Characterization and validation of long-term cultured TERT-immortalized human Wharton’s jelly–derived mesenchymal stromal cells

Characterization and validation of long-term cultured TERT-immortalized human Wharton’s jelly–derived mesenchymal stromal cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Characterization and validation of long-term cultured TERT-immortalized human Wharton’s jelly–derived mesenchymal stromal cells Young Eun Kim, So Yoon Ahn, Yeon Ju Lee, Sein Hwang, Yun Sil Chang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8322013/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Apr, 2026 Read the published version in Stem Cell Reviews and Reports → Version 1 posted 7 You are reading this latest preprint version Abstract Mesenchymal stromal cells (MSCs) have demonstrated efficacy and feasibility in numerous preclinical and clinical studies. However, their application is limited at early passages owing to replicative senescence. This study investigated whether MSCs immortalized via human telomerase reverse transcriptase (TERT) overexpression (TERT-MSCs) maintain their characteristics and efficacy after extended passaging. Human Wharton’s jelly-derived MSCs at passage (P) 6 were immortalized by retroviral transfection with human TERT. TERT-MSCs at mid (P13) and late (P30) passages were analyzed. Their morphology, differentiation potential, and surface marker expression were preserved throughout extended passaging. Single-cell RNA sequencing reconfirmed the characteristics of TERT-MSCs and compared their gene expression profiles with those of P8 parental (control) cells. As passage number increased, control MSCs exhibited gradual declines in TERT expression, telomere length, and cytoprotective effects, whereas both P13 and P30 TERT-MSCs retained these properties. Expression levels of paracrine-related genes and enriched Gene Ontology terms in extended-passage TERT-MSCs were comparable to those of control MSCs. These findings suggest that TERT-MSCs maintain the key characteristics and therapeutic potential of early-passage MSCs through extended culture, offering a promising approach to overcome the limitations of early-passage MSCs in clinical applications. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Mesenchymal stromal/stem cells (MSCs) have been extensively investigated for more than a decade. MSCs are a subset of cells that naturally reside in various tissues, including cord blood, adipose tissue, and bone marrow. These cells play critical roles in modulating surrounding cells, regulating immune responses, repairing damaged tissues, and maintaining homeostasis in the body. Numerous studies have demonstrated the anti-inflammatory, anti-apoptotic, and cytoprotective effects of MSCs in both preclinical and clinical studies targeting various incurable diseases [ 1 ]. Despite their therapeutic potential, MSCs are present in low quantities within their native niches, and their functionality is highly donor-dependent. Although MSCs can be easily expanded in vitro, their use is generally recommended within approximately six passages, ideally below ten passages, to preserve therapeutic potential. Later passages are typically considered unsuitable for clinical applications due to decreased efficacy and increased risk of cellular dysfunction resulting from replicative senescence [ 2 ]. Previous studies have also reported that MSCs undergo age-related alterations in protein profiles, including reduced levels of immunomodulatory [ 3 ], pro-angiogenic [ 4 ], and cytoskeletal proteins essential for migration [ 5 ]. Therefore, a new conceptual approach is required to address these limitations. In general, telomere length progressively shortens and cell doubling time increases following population division [ 6 ]. Over the past decade, it has been reported that introducing the telomere reverse transcriptase (TERT) gene can immortalize cells, and the technique for establishing immortalized cell lines is not new. However, only a few studies have compared the functional and phenotypic differences in MSCs before and after TERT-induced immortalization during extended passaging. For MSC-based therapies, such as those using MSC-derived extracellular vesicles (EVs), the initially limited MSC population must be expanded to a large scale, and minimizing variability across different passage stages is critically important. Immortalized MSC-based therapy may offer a promising strategy for exploring the feasibility of large-scale manufacturing and ensuring consistent product consistency. In this study, we generated TERT-induced immortalized MSCs at passage (P) 6, cultured them until P30, and analyzed them under three conditions: un-immortalized early-passage control (CTRL) (P8), mid-passage post-immortalization (P13), and late-passage post-immortalization (P30) MSCs. We aimed to determine whether MSCs retained their cytoprotective and anti-inflammatory properties upto P30 in an in-vitro cellular stress model and employed single-cell RNA sequencing (scRNA-seq) to characterize the associated transcriptional changes. Specifically, we examined whether the proportions of distinct cell clusters were altered, which clusters were affected by TERT-induced immortalization, and what molecular features defined these altered clusters. Materials and methods MSC isolation and TERT-transfection MSCs were provided by the Samsung Medical Center GMP facility under standard quality controls, including mycoplasma and sterility testing; these cells were isolated from human Wharton’s jelly obtained from a single healthy donor (female, term delivery) and used exclusively for research under Institutional Review Board approval (IRB No: 2016-07-102-047) with written informed consent. The human TERT (h TERT ) gene was overexpressed by transducing MSCs with the pBabe-puro-hTERT retroviral vector (Addgene plasmid #1771) at P6. Viral particles were produced in 293T cells via co-transfection of pBabe-puro-hTERT with the packaging plasmids pCMV-GAG-pol and pCMV-VSV-G. MSCs were then transduced with the retroviral particles carrying the hTERT expression cassette. Successfully transduced MSCs (TERT-MSCs) were selected using puromycin (1 µg/mL; Thermo Fisher Scientific, Waltham, MA, USA), resulting in the formation of resistant colonies. Individual colonies were picked using a micropipette tip, transferred to separate cell culture dishes, and expanded in minimum essential medium alpha (MEMα; Gibco, CA, USA) supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France) and 1% gentamicin (Gibco). MSCs were seeded at 3–4 × 10⁴ cells per 100-mm culture dish and maintained under standard culture conditions, with serial passaging performed continuously until cells reached P30. No morphological alterations were observed after transduction (Figure S1 A). Short tandem repeat genotyping using a 3730xl DNA Analyzer confirmed complete (100%) genetic concordance between TERT-MSCs and parental cells through P30 (Figure S1 B). MSC characterization Cell morphology—characterized by spindle-shaped, adherent growth—was monitored daily using phase-contrast microscopy (Eclipse Ts2, Nikon, Tokyo, Japan) throughout the experimental period. Positive expression of CD90, CD105, and CD73, and negative expression of CD14, CD45, and HLA-DR were confirmed via flow cytometry (BD FACS Verse, BD Biosciences, NJ, USA). The differentiation potential into three lineages (osteogenic, adipogenic, and chondrogenic) was verified after three weeks of induction using the StemPro® Osteogenesis, Adipogenesis, and Chondrogenesis Differentiation Kits (Gibco). Differentiated osteocytes, adipocytes, and chondrocytes were fixed with 4% paraformaldehyde, stained with Alizarin Red S, Oil-Red O, and Alcian Blue, respectively, and examined microscopically. Enzyme-linked immunosorbent assay (ELISA) TERT protein levels in cell lysates and conditioned media were quantified using a commercial ELISA kit (MyBioSource, San Diego, CA, USA). Levels of brain-derived neurotrophic facor (BDNF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) were determined using commercial ELISA kits (R&D Systems, Minneapolis, MN, USA), following the manufacturers’ protocols. Each independently seeded well was treated as an experimental unit, representing an independent biological replicate. Quantification of TERT mRNA expression and telomere length via real-time quantitative PCR (qPCR) To quantify TERT mRNA expression, total RNA was extracted using the Xenopure Total RNA Purification Kit (Xenohelix, Incheon, Republic of Korea), and complementary DNA (cDNA) was synthesized from using the Primescript RT reagent kit (Takara, Shiga, Japan) according to the manufacturer’s protocols. qPCR was performed using the AccuPower® 2X Greenstar qPCR Mater Mix (Bioneer, Daejeon, Republic of Korea). The following primer sequences were used for h TERT : forward 5′-ACTTTGTCAAGGTGGATGTGACGG-3′ and reverse 5′-AAGAAATCATCCACCAAACGCAGG-3′. The human β-actin gene was used as a housekeeping gene with the following primers: forward 5′-CCTGCTTGCTGATCCACATC-3′ and reverse 5′-CATCCGCAAAGACCTGTACG-3′. Relative telomere length was determined using a qPCR-based method as previously described [ 7 , 8 ]. Briefly, genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. qPCR was performed using the AccuPower® 2X Greenstar qPCR Mater Mix (Bioneer) with the following primer sequences for telomeres: forward 5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3′ and reverse 5′- GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3′. The single-copy gene 36B4F was used as a reference, with primers: forward 5′- CAGCAAGTGGGAAGGTGTAATCC-3′ and reverse 5′- CCCATTCTATCATCAACGGGTACAA-3′. The relative telomere length was expressed as the ratio of telomere repeat copy number to that of the single-copy reference gene. All qPCR reactions were performed on the Applied Biosystems QuantStudio 6 Flex Real-Time PCR System (Invitrogen). Each independently seeded well was treated as an experimental unit, representing an independent biological replicate. In vitro efficacy study To establish an oxidative stress-induced cell death model, L2 pulmonary epithelial cells were seeded one day before the experiment. Upon reaching 80–90% confluence, cells were exposed to H₂O₂ (100 µM; Sigma-Aldrich) to induce oxidative stress. CTRL MSCs or TERT-MSCs were added at an MSC:L2 ratio of 1:10. In the MSC-untreated groups (normal control and H₂O₂-only control), an equivalent number of L2 cells was added to equalize total cell numbers across groups. Cell viability was measured using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan), according to the manufacturer’s instructions. To establish an inflammation model, RAW264.7 alveolar macrophages and BV2 microglia were seeded one day before the experiment. At 60–70% confluence, cells were exposed to LPS (0.1 µg/ml; Sigma-Aldrich) or thrombin (40 U; Reyon Pharmaceutical Co., Ltd., Seoul, Korea). CTRL MSCs or TERT-MSCs were then added at a ratio of 1:10 relative to the total number of RAW264.7 or BV2 cells. After 24 h, conditioned media were collected, and inflammatory cytokine concentrations were measured using ELISA kits (R&D Systems, Minneapolis, MN, USA). Each independently seeded well was treated as an experimental unit, representing an independent biological replicate. scRNA-seq and data analysis scRNA-seq was performed on TERT-MSCs, following retroviral transduction and stabilization. TERT-MSCs at P13 were used to represent mid-passage TERT-MSCs, and those at P30 were used as late-passage cells. Untransduced MSCs at P8 were used as the controls. Following reverse transcription and cDNA amplification, scRNA libraries were constructed according to the manufacturer’s protocol. Libraries were sequenced on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA), and raw sequencing data were processed using the Cell Ranger pipeline (10x Genomics) for demultiplexing, alignment to the human reference genome (GRCh38), unique molecular identifier counting, and generation of gene-barcode matrices. Downstream analysis was performed using the “Seurat” package in R (version 4.4.1). Cells with fewer than 200 or more than 6,000 detected genes, or with > 5% mitochondrial transcript content, were excluded. Genes expressed in fewer than three cells were also filtered out. Seurat’s standard preprocessing workflow was applied, including normalization, data scaling, Harmony integration for batch-effect correction, dimensionality reduction, and clustering based on the first 30 principal components. The average expression of selected gene sets— paracrine signaling-related (RAB27A, VAMP1, SNAP25, and STX1A) and EV marker (FLOT1, CLTC, TSG101, and CD81) genes was calculated for each cell in the scRNA-seq datasets using the “AddModuleScore” function. Data visualization was performed using the “UMAP”, “DimPlot”, “FeaturePlot”, “VlnPlot” and “DotPlot” functions in “Seurat”. Differential gene expression and pathway enrichment analyses Differentially expressed genes (DEGs) between clusters or sample groups were identified using the “FindMarkers,” “MAST,” and “FindAllMarkers” functions in Seurat, applying the Wilcoxon rank-sum test. Genes with an adjusted p-value < 0.05 and log fold-change ≥ 0.25 were considered statistically significant. Gene Ontology (GO) enrichment analysis for DEGs was performed using the “ClusterProfiler” R package (version 4.4.1) to identify enriched biological processes and molecular functions. Statistical Analysis Data were presented as the means ± standard errors of the mean. Statistical comparisons between groups were performed using one-way ANOVA followed by Tukey’s post-hoc test. All data were analyzed using GraphPad Prime 8 software (GraphPad, San Diego, CA, USA), and p-values less than 0.05 were considered statistically significant. Results Cell characterization TERT-MSCs retained their characteristic spindle-shaped or polygonal morphology at P13 and P30. Microscopic examination revealed no notable morphological differences between TERT-MSCs across P8 to P30 and P8 control MSCs (CTRL MSC-P8) (Fig. 1 A and Figure S1 A). After the stabilization period, TERT-MSCs maintained a doubling time of approximately 40–60 h and showed a consistent cumulative population doubling curve (R 2 = 0.998), comparable to that of early-passage MSCs [ 9 , 10 ] (Fig. 1 B). The trilineage differentiation potential—chondrogenic, osteogenic and adipogenic—was preserved in TERT-MSCs at P13 and P30, comparable to that of CTRL MSCs at P8 (Fig. 1 C). Flow cytometric analysis of TERT-MSCs at P13, P20, and P30 demonstrated consistent positive expression of CD90, CD105, and CD73, alongside negative expression of CD14, CD45, and HLA-DR, comparable to that in CTRL MSCs at P8 (Fig. 1 D). Karyotypic analysis revealed no detectable chromosomal abnormalities in either CTRL MSCs or TERT-MSCs at P30 (Fig. 1 E). TERT-over expression validation At the protein level, TERT expression in untransduced CTRL MSCs was not significantly different between passages P7 and P9, but gradually declined with passaging, particularly after P10, reflecting replicative senescence (Fig. 2 A). Specifically, TERT levels at P12, P13, and P28 were significantly lower than those in CTRL MSCs at P10. In contrast, TERT expression in TERT-MSCs remained stable up to P30, with levels at P13, P19, and P30 not significantly different from those in CTRL MSC at P10 (Fig. 2 A). Additionally, telomere length in TERT-MSCs was significantly longer than that in CTRL MSCs and was stably maintained up to P30 (Fig. 2 B). Conversely, CTRL MSCs exhibited progressive telomere shortening with extended passages. At the mRNA level, both scRNA-seq and qPCR analyses revealed markedly elevated TERT expression in TERT-MSCs at P13 and P30, whereas it was barely detectable in parental CTRL MSCs (Fig. 2 C and 2 D). Cytoprotective and anti-inflammatory effects of TERT-MSCs To evaluate changes in the cytoprotective capacity of CTRL MSCs during passaging, an H 2 O 2 -induced L2 lung epithelial cell injury model was employed (Fig. 3 A). Co-treatment with early-passage CTRL MSCs (P6 and P9) significantly enhanced cell viability in this model; however, this protective effect progressively declined in MSCs from later passages (P15 and P19). Brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) have been identified as key paracrine mediators with therapeutic potential in inflammatory diseases affecting various organs, including the brain and lungs, as shown in our previous studies [ 11 , 12 ]. The levels of BDNF, VEGF, and HGF progressively declined with passaging, in both cell lysates and conditioned medium. (Fig. 3 B). In contrast, TERT-MSCs retained their cytoprotective effect at both P13 and P30, comparable to that observed in CTRL MSCs at P8 (Fig. 3 C). In the LPS-induced inflammation model using RAW264.7 macrophages and BV2 microglia, the elevated levels of pro-inflammatory cytokines IL-1β and IL-6 were significantly reduced by co-treatment with CTRL MSC-P8. A comparable anti-inflammatory effect was also observed with TERT-MSCs at P13 and P30 (Fig. 3 D). A similar trend was observed in the thrombin-induced inflammation model using RAW264.7 and BV2 cells (Fig. 3 E). scRNA-seq analysis revealed that the proportions of BDNF- and VEGF- expressing cells were comparable across CTRL MSCs and TERT-MSCs at P13 and P30 (Fig. 3 F). Similarly, the concentrations of secreted BDNF and VEGF in conditioned media were consistent among all groups (Fig. 3 G). Module scores for paracrine signaling-related genes (RAB27A, VAMP1, SNAP25, and STX1A) and exosome markers (FLOT1, CLTC, TSG101, and CD81) showed no significant differences between groups (Fig. 3 H). Correspondingly, the cytoprotective effects did not differ significantly among the groups (Fig. 3 I). Physiological changes in TERT-MSCs To assess gene expression changes following exogenous TERT trasnduction, DEGs were identified for TERT-MSCs at P13 versus CTRL MSCs at P8, visualized using a volcano plot (Fig. 4 A), and subjected to GO enrichment analysis (Fig. 4 B). In this analysis, TERT-MSCs displayed a significant enrichment of terms related to DNA metabolic process, mitotic spindle organization, and RNA synthesis within the “Biological Process” category; nucleus and intracellular organelle within the “Cellular Component” category; and RNA binding within the “Molecular Function” category, compared with parental CTRL MSCs. These enriched GO terms collectively indicate enhanced nuclear activities that promote cell division and gene expression. Cell proliferation and senescence were evaluated using Ki-67 and CDKN2A as markers, respectively (Fig. 4 C). Following TERT overexpression, the proportion of Ki67-positive cells increased, whereas the population of CDKN2A-positive cells decreased compared to CTRL MSCs. The proportion of Ki-67- and CDKN2A-expressing cells in TERT-MSCs at P30 remained comparable to those of TERT-MSCs at P13. UMAP clustering patterns and cluster-specific GO term enrichment in TERT-MSCs UMAP visualization revealed that TERT overexpression altered clustering patterns (Fig. 5 A). Among the ten clusters, a GO bar chart for the three principal clusters (Clusters 0–2) is shown in Fig. 5 B. In untransduced CTRL MSCs, Cluster 0 (C0) predominant, accounting for over 75% of all cells. Following TERT transduction, the proportion of C0 decreased, whereas that of C1 and C2 increased. Nevertheless, among the top GO terms, no significant differences were observed across groups—control CTRL MSCs and TERT-MSCs at P13 and P30—within each GO category (Fig. 5 C). In the cluster-specific GO term enrichment analysis for C0–C2, the top two GO terms–cytoplasmic translation and oxidative phosphorylation–remained largely unchanged. Discussion The therapeutic potential of MSCs has been demonstrated over many years in multiple studies, showing anti-apoptotic, anti-inflammatory, immunomodulatory, pro-antigenic and regenerative effects in various in-vitro and in-vivo disease models. In most clinical applications, researchers use primary-cultured MSCs derived from individual donor tissues such as bone marrow, umbilical cord blood, and Wharton’s jelly. Once the master cell bank (MCB) from a donor is exhausted—typically after 5–8 passages due to replicative senescence—investigators must secure a new tissue sample from a different donor and establish a fresh MCB. This process requires extensive phenotypic and functional validation, leading to considerable time delays and financial costs before the experiments or clinical trials can proceed. In this study, we evaluated whether TERT-MSCs retained their original phenotypic and functional properties after extended passaging up to P30. Cell proliferation, morphology, MSC surface marker expression (positive for CD90, CD73, and CD105; negative for CD14, CD45, and HLA-DR), differentiation potential, and therapeutic effects on cytoprotection and anti-inflammation were preserved in extended passage (P30) cells compared with early-passage primary cultured (naïve) MSCs. In single-cell RNA-seq analysis, we confirmed sustained TERT overexpression, preservation of MSC surface marker expression, and continued expression of key paracrine factors such as BDNF and VEGF in TERT-MSCs up to P30. The establishment of stable MSC lines offers distinct advantages over primary MSC preparations, particularly in the context of GMP-compliant therapeutic production. First, they provide batch-to-batch consistency, ensuring that critical attributes—such as surface marker expression, differentiation potential, and secretome profile—remain constant throughout the production process. This consistency simplifies process validation, thereby reducing time delays for bioequivalence testing. Stable cell lines may also reduce manufacturing costs, including those associated with acquiring new MSC sources and performing quality control assays. The use of a standardized and immortalized MCB eliminates donor-to-donor variability while ensuring reproducible efficacy and safety profiles. Additionally, scaling up MSC culture is essential for MSC-based cell-free therapies, such as exosome or EV therapy, because large-scale culture is required to obtain sufficient conditioned media for exosome concentration and purification. The therapeutic effects of MSCs are primarily mediated by paracrine signaling through their secretome including exosomes and EVs, which reflect the characteristics of the parental cells. A previous study reported that EVs derived from TERT-immortalized MSCs maintained consistent quality in terms of particle size, concentration, and protein markers across two independent production batches and demonstrated a wound healing effect comparable to EVs derived from a control (cancer cell line) [ 13 ]. However, a direct comparison between TERT-immortalized MSCs and their parental cells, as well as an investigation of stemness retention during long-term passaging, has not yet been conducted. In this study, we directly compared TERT-MSCs with their parental cells and found that TERT-MSCs represents a promising source of EV, as late-passage TERT-MSC-derived EVs were as effective as those from early-passage parental cells (CTRL MSCs) in protecting lung epithelial cells from H 2 O 2 -induced injury. With respect to safety, although elevated TERT expression was detected in TERT-MSC lysates, TERT levels in both the conditioned medium and EVs were below the limit of detection (LOD) (Figure S2). Furthermore, no tumorigenic potential was observed in the soft agar assay (Figure S3). Collectively, these findings suggest that therapeutic approaches employing TERT-overexpressing MSCs could mitigate safety concerns—such as tumorigenicity—by utilizing MSC-derived EVs rather than administering the cells themselves. However, to ensure consistent quality of TERT-MSC-derived EVs, further investigations into EV characterization and cargo profiling across independent production batches and passage numbers are essential to control batch-to-batch and passage-to-passage variability. Additionally, although TERT-MSCs retained stemness and beneficial properties (including cytoprotective and anti-inflammatory effects), alterations in the proportions of cellular subpopulations were observed via single-cell RNA seq. Following TERT-induced immortalization, cluster 0—characterized by low expression of CDKN2A, a senescence marker—decreased in proportion, whereas clusters 1 and 2—characterized by high expression of Ki67, a proliferation marker—increased compared to untransduced CTRL MSCs. Overall GO term categories were comparable across samples (untransduced CTRL MSCs, P13 TERT-MSCs and P30 TERT-MSCs). However, the cytoplasmic translation category was slightly enriched in P13 and P30 TERT-MSCs compared to untransduced CTRL MSCs, suggesting that protein synthesis—associated processes became active. Based on previous reports, TERT is essential for telomere maintenance but also functions as a transcriptional regulator that interacts with multiple proteins to activate ribosomal RNA biogenesis and stimulate protein translation [ 14 ]. TERT can directly bind ribosomal DNA promoter and coding regions under specific conditions, such as highly proliferative cells [ 14 , 15 ]. Given that the non-telomeric functions of TERT remain incompletely understood, further preclinical studies are needed to investigate the potential side effects of TERT overexpression. In conclusion, we observed that MSCs exhibited diminished therapeutic efficacy, likely attributable to replicative senescence. In contrast, TERT overexpression maintained TERT expression and telomere length during extended passaging, preserved MSC identity and paracrine functionality, and showed no evidence of tumorigenicity, such as abnormal proliferation. The cytoprotection and anti-inflammatory effects of MSCs are primarily mediated through paracrine mechanisms. Even after passaging to P30, TERT-MSCs retained expression of paracrine-related genes, and the levels of key factors such as BDNF and VEGF in conditioned media and EVs were comparable to those of naïve MSCs at early passages. Although TERT protein was detected in cell lysates, it remained below the limit of LOD in both conditioned media and EVs. Collectively, these findings indicate that TERT-MSCs represent a promising source for MSC-based, cell-free therapeutic applications such as EV therapy. Nevertheless, comprehensive preclinical safety evaluations are necessary prior to clinical translation. Declarations Data Availability The data from the study are available from the corresponding authors upon reasonable request. Ethics Approval Clinical trial number: not applicable Funding This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (KH129441 and RS-2024-00436750); by the Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korean government (Ministry of Science and ICT, Ministry of Health & Welfare) (23C0119L1); the SMC-Ottogi Research Fund (#SMX1210881); and the Future Medicine 2030 Project of Samsung Medical Centre (#SMX1240621). Author Contribution Conceptualization, Y.S.C.; Methodology and Formal Analysis, Y.E.K., Y.J.L., and S.H.; Investigation, Y.E.K. and S.Y.A.; Writing—Original Draft Preparation, Y.E.K. and S.Y.A.; Writing—Review & Editing, Y.S.C.; Funding Acquisition, Y.S.C. and S.Y.A. All authors have read and agreed to the published version of the manuscript. Acknowledgement We thank BioRender for providing the online illustration tools used to create the graphical abstract. The image created with BioRender is used under license. References Patel, D. M., Shah, J., & Srivastava, A. S. (2013). Therapeutic potential of mesenchymal stem cells in regenerative medicine. Stem Cells Int, 2013: p. 496218. Lee, C. W., et al. (2018). Improvement of Cell Cycle Lifespan and Genetic Damage Susceptibility of Human Mesenchymal Stem Cells by Hypoxic Priming. Int J Stem Cells , 11 (1), 61–67. Gnani, D., et al. (2019). An early-senescence state in aged mesenchymal stromal cells contributes to hematopoietic stem and progenitor cell clonogenic impairment through the activation of a pro-inflammatory program. Aging Cell , 18 (3), e12933. Efimenko, A., et al. (2011). Angiogenic properties of aged adipose derived mesenchymal stem cells after hypoxic conditioning. J Transl Med , 9 , 10. Kasper, G., et al. (2009). Insights into mesenchymal stem cell aging: involvement of antioxidant defense and actin cytoskeleton. Stem Cells , 27 (6), 1288–1297. Baxter, M. A., et al. (2004). Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion. Stem Cells , 22 (5), 675–682. Joglekar, M. V. (2020). An Optimised Step-by-Step Protocol for Measuring Relative Telomere Length . Methods Protoc , 3(2). O'Callaghan, N. J., & Fenech, M. (2011). A quantitative PCR method for measuring absolute telomere length. Biol Proced Online , 13 , 3. Yoon, J. H. (2013). Comparison of explant-derived and enzymatic digestion-derived MSCs and the growth factors from Wharton's jelly. Biomed Res Int, 2013: p. 428726. Vellasamy, S., et al. (2012). Isolation and characterisation of mesenchymal stem cells derived from human placenta tissue. World J Stem Cells , 4 (6), 53–61. Ahn, S. Y., et al. (2017). Pivotal Role of Brain-Derived Neurotrophic Factor Secreted by Mesenchymal Stem Cells in Severe Intraventricular Hemorrhage in Newborn Rats. Cell Transplantation , 26 (1), 145–156. Ahn, S. Y., et al. (2018). Vascular endothelial growth factor mediates the therapeutic efficacy of mesenchymal stem cell-derived extracellular vesicles against neonatal hyperoxic lung injury. Experimental & Molecular Medicine , 50 (4), 1–12. Hindle, J. (2024). hTERT-Immortalized Mesenchymal Stem Cell-Derived Extracellular Vesicles: Large-Scale Manufacturing, Cargo Profiling, and Functional Effects in Retinal Epithelial Cells . Cells , 13(10). Yu, P., et al. (2023). TERT accelerates BRAF mutant-induced thyroid cancer dedifferentiation and progression by regulating ribosome biogenesis. Science Advances , 9 (35), eadg7125. Gonzalez, O. G., et al. (2014). Telomerase stimulates ribosomal DNA transcription under hyperproliferative conditions. Nature Communications , 5 , 4599. Additional Declarations No competing interests reported. Supplementary Files TERTMSCsSupplementarymaterial.pdf Cite Share Download PDF Status: Published Journal Publication published 11 Apr, 2026 Read the published version in Stem Cell Reviews and Reports → Version 1 posted Editorial decision: Revision requested 02 Feb, 2026 Reviews received at journal 02 Jan, 2026 Reviewers agreed at journal 13 Dec, 2025 Reviewers invited by journal 12 Dec, 2025 Editor assigned by journal 11 Dec, 2025 Submission checks completed at journal 11 Dec, 2025 First submitted to journal 09 Dec, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8322013","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":559727574,"identity":"69ccf1bc-7e78-480b-bb06-b0ca8938fecc","order_by":0,"name":"Young Eun Kim","email":"","orcid":"","institution":"Samsung Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Young","middleName":"Eun","lastName":"Kim","suffix":""},{"id":559727576,"identity":"c66b98db-17e8-42f2-bf0f-a180162a8847","order_by":1,"name":"So Yoon Ahn","email":"","orcid":"","institution":"Samsung Medical 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17:09:22","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":61665,"visible":true,"origin":"","legend":"","description":"","filename":"2465df0caa194f4d9c65fb1732120bc11structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8322013/v1/baadd9e7416adb76fe80f41e.xml"},{"id":98546795,"identity":"ea68d904-d9ec-4de3-b827-d686afbf2f16","added_by":"auto","created_at":"2025-12-18 19:18:42","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69169,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8322013/v1/e1e6762437afc60ac20b83af.html"},{"id":98546768,"identity":"3bbc14e3-8e4e-4a87-9f4a-9744fad507c2","added_by":"auto","created_at":"2025-12-18 19:18:42","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":827825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of MSCs.\u003c/strong\u003e (A) The typical morphology of P8 CTRL MSC and P13 \u0026amp; P30 TERT-MSC. (B) Cell doubling time and cumulative population doubling level. (C) MSC tri-lineage differentiation into chondrogenic, adipogenic, and osteogenic lineages, confirmed by Alcian blue, Oil Red O, and Alizarin S staining. (D) Surface antigen profiling of MSCs by flow cytometry showing positivity for CD90, CD105, and CD73, and negativity for CD14, CD45, and HLA-DR. (E) Representative karyotype images showing a normal chromosomal pattern in P8 CTRL MSC and P30 TERT-MSC.\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8322013/v1/a07937fe9fe5633a734d0761.jpg"},{"id":98625698,"identity":"e5e6cf1b-5b82-4e07-9fd6-f0da8391146c","added_by":"auto","created_at":"2025-12-19 17:09:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":267635,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of TERT transduction in MSCs.\u003c/strong\u003e (A) Quantification of TERT protein levels by ELISA in cell lysates of CTRL MSCs and TERT-MSCs. TERT levels exhibited a passage-dependent decline in CTRL MSCs, whereas they were retained in TERT-MSCs. * p \u0026lt; 0.05 vs. CTRL MSC (P10); # p \u0026lt; 0.05 vs. CTRL MSC (P12); $ p \u0026lt; 0.05 vs. CTRL MSC (P19). No significant differences were observed among passages P7–P9 in CTRL MSCs or among P13, P19, and P30 in TERT-MSCs. (n = 3 per group) (B) Relative quantification of telomere length by PCR in CTRL MSCs and TERT-MSCs. * p \u0026lt; 0.05 vs. CTRL MSC (P8); # p \u0026lt; 0.05 vs. CTRL MSC (P13); $ p \u0026lt; 0.05 vs. CTRL MSC (P19). (n = 3 per group). (C–D) Validation of TERT overexpression at the mRNA level: (C) FeaturePlot visualization of TERT expression from single-cell RNA-seq (purple indicates the normalized gene expression level). (D) Quantification of TERT mRNA levels by real-time PCR. * p \u0026lt; 0.05 vs. CTRL MSC. (n = 3 per group)\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8322013/v1/c81eea8132224f17bd4954a6.jpg"},{"id":98546775,"identity":"e33637be-8190-4778-a05a-2de819684c24","added_by":"auto","created_at":"2025-12-18 19:18:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":734255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of MSC efficacy and beneficial factor expression.\u003c/strong\u003e (A) Passage-dependent reduction in the cell-protective effect of CTRL MSCs in an H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced L2 cell injury model. *, p \u0026lt; 0.05 vs. Normal control; #, p \u0026lt; 0.05 vs. H2O2 control; $, p \u0026lt; 0.05 vs. H2O2+CTRL-MSC_P6; %, p \u0026lt; 0.05 vs. H2O2+CTRL-MSC_P9. (n = 8 per group) (B) Passage-dependent decline in the levels of beneficial factors—brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF)—in both cell lysates and in conditioned media. (n = 4 per group) (C) The cell-protective effects of P13 and P30 TERT-MSCs were comparable to those of P8 CTRL MSCs in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced L2 cell injury model. *, p \u0026lt; 0.05 vs. Normal control; #, p \u0026lt; 0.05 vs. H2O2 control (n = 8 per group). (D–E) The anti-inflammatory effects of P13 and P30 TERT-MSCs were comparable to those of P8 CTRL MSCs in BV2 microglia and RAW264.7 alveolar macrophages under (D) LPS-induced and (E) thrombin-induced inflammatory conditions, respectively. *, p \u0026lt; 0.05 vs. Normal control; #, p \u0026lt; 0.05 vs LPS (or thrombin) control; $, p \u0026lt; 0.05 vs. induced + CTRL MSCs (n = 5 per group). (F) FeaturePlot and boxplot of BDNF and VEGF expression in P8 CTRL MSC, P13 TERT-MSC, and P30 TERT-MSC, from single-cell RNA-sequencing. (G) Quantification of BDNF and VEGF levels in conditioned media. (H) Module scores of paracrine signaling-related genes (RAB27A, VAMP1, SNAP25, STX1A) and extracellular vesicles (EV) markers (FLOT1, CLTC, TSG101, CD81) (I) Comparable cell-protective effects of EVs isolated from P8 CTRL MSCs, P13 TERT-MSCs, and P30 TERT-MSCs in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced L2 cell injury model. *, p \u0026lt; 0.05 vs. Normal control; #, p \u0026lt; 0.05 vs. H2O2 control (n = 8 per group).\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8322013/v1/404b68389f8cd7ae4cd9e3bf.jpg"},{"id":98625642,"identity":"846b750d-f170-49a3-b610-d2357a796572","added_by":"auto","created_at":"2025-12-19 17:09:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":866339,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhysiological changes of TERT-MSCs, in scRNA-seq. \u003c/strong\u003e(A) Differentially expression genes (DEGs) between TERT-MSC (P13) vs. CTRL MSC. (B) Enriched gene ontology (GO) enrichment analysis of TERT-MSC (P13) compared with CTRL MSC. (C) FeaturePlot visualization of Ki67 and CDKN2A—markers of proliferation and senescence, respectively—from single-cell RNA-seq (purple indicates the normalized gene expression level).\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8322013/v1/f5f77b4b93e34d665c4e0714.jpg"},{"id":98625662,"identity":"8bd1162b-5a12-4eea-89ed-38a327f4de4d","added_by":"auto","created_at":"2025-12-19 17:09:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1055323,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-cell transcriptomic profiling, cluster characterization, and GO enrichment analysis of TERT-MSCs compared with CTRL MSCs. \u003c/strong\u003e(A) UMAP visualization of single-cell RNA-seq data from CTRL MSCs (P8) and TERT-MSCs (P13 and P30). Ten transcriptionally distinct clusters (C0–C9) were identified across samples, and their proportions are shown for each condition. (B) GO bar charts showing the top enriched biological process in the three major clusters (C0, C1, and C2). (C) GO term comparison across CTRL MSC, TERT-MSC_P13, and TERT-MSC_P30. Left panel: GO enrichment comparison across all clusters combined. Right panel: GO enrichment comparison focused on the three major clusters in the three main clusters (C0, C1, and C2).\u003c/p\u003e","description":"","filename":"figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8322013/v1/f18ef7da0a653ed957c2a7d3.jpg"},{"id":106809342,"identity":"6125464d-4997-4157-96c2-173951773bae","added_by":"auto","created_at":"2026-04-13 16:09:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4526829,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8322013/v1/adb0cb00-e59f-4443-95b4-5bd35feab9cc.pdf"},{"id":98626193,"identity":"62e6353e-0c71-45b9-8b50-ee276b0c3809","added_by":"auto","created_at":"2025-12-19 17:09:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":459517,"visible":true,"origin":"","legend":"","description":"","filename":"TERTMSCsSupplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8322013/v1/ae788d60e807fc32d525ade4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization and validation of long-term cultured TERT-immortalized human Wharton’s jelly–derived mesenchymal stromal cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMesenchymal stromal/stem cells (MSCs) have been extensively investigated for more than a decade. MSCs are a subset of cells that naturally reside in various tissues, including cord blood, adipose tissue, and bone marrow. These cells play critical roles in modulating surrounding cells, regulating immune responses, repairing damaged tissues, and maintaining homeostasis in the body. Numerous studies have demonstrated the anti-inflammatory, anti-apoptotic, and cytoprotective effects of MSCs in both preclinical and clinical studies targeting various incurable diseases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite their therapeutic potential, MSCs are present in low quantities within their native niches, and their functionality is highly donor-dependent. Although MSCs can be easily expanded in vitro, their use is generally recommended within approximately six passages, ideally below ten passages, to preserve therapeutic potential. Later passages are typically considered unsuitable for clinical applications due to decreased efficacy and increased risk of cellular dysfunction resulting from replicative senescence [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Previous studies have also reported that MSCs undergo age-related alterations in protein profiles, including reduced levels of immunomodulatory [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], pro-angiogenic [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and cytoskeletal proteins essential for migration [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, a new conceptual approach is required to address these limitations. In general, telomere length progressively shortens and cell doubling time increases following population division [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Over the past decade, it has been reported that introducing the telomere reverse transcriptase (TERT) gene can immortalize cells, and the technique for establishing immortalized cell lines is not new. However, only a few studies have compared the functional and phenotypic differences in MSCs before and after TERT-induced immortalization during extended passaging. For MSC-based therapies, such as those using MSC-derived extracellular vesicles (EVs), the initially limited MSC population must be expanded to a large scale, and minimizing variability across different passage stages is critically important. Immortalized MSC-based therapy may offer a promising strategy for exploring the feasibility of large-scale manufacturing and ensuring consistent product consistency.\u003c/p\u003e \u003cp\u003eIn this study, we generated TERT-induced immortalized MSCs at passage (P) 6, cultured them until P30, and analyzed them under three conditions: un-immortalized early-passage control (CTRL) (P8), mid-passage post-immortalization (P13), and late-passage post-immortalization (P30) MSCs. We aimed to determine whether MSCs retained their cytoprotective and anti-inflammatory properties upto P30 in an in-vitro cellular stress model and employed single-cell RNA sequencing (scRNA-seq) to characterize the associated transcriptional changes. Specifically, we examined whether the proportions of distinct cell clusters were altered, which clusters were affected by TERT-induced immortalization, and what molecular features defined these altered clusters.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003eMSC isolation and TERT-transfection\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMSCs were provided by the Samsung Medical Center GMP facility under standard quality controls, including mycoplasma and sterility testing; these cells were isolated from human Wharton\u0026rsquo;s jelly obtained from a single healthy donor (female, term delivery) and used exclusively for research under Institutional Review Board approval (IRB No: 2016-07-102-047) with written informed consent. The human TERT (h\u003cem\u003eTERT\u003c/em\u003e) gene was overexpressed by transducing MSCs with the pBabe-puro-hTERT retroviral vector (Addgene plasmid #1771) at P6. Viral particles were produced in 293T cells via co-transfection of pBabe-puro-hTERT with the packaging plasmids pCMV-GAG-pol and pCMV-VSV-G. MSCs were then transduced with the retroviral particles carrying the hTERT expression cassette. Successfully transduced MSCs (TERT-MSCs) were selected using puromycin (1 \u0026micro;g/mL; Thermo Fisher Scientific, Waltham, MA, USA), resulting in the formation of resistant colonies. Individual colonies were picked using a micropipette tip, transferred to separate cell culture dishes, and expanded in minimum essential medium alpha (MEMα; Gibco, CA, USA) supplemented with 10% fetal bovine serum (Biowest, Nuaill\u0026eacute;, France) and 1% gentamicin (Gibco). MSCs were seeded at 3\u0026ndash;4 \u0026times; 10⁴ cells per 100-mm culture dish and maintained under standard culture conditions, with serial passaging performed continuously until cells reached P30. No morphological alterations were observed after transduction (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Short tandem repeat genotyping using a 3730xl DNA Analyzer confirmed complete (100%) genetic concordance between TERT-MSCs and parental cells through P30 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMSC characterization\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCell morphology\u0026mdash;characterized by spindle-shaped, adherent growth\u0026mdash;was monitored daily using phase-contrast microscopy (Eclipse Ts2, Nikon, Tokyo, Japan) throughout the experimental period. Positive expression of CD90, CD105, and CD73, and negative expression of CD14, CD45, and HLA-DR were confirmed via flow cytometry (BD FACS Verse, BD Biosciences, NJ, USA). The differentiation potential into three lineages (osteogenic, adipogenic, and chondrogenic) was verified after three weeks of induction using the StemPro\u0026reg; Osteogenesis, Adipogenesis, and Chondrogenesis Differentiation Kits (Gibco). Differentiated osteocytes, adipocytes, and chondrocytes were fixed with 4% paraformaldehyde, stained with Alizarin Red S, Oil-Red O, and Alcian Blue, respectively, and examined microscopically.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eTERT protein levels in cell lysates and conditioned media were quantified using a commercial ELISA kit (MyBioSource, San Diego, CA, USA). Levels of brain-derived neurotrophic facor (BDNF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) were determined using commercial ELISA kits (R\u0026amp;D Systems, Minneapolis, MN, USA), following the manufacturers\u0026rsquo; protocols. Each independently seeded well was treated as an experimental unit, representing an independent biological replicate.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantification of\u003c/b\u003e \u003cb\u003eTERT\u003c/b\u003e \u003cb\u003emRNA expression and telomere length via real-time quantitative PCR (qPCR)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo quantify TERT mRNA expression, total RNA was extracted using the Xenopure Total RNA Purification Kit (Xenohelix, Incheon, Republic of Korea), and complementary DNA (cDNA) was synthesized from using the Primescript RT reagent kit (Takara, Shiga, Japan) according to the manufacturer\u0026rsquo;s protocols. qPCR was performed using the AccuPower\u0026reg; 2X Greenstar qPCR Mater Mix (Bioneer, Daejeon, Republic of Korea). The following primer sequences were used for h\u003cem\u003eTERT\u003c/em\u003e: forward 5\u0026prime;-ACTTTGTCAAGGTGGATGTGACGG-3\u0026prime; and reverse 5\u0026prime;-AAGAAATCATCCACCAAACGCAGG-3\u0026prime;. The human β-actin gene was used as a housekeeping gene with the following primers: forward 5\u0026prime;-CCTGCTTGCTGATCCACATC-3\u0026prime; and reverse 5\u0026prime;-CATCCGCAAAGACCTGTACG-3\u0026prime;. Relative telomere length was determined using a qPCR-based method as previously described [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Briefly, genomic DNA was extracted using the DNeasy Blood \u0026amp; Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer\u0026rsquo;s instructions. qPCR was performed using the AccuPower\u0026reg; 2X Greenstar qPCR Mater Mix (Bioneer) with the following primer sequences for telomeres: forward 5\u0026prime;-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3\u0026prime; and reverse 5\u0026prime;- GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3\u0026prime;. The single-copy gene 36B4F was used as a reference, with primers: forward 5\u0026prime;- CAGCAAGTGGGAAGGTGTAATCC-3\u0026prime; and reverse 5\u0026prime;- CCCATTCTATCATCAACGGGTACAA-3\u0026prime;. The relative telomere length was expressed as the ratio of telomere repeat copy number to that of the single-copy reference gene. All qPCR reactions were performed on the Applied Biosystems QuantStudio 6 Flex Real-Time PCR System (Invitrogen). Each independently seeded well was treated as an experimental unit, representing an independent biological replicate.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIn vitro efficacy study\u003c/h3\u003e\n\u003cp\u003eTo establish an oxidative stress-induced cell death model, L2 pulmonary epithelial cells were seeded one day before the experiment. Upon reaching 80\u0026ndash;90% confluence, cells were exposed to H₂O₂ (100 \u0026micro;M; Sigma-Aldrich) to induce oxidative stress. CTRL MSCs or TERT-MSCs were added at an MSC:L2 ratio of 1:10. In the MSC-untreated groups (normal control and H₂O₂-only control), an equivalent number of L2 cells was added to equalize total cell numbers across groups. Cell viability was measured using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan), according to the manufacturer\u0026rsquo;s instructions. To establish an inflammation model, RAW264.7 alveolar macrophages and BV2 microglia were seeded one day before the experiment. At 60\u0026ndash;70% confluence, cells were exposed to LPS (0.1 \u0026micro;g/ml; Sigma-Aldrich) or thrombin (40 U; Reyon Pharmaceutical Co., Ltd., Seoul, Korea). CTRL MSCs or TERT-MSCs were then added at a ratio of 1:10 relative to the total number of RAW264.7 or BV2 cells. After 24 h, conditioned media were collected, and inflammatory cytokine concentrations were measured using ELISA kits (R\u0026amp;D Systems, Minneapolis, MN, USA). Each independently seeded well was treated as an experimental unit, representing an independent biological replicate.\u003c/p\u003e\n\u003ch3\u003escRNA-seq and data analysis\u003c/h3\u003e\n\u003cp\u003escRNA-seq was performed on TERT-MSCs, following retroviral transduction and stabilization. TERT-MSCs at P13 were used to represent mid-passage TERT-MSCs, and those at P30 were used as late-passage cells. Untransduced MSCs at P8 were used as the controls. Following reverse transcription and cDNA amplification, scRNA libraries were constructed according to the manufacturer\u0026rsquo;s protocol. Libraries were sequenced on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA), and raw sequencing data were processed using the Cell Ranger pipeline (10x Genomics) for demultiplexing, alignment to the human reference genome (GRCh38), unique molecular identifier counting, and generation of gene-barcode matrices.\u003c/p\u003e \u003cp\u003eDownstream analysis was performed using the \u0026ldquo;Seurat\u0026rdquo; package in R (version 4.4.1). Cells with fewer than 200 or more than 6,000 detected genes, or with \u0026gt;\u0026thinsp;5% mitochondrial transcript content, were excluded. Genes expressed in fewer than three cells were also filtered out. Seurat\u0026rsquo;s standard preprocessing workflow was applied, including normalization, data scaling, Harmony integration for batch-effect correction, dimensionality reduction, and clustering based on the first 30 principal components. The average expression of selected gene sets\u0026mdash; paracrine signaling-related (RAB27A, VAMP1, SNAP25, and STX1A) and EV marker (FLOT1, CLTC, TSG101, and CD81) genes was calculated for each cell in the scRNA-seq datasets using the \u0026ldquo;AddModuleScore\u0026rdquo; function. Data visualization was performed using the \u0026ldquo;UMAP\u0026rdquo;, \u0026ldquo;DimPlot\u0026rdquo;, \u0026ldquo;FeaturePlot\u0026rdquo;, \u0026ldquo;VlnPlot\u0026rdquo; and \u0026ldquo;DotPlot\u0026rdquo; functions in \u0026ldquo;Seurat\u0026rdquo;.\u003c/p\u003e\n\u003ch3\u003eDifferential gene expression and pathway enrichment analyses\u003c/h3\u003e\n\u003cp\u003eDifferentially expressed genes (DEGs) between clusters or sample groups were identified using the \u0026ldquo;FindMarkers,\u0026rdquo; \u0026ldquo;MAST,\u0026rdquo; and \u0026ldquo;FindAllMarkers\u0026rdquo; functions in Seurat, applying the Wilcoxon rank-sum test. Genes with an adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and log fold-change\u0026thinsp;\u0026ge;\u0026thinsp;0.25 were considered statistically significant. Gene Ontology (GO) enrichment analysis for DEGs was performed using the \u0026ldquo;ClusterProfiler\u0026rdquo; R package (version 4.4.1) to identify enriched biological processes and molecular functions.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData were presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors of the mean. Statistical comparisons between groups were performed using one-way ANOVA followed by Tukey\u0026rsquo;s post-hoc test. All data were analyzed using GraphPad Prime 8 software (GraphPad, San Diego, CA, USA), and p-values less than 0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell characterization\u003c/h2\u003e \u003cp\u003eTERT-MSCs retained their characteristic spindle-shaped or polygonal morphology at P13 and P30. Microscopic examination revealed no notable morphological differences between TERT-MSCs across P8 to P30 and P8 control MSCs (CTRL MSC-P8) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). After the stabilization period, TERT-MSCs maintained a doubling time of approximately 40\u0026ndash;60 h and showed a consistent cumulative population doubling curve (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.998), comparable to that of early-passage MSCs [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The trilineage differentiation potential\u0026mdash;chondrogenic, osteogenic and adipogenic\u0026mdash;was preserved in TERT-MSCs at P13 and P30, comparable to that of CTRL MSCs at P8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Flow cytometric analysis of TERT-MSCs at P13, P20, and P30 demonstrated consistent positive expression of CD90, CD105, and CD73, alongside negative expression of CD14, CD45, and HLA-DR, comparable to that in CTRL MSCs at P8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Karyotypic analysis revealed no detectable chromosomal abnormalities in either CTRL MSCs or TERT-MSCs at P30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTERT-over expression validation\u003c/h3\u003e\n\u003cp\u003eAt the protein level, TERT expression in untransduced CTRL MSCs was not significantly different between passages P7 and P9, but gradually declined with passaging, particularly after P10, reflecting replicative senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Specifically, TERT levels at P12, P13, and P28 were significantly lower than those in CTRL MSCs at P10. In contrast, TERT expression in TERT-MSCs remained stable up to P30, with levels at P13, P19, and P30 not significantly different from those in CTRL MSC at P10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Additionally, telomere length in TERT-MSCs was significantly longer than that in CTRL MSCs and was stably maintained up to P30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Conversely, CTRL MSCs exhibited progressive telomere shortening with extended passages. At the mRNA level, both scRNA-seq and qPCR analyses revealed markedly elevated \u003cem\u003eTERT\u003c/em\u003e expression in TERT-MSCs at P13 and P30, whereas it was barely detectable in parental CTRL MSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCytoprotective and anti-inflammatory effects of TERT-MSCs\u003c/h2\u003e \u003cp\u003eTo evaluate changes in the cytoprotective capacity of CTRL MSCs during passaging, an H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced L2 lung epithelial cell injury model was employed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Co-treatment with early-passage CTRL MSCs (P6 and P9) significantly enhanced cell viability in this model; however, this protective effect progressively declined in MSCs from later passages (P15 and P19). Brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) have been identified as key paracrine mediators with therapeutic potential in inflammatory diseases affecting various organs, including the brain and lungs, as shown in our previous studies [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The levels of BDNF, VEGF, and HGF progressively declined with passaging, in both cell lysates and conditioned medium. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In contrast, TERT-MSCs retained their cytoprotective effect at both P13 and P30, comparable to that observed in CTRL MSCs at P8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In the LPS-induced inflammation model using RAW264.7 macrophages and BV2 microglia, the elevated levels of pro-inflammatory cytokines IL-1β and IL-6 were significantly reduced by co-treatment with CTRL MSC-P8. A comparable anti-inflammatory effect was also observed with TERT-MSCs at P13 and P30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). A similar trend was observed in the thrombin-induced inflammation model using RAW264.7 and BV2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). scRNA-seq analysis revealed that the proportions of BDNF- and VEGF- expressing cells were comparable across CTRL MSCs and TERT-MSCs at P13 and P30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Similarly, the concentrations of secreted BDNF and VEGF in conditioned media were consistent among all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Module scores for paracrine signaling-related genes (RAB27A, VAMP1, SNAP25, and STX1A) and exosome markers (FLOT1, CLTC, TSG101, and CD81) showed no significant differences between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Correspondingly, the cytoprotective effects did not differ significantly among the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological changes in TERT-MSCs\u003c/h2\u003e \u003cp\u003eTo assess gene expression changes following exogenous TERT trasnduction, DEGs were identified for TERT-MSCs at P13 versus CTRL MSCs at P8, visualized using a volcano plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), and subjected to GO enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In this analysis, TERT-MSCs displayed a significant enrichment of terms related to DNA metabolic process, mitotic spindle organization, and RNA synthesis within the \u0026ldquo;Biological Process\u0026rdquo; category; nucleus and intracellular organelle within the \u0026ldquo;Cellular Component\u0026rdquo; category; and RNA binding within the \u0026ldquo;Molecular Function\u0026rdquo; category, compared with parental CTRL MSCs. These enriched GO terms collectively indicate enhanced nuclear activities that promote cell division and gene expression. Cell proliferation and senescence were evaluated using Ki-67 and CDKN2A as markers, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Following TERT overexpression, the proportion of Ki67-positive cells increased, whereas the population of CDKN2A-positive cells decreased compared to CTRL MSCs. The proportion of Ki-67- and CDKN2A-expressing cells in TERT-MSCs at P30 remained comparable to those of TERT-MSCs at P13.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eUMAP clustering patterns and cluster-specific GO term enrichment in TERT-MSCs\u003c/h2\u003e \u003cp\u003eUMAP visualization revealed that TERT overexpression altered clustering patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Among the ten clusters, a GO bar chart for the three principal clusters (Clusters 0\u0026ndash;2) is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. In untransduced CTRL MSCs, Cluster 0 (C0) predominant, accounting for over 75% of all cells. Following TERT transduction, the proportion of C0 decreased, whereas that of C1 and C2 increased. Nevertheless, among the top GO terms, no significant differences were observed across groups\u0026mdash;control CTRL MSCs and TERT-MSCs at P13 and P30\u0026mdash;within each GO category (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In the cluster-specific GO term enrichment analysis for C0\u0026ndash;C2, the top two GO terms\u0026ndash;cytoplasmic translation and oxidative phosphorylation\u0026ndash;remained largely unchanged.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe therapeutic potential of MSCs has been demonstrated over many years in multiple studies, showing anti-apoptotic, anti-inflammatory, immunomodulatory, pro-antigenic and regenerative effects in various in-vitro and in-vivo disease models. In most clinical applications, researchers use primary-cultured MSCs derived from individual donor tissues such as bone marrow, umbilical cord blood, and Wharton\u0026rsquo;s jelly. Once the master cell bank (MCB) from a donor is exhausted\u0026mdash;typically after 5\u0026ndash;8 passages due to replicative senescence\u0026mdash;investigators must secure a new tissue sample from a different donor and establish a fresh MCB. This process requires extensive phenotypic and functional validation, leading to considerable time delays and financial costs before the experiments or clinical trials can proceed. In this study, we evaluated whether TERT-MSCs retained their original phenotypic and functional properties after extended passaging up to P30. Cell proliferation, morphology, MSC surface marker expression (positive for CD90, CD73, and CD105; negative for CD14, CD45, and HLA-DR), differentiation potential, and therapeutic effects on cytoprotection and anti-inflammation were preserved in extended passage (P30) cells compared with early-passage primary cultured (na\u0026iuml;ve) MSCs. In single-cell RNA-seq analysis, we confirmed sustained TERT overexpression, preservation of MSC surface marker expression, and continued expression of key paracrine factors such as BDNF and VEGF in TERT-MSCs up to P30.\u003c/p\u003e \u003cp\u003eThe establishment of stable MSC lines offers distinct advantages over primary MSC preparations, particularly in the context of GMP-compliant therapeutic production. First, they provide batch-to-batch consistency, ensuring that critical attributes\u0026mdash;such as surface marker expression, differentiation potential, and secretome profile\u0026mdash;remain constant throughout the production process. This consistency simplifies process validation, thereby reducing time delays for bioequivalence testing. Stable cell lines may also reduce manufacturing costs, including those associated with acquiring new MSC sources and performing quality control assays. The use of a standardized and immortalized MCB eliminates donor-to-donor variability while ensuring reproducible efficacy and safety profiles. Additionally, scaling up MSC culture is essential for MSC-based cell-free therapies, such as exosome or EV therapy, because large-scale culture is required to obtain sufficient conditioned media for exosome concentration and purification. The therapeutic effects of MSCs are primarily mediated by paracrine signaling through their secretome including exosomes and EVs, which reflect the characteristics of the parental cells. A previous study reported that EVs derived from TERT-immortalized MSCs maintained consistent quality in terms of particle size, concentration, and protein markers across two independent production batches and demonstrated a wound healing effect comparable to EVs derived from a control (cancer cell line) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, a direct comparison between TERT-immortalized MSCs and their parental cells, as well as an investigation of stemness retention during long-term passaging, has not yet been conducted. In this study, we directly compared TERT-MSCs with their parental cells and found that TERT-MSCs represents a promising source of EV, as late-passage TERT-MSC-derived EVs were as effective as those from early-passage parental cells (CTRL MSCs) in protecting lung epithelial cells from H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced injury. With respect to safety, although elevated TERT expression was detected in TERT-MSC lysates, TERT levels in both the conditioned medium and EVs were below the limit of detection (LOD) (Figure S2). Furthermore, no tumorigenic potential was observed in the soft agar assay (Figure S3). Collectively, these findings suggest that therapeutic approaches employing TERT-overexpressing MSCs could mitigate safety concerns\u0026mdash;such as tumorigenicity\u0026mdash;by utilizing MSC-derived EVs rather than administering the cells themselves.\u003c/p\u003e \u003cp\u003eHowever, to ensure consistent quality of TERT-MSC-derived EVs, further investigations into EV characterization and cargo profiling across independent production batches and passage numbers are essential to control batch-to-batch and passage-to-passage variability. Additionally, although TERT-MSCs retained stemness and beneficial properties (including cytoprotective and anti-inflammatory effects), alterations in the proportions of cellular subpopulations were observed via single-cell RNA seq.\u0026nbsp;Following TERT-induced immortalization, cluster 0\u0026mdash;characterized by low expression of CDKN2A, a senescence marker\u0026mdash;decreased in proportion, whereas clusters 1 and 2\u0026mdash;characterized by high expression of Ki67, a proliferation marker\u0026mdash;increased compared to untransduced CTRL MSCs. Overall GO term categories were comparable across samples (untransduced CTRL MSCs, P13 TERT-MSCs and P30 TERT-MSCs). However, the cytoplasmic translation category was slightly enriched in P13 and P30 TERT-MSCs compared to untransduced CTRL MSCs, suggesting that protein synthesis\u0026mdash;associated processes became active. Based on previous reports, TERT is essential for telomere maintenance but also functions as a transcriptional regulator that interacts with multiple proteins to activate ribosomal RNA biogenesis and stimulate protein translation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. TERT can directly bind ribosomal DNA promoter and coding regions under specific conditions, such as highly proliferative cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Given that the non-telomeric functions of TERT remain incompletely understood, further preclinical studies are needed to investigate the potential side effects of TERT overexpression.\u003c/p\u003e \u003cp\u003eIn conclusion, we observed that MSCs exhibited diminished therapeutic efficacy, likely attributable to replicative senescence. In contrast, TERT overexpression maintained TERT expression and telomere length during extended passaging, preserved MSC identity and paracrine functionality, and showed no evidence of tumorigenicity, such as abnormal proliferation. The cytoprotection and anti-inflammatory effects of MSCs are primarily mediated through paracrine mechanisms. Even after passaging to P30, TERT-MSCs retained expression of paracrine-related genes, and the levels of key factors such as BDNF and VEGF in conditioned media and EVs were comparable to those of na\u0026iuml;ve MSCs at early passages. Although TERT protein was detected in cell lysates, it remained below the limit of LOD in both conditioned media and EVs. Collectively, these findings indicate that TERT-MSCs represent a promising source for MSC-based, cell-free therapeutic applications such as EV therapy. Nevertheless, comprehensive preclinical safety evaluations are necessary prior to clinical translation.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe data from the study are available from the corresponding authors upon reasonable request.\u003c/p\u003e \u003c/div\u003e\u003cp\u003e \u003ch2\u003eEthics Approval\u003c/h2\u003e \u003cp\u003eClinical trial number: not applicable\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by a grant from the Korea Health Technology R\u0026amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health \u0026amp; Welfare, Republic of Korea (KH129441 and RS-2024-00436750); by the Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korean government (Ministry of Science and ICT, Ministry of Health \u0026amp; Welfare) (23C0119L1); the SMC-Ottogi Research Fund (#SMX1210881); and the Future Medicine 2030 Project of Samsung Medical Centre (#SMX1240621).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, Y.S.C.; Methodology and Formal Analysis, Y.E.K., Y.J.L., and S.H.; Investigation, Y.E.K. and S.Y.A.; Writing\u0026mdash;Original Draft Preparation, Y.E.K. and S.Y.A.; Writing\u0026mdash;Review \u0026amp; Editing, Y.S.C.; Funding Acquisition, Y.S.C. and S.Y.A. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eWe thank BioRender for providing the online illustration tools used to create the graphical abstract. The image created with BioRender is used under license.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePatel, D. M., Shah, J., \u0026amp; Srivastava, A. S. (2013). \u003cem\u003eTherapeutic potential of mesenchymal stem cells in regenerative medicine.\u003c/em\u003e Stem Cells Int, 2013: p. 496218.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, C. W., et al. (2018). Improvement of Cell Cycle Lifespan and Genetic Damage Susceptibility of Human Mesenchymal Stem Cells by Hypoxic Priming. \u003cem\u003eInt J Stem Cells\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(1), 61\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGnani, D., et al. (2019). An early-senescence state in aged mesenchymal stromal cells contributes to hematopoietic stem and progenitor cell clonogenic impairment through the activation of a pro-inflammatory program. \u003cem\u003eAging Cell\u003c/em\u003e, \u003cem\u003e18\u003c/em\u003e(3), e12933.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEfimenko, A., et al. (2011). Angiogenic properties of aged adipose derived mesenchymal stem cells after hypoxic conditioning. \u003cem\u003eJ Transl Med\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e, 10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKasper, G., et al. (2009). Insights into mesenchymal stem cell aging: involvement of antioxidant defense and actin cytoskeleton. \u003cem\u003eStem Cells\u003c/em\u003e, \u003cem\u003e27\u003c/em\u003e(6), 1288\u0026ndash;1297.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaxter, M. A., et al. (2004). Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion. \u003cem\u003eStem Cells\u003c/em\u003e, \u003cem\u003e22\u003c/em\u003e(5), 675\u0026ndash;682.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoglekar, M. V. (2020). \u003cem\u003eAn Optimised Step-by-Step Protocol for Measuring Relative Telomere Length\u003c/em\u003e. \u003cem\u003eMethods Protoc\u003c/em\u003e, 3(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO'Callaghan, N. J., \u0026amp; Fenech, M. (2011). A quantitative PCR method for measuring absolute telomere length. \u003cem\u003eBiol Proced Online\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e, 3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoon, J. H. (2013). \u003cem\u003eComparison of explant-derived and enzymatic digestion-derived MSCs and the growth factors from Wharton's jelly.\u003c/em\u003e Biomed Res Int, 2013: p. 428726.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVellasamy, S., et al. (2012). Isolation and characterisation of mesenchymal stem cells derived from human placenta tissue. \u003cem\u003eWorld J Stem Cells\u003c/em\u003e, \u003cem\u003e4\u003c/em\u003e(6), 53\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhn, S. Y., et al. (2017). Pivotal Role of Brain-Derived Neurotrophic Factor Secreted by Mesenchymal Stem Cells in Severe Intraventricular Hemorrhage in Newborn Rats. \u003cem\u003eCell Transplantation\u003c/em\u003e, \u003cem\u003e26\u003c/em\u003e(1), 145\u0026ndash;156.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhn, S. Y., et al. (2018). Vascular endothelial growth factor mediates the therapeutic efficacy of mesenchymal stem cell-derived extracellular vesicles against neonatal hyperoxic lung injury. \u003cem\u003eExperimental \u0026amp; Molecular Medicine\u003c/em\u003e, \u003cem\u003e50\u003c/em\u003e(4), 1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHindle, J. (2024). \u003cem\u003ehTERT-Immortalized Mesenchymal Stem Cell-Derived Extracellular Vesicles: Large-Scale Manufacturing, Cargo Profiling, and Functional Effects in Retinal Epithelial Cells\u003c/em\u003e. \u003cem\u003eCells\u003c/em\u003e, 13(10).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, P., et al. (2023). TERT accelerates BRAF mutant-induced thyroid cancer dedifferentiation and progression by regulating ribosome biogenesis. \u003cem\u003eScience Advances\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(35), eadg7125.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonzalez, O. G., et al. (2014). Telomerase stimulates ribosomal DNA transcription under hyperproliferative conditions. \u003cem\u003eNature Communications\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e, 4599.\u003c/span\u003e\u003c/li\u003e\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":"stem-cell-reviews-and-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"stcr","sideBox":"Learn more about [Stem Cell Reviews and Reports](https://www.springer.com/journal/12015)","snPcode":"12015","submissionUrl":"https://submission.nature.com/new-submission/12015/3","title":"Stem Cell Reviews and Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8322013/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8322013/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMesenchymal stromal cells (MSCs) have demonstrated efficacy and feasibility in numerous preclinical and clinical studies. However, their application is limited at early passages owing to replicative senescence. This study investigated whether MSCs immortalized via human telomerase reverse transcriptase (TERT) overexpression (TERT-MSCs) maintain their characteristics and efficacy after extended passaging. Human Wharton\u0026rsquo;s jelly-derived MSCs at passage (P) 6 were immortalized by retroviral transfection with human TERT. TERT-MSCs at mid (P13) and late (P30) passages were analyzed. Their morphology, differentiation potential, and surface marker expression were preserved throughout extended passaging. Single-cell RNA sequencing reconfirmed the characteristics of TERT-MSCs and compared their gene expression profiles with those of P8 parental (control) cells. As passage number increased, control MSCs exhibited gradual declines in TERT expression, telomere length, and cytoprotective effects, whereas both P13 and P30 TERT-MSCs retained these properties. Expression levels of paracrine-related genes and enriched Gene Ontology terms in extended-passage TERT-MSCs were comparable to those of control MSCs. These findings suggest that TERT-MSCs maintain the key characteristics and therapeutic potential of early-passage MSCs through extended culture, offering a promising approach to overcome the limitations of early-passage MSCs in clinical applications.\u003c/p\u003e","manuscriptTitle":"Characterization and validation of long-term cultured TERT-immortalized human Wharton’s jelly–derived mesenchymal stromal cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-18 19:18:37","doi":"10.21203/rs.3.rs-8322013/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-02T09:30:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-02T20:57:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166616677435381842269087959950022204418","date":"2025-12-13T08:07:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-12T16:02:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-11T07:09:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-11T07:07:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Reviews and Reports","date":"2025-12-10T01:38:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-reviews-and-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"stcr","sideBox":"Learn more about [Stem Cell Reviews and Reports](https://www.springer.com/journal/12015)","snPcode":"12015","submissionUrl":"https://submission.nature.com/new-submission/12015/3","title":"Stem Cell Reviews and Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"99dc5b25-303e-44ee-bf72-78c8ae036a36","owner":[],"postedDate":"December 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:05:54+00:00","versionOfRecord":{"articleIdentity":"rs-8322013","link":"https://doi.org/10.1007/s12015-026-11118-4","journal":{"identity":"stem-cell-reviews-and-reports","isVorOnly":false,"title":"Stem Cell Reviews and Reports"},"publishedOn":"2026-04-11 15:59:05","publishedOnDateReadable":"April 11th, 2026"},"versionCreatedAt":"2025-12-18 19:18:37","video":"","vorDoi":"10.1007/s12015-026-11118-4","vorDoiUrl":"https://doi.org/10.1007/s12015-026-11118-4","workflowStages":[]},"version":"v1","identity":"rs-8322013","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8322013","identity":"rs-8322013","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}