003e. RT-qPCR was performed in a CFX96 (Bio-Rad) thermal cycler using standard reaction set up that comprised an activation at 95\u003csup\u003eo\u003c/sup\u003e C for 2 min, 40 cycles of 95\u003csup\u003eo\u003c/sup\u003eC for 1 min and 60\u003csup\u003eo\u003c/sup\u003eC 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\u003csup\u003e\u0026ndash;∆∆Ct\u003c/sup\u003e) as described previously [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eTelomere length analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe relative telomere lengths of BM-MSCs from multiple donors were quantified RT-qPCR using previously established protocols [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Genomic DNA of BM-MSCs at multiple passages was isolated using the DNeasy Blood \u0026amp; Tissue Kit (Qiagen, cat.no. 69504) following manufacturer\u0026rsquo;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 \u003cb\u003eSupplemental Table\u0026nbsp;6\u003c/b\u003e. The thermal cycler CFX96 (BioRad) set up involved activation at 95\u003csup\u003eo\u003c/sup\u003eC for 1 min, followed by 40 cycles at 95\u003csup\u003eo\u003c/sup\u003eC (1min), 60\u003csup\u003eo\u003c/sup\u003eC (30 s), and 2 min at 54\u003csup\u003eo\u003c/sup\u003eC. 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\u003csup\u003eo\u003c/sup\u003eC, 30 sec at 60\u003csup\u003eo\u003c/sup\u003eC, and 1 min at 58\u003csup\u003eo\u003c/sup\u003eC. 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 \u003cem\u003eTel\u003c/em\u003e primers and \u003cem\u003e36B4\u003c/em\u003e 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\u003csup\u003e\u0026ndash;∆∆Ct\u003c/sup\u003e as described previously [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003ehTERT gene expression analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe 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 \u003cem\u003ehTERT\u003c/em\u003e gene are provided in \u003cb\u003eSupplemental Table\u0026nbsp;6\u003c/b\u003e. 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\u003csup\u003eo\u003c/sup\u003eC for 2 min, 40 cycles of 95\u003csup\u003eo\u003c/sup\u003eC for 1 min and 60\u003csup\u003eo\u003c/sup\u003eC for 30 sec) in an CFX96 (BioRad) thermal cycler, followed by melt curve analysis. Data was analyzed using 2\u003csup\u003e\u0026ndash;∆∆Ct\u003c/sup\u003e as described previously [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] using RPLP0 as the housekeeping gene.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSenescence-associated Beta-galactosidase (SA-b-gal) Staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRepresentative BM-MSCs from each GSTT1 genotype (BM-MSC 164\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, BM-MSC 310277\u003csup\u003e+/+\u003c/sup\u003e, and BM-MSC 257\u003csup\u003e+/\u0026minus;\u003c/sup\u003e) 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 10Gy or were left non-irradiated in preparation for assessing SA-β-gal activity using the senescence-associated b-galactosidase staining kit (Cell Signaling, cat.no. 9860) following manufacturer\u0026rsquo;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\u0026deg;C overnight in a dry incubator (no CO\u003csub\u003e2\u003c/sub\u003e). 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-b-gal activity was assessed in continuously expanded cells until passage 15 to assess in vitro replicative senescence. Stained images were examined under bright field microscopy. Cells stained positively were counted to estimate the percentage of \u003cem\u003eSA-b-gal\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e cells.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eTo compare phenotypic measurements and gene expression between the three genotypes, one-way ANOVA, two-way ANOVA, and \u003cem\u003et\u003c/em\u003e-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\u0026thinsp;\u0026le;\u0026thinsp;0.05, ** for a p-value\u0026thinsp;\u0026le;\u0026thinsp;0.01, *** for a p-value\u0026thinsp;\u0026le;\u0026thinsp;0.001, and **** for a p-value\u0026thinsp;\u0026le;\u0026thinsp;0.0001.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING SUPPORT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe 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, University of Arkansas Women\u0026apos;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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI.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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADDITIONAL INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eV. V. 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Cool, \u0026quot;Telomere length analysis of human mesenchymal stem cells by quantitative PCR,\u0026quot; \u003cem\u003eGene, \u003c/em\u003evol. 519, no. 2, pp. 348-355, 2013, doi: 10.1016/j.gene.2013.01.039.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mesenchymal stem cells, Senescence, Biomarker, GSTT1 polymorphism, Cell expansion, Cell therapies","lastPublishedDoi":"10.21203/rs.3.rs-7115326/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7115326/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEx 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. First, we identified the GSTT1 genotype (either homozygous positive, heterozygous, or homozygous negative) of nine BM-MSC groups. Then, we performed long-term in vitro culture and exposed cells to irradiation as senescence models. After that, their proliferative potential, their SASP, and the expression of key genes were investigated. The results show that GSTT1 null BM-MSCs have a higher proliferative potential and exhibit fewer senescent cells in culture when compared to the other genotypes. Additionally, these cells exhibit a lower expression of p21 Waf1, p14ARF, IL-6, PDXN, and 53BP1 and a higher expression of TWIST1 and ACTA2 genes, especially at low passages. A GSTT1 null genotype can serve as a potential biomarker to identify BM-MSC populations with higher resistance to senesce.\u003c/p\u003e","manuscriptTitle":"Validation of a Novel Genomic Biomarker of Mesenchymal Stem Cell Scalability and Implications of Genotype Status on Cellular Senescence Phenotypes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-31 18:48:09","doi":"10.21203/rs.3.rs-7115326/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-19T17:48:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-16T16:15:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-08T13:16:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81402892202014185130827607851476230900","date":"2025-08-22T09:02:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"205318565510146228015037045671062255842","date":"2025-08-20T13:18:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"244753695571017805473799667016621472525","date":"2025-08-18T15:41:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-04T15:03:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"132850159449199479905488470316703325004","date":"2025-07-30T10:40:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-29T10:34:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-29T10:20:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-25T11:14:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-23T13:24:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-23T13:20:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"25472a7f-8978-45f7-a82c-8773c46f77c5","owner":[],"postedDate":"July 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52362571,"name":"Biological sciences/Cell biology"},{"id":52362572,"name":"Biological sciences/Genetics"},{"id":52362573,"name":"Biological sciences/Molecular biology"},{"id":52362574,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-02-02T16:02:24+00:00","versionOfRecord":{"articleIdentity":"rs-7115326","link":"https://doi.org/10.1038/s41598-026-37517-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-26 15:59:25","publishedOnDateReadable":"January 26th, 2026"},"versionCreatedAt":"2025-07-31 18:48:09","video":"","vorDoi":"10.1038/s41598-026-37517-8","vorDoiUrl":"https://doi.org/10.1038/s41598-026-37517-8","workflowStages":[]},"version":"v1","identity":"rs-7115326","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7115326","identity":"rs-7115326","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}