Methodological Validation of RQ-TRAP for Telomerase Activity Detection and Its Applications in Human Mesenchymal Stem Cells

doi:10.19803/j.1672-8629.20250916

Abstract

Abstract: Objective To validate the methodological performance of the real-time quantitative telomeric repeat amplification protocol (RQ-TRAP), including the specificity, accuracy, precision, linearity, amplification efficiency, and lower limit of quantification, and detect telomerase activity in human mesenchymal stem cells (hMSCs) using this method in order to assess the proliferative potential, senescence process, and potential tumorigenic risk of hMSCs. Methods Telomerase activity in hMSCs was detected using the RQ-TRAP method. hMSCs derived from human umbilical cords were cultured in MEM-α medium. Samples were prepared via cell lysis and extraction, and absolute quantification was performed on a real-time polymerase chain reaction system using TRAP reaction buffer. Methodological validation was conducted using TSR8 control templates to evaluate the specificity, accuracy (recovery rate), intermediate precision (RSD), linearity (R² and amplification efficiency), and LLOQ (confidence interval of recovery). For hMSCs assessment, telomerase activity was calculated based on standard curve fitting and Ct values. Results Methodological validation results indicated a good specificity with no interference from heat-inactivated cell matrices. The 95% confidence interval (CI) for accuracy recovery ranged from 91.37% to 111.0% (meeting the standard of 75%-125%). The intermediate precision (RSD) was less than 25% for all samples. Linearity assessment showed an R²>0.99 with an amplification efficiency of 104.1% (within the 90%-110% range). The LLOQ was determined to be 0.2 TPG Units (recovery CI: 70%-130%). The telomerase activity in hMSCs was measured at 1.432 TPG per 10 000 cells and the amplification efficiency of the standard curve in this assay was 102.6%. Conclusion The RQ-TRAP method has proved to be reliable and sensitive, which can be used for quantitative detection of telomerase activity in hMSCs and for effective assessment of cell senescence and tumorigenic risks. This method fills the gap in standardized testing for hMSCs and supports safe applications of regenerative medicine.

Key words: Telomerase Activity, Mesenchymal Stem Cells, Methodological Validation, Real-time Quantitative PCR, Tumorigenicity Assessment

CLC Number:

R917

ZHANG Zhen, YU Xie'an, XU Chunqi, CHEN Ning, QIN Meirong, WANG Ping. Methodological Validation of RQ-TRAP for Telomerase Activity Detection and Its Applications in Human Mesenchymal Stem Cells[J]. Chinese Journal of Pharmacovigilance, 2026, 23(2): 121-126.

Add to citation manager EndNote|Ris|BibTeX

URL: https://zgywjj.magtechjournal.com/EN/10.19803/j.1672-8629.20250916

https://zgywjj.magtechjournal.com/EN/Y2026/V23/I2/121

References

[1] CHENG X, FU QY, ZHENG YL, et al.Telomerase-Active Urine-Derived Stem Cells: Regenerative Solutions for Aging[J]. Ageing Res Rev, 2026, 113: 102939.
[2] APETROAEI MM, FRAGKIADAKI P, VELESCU BȘ, et al.Pharmacotherapeutic Considerations on Telomere Biology: the Positive Effect of Pharmacologically Active Substances on Telomere Length[J]. Int J Mol Sci, 2024, 25(14): 7694.
[3] AFSHARIf N, Al-GAZALLY ME, RASULOVA I, et al.Sensitive Bioanalytical Methods for Telomerase Activity Detection: a Cancer Biomarker[J]. Anal Methods, 2022, 14(42): 4174-4184.
[4] CASADIO V, BRAVACCINI S.Telomerase Activity Analysis in Urine Sediment for Bladder Cancer[J]. Methods Mol Biol, 2021, 2292: 133-141.
[5] HE Y, CUI Y, WANG L, et al.Extracellular Vesicles for Delivery of Functional Telomerase[J]. Cell Commun Signal, 2025, 23(1): 509.
[6] KHALIFEZADEH M, MASOUMI S, NAJAFI K, et al.Telomerase Biosensors: a Promising Approach in Cancer Diagnostics[J]. Clin Chim Acta, 2026, 579: 120668.
[7] CARLUND O, NORBERG A, OSTERMAN P, et al.Telomerase Activity in T-Cells as a Functional Test for Pathogenicity Assessment of Novel Genetic Variants in Telomere Biology Disorders[J]. Sci Rep, 2025, 15(1): 29048.
[8] NAKAMURA K.Key Mediators of the Efficacy of Mesenchymal Stem Cells on in vivo Disease Models[J]. Cell Transplant, 2025, 34: 9636897251348566.
[9] YAMAGUCHI N, HORIO E, SONODA J, et al.Immortalization of Mesenchymal Stem Cells for Application in Regenerative Medicine and Their Potential Risks of Tumorigenesis[J]. Int J Mol Sci, 2024, 25(24): 13562.
[10] HU L, WANG J, ZHOU X, et al.Exosomes Derived from Human Mesenchymal Stem Cells as Nanocarriers for Drug Delivery[J]. J Nanobiotechnology, 2021, 19(1): 107.
[11] AFKHAMI H, MAHMOUDVAND G, FAKOURI A, et al.New Insights in Application of Mesenchymal Stem Cells Therapy in Tumor Microenvironment: Pros and Cons[J]. Front Cell Dev Biol, 2023, 11: 1255697.
[12] KARAGYAUR M, PRIMAK A, BASALOVA N, et al.Safety and Regenerative Properties of Immortalized Human Mesenchymal Stromal Cell Secretome[J]. Int J Mol Sci, 2025, 26(19): 9322.
[13] CHIN SP, SAFFERY NS, THEN KY, et al.Preclinical Assessments of Safety and Tumorigenicity of Very High Doses of Allogeneic Human Umbilical Cord Mesenchymal Stem Cells[J]. In vitro Cell Dev Biol Anim, 2024, 60(3): 307-319.
[14] LEONOV GE, GRINCHEVSKAYA LR, MAKHNACH OV, et al.Safety Assessment of Stem Cell-Based Therapies: Current Standards and Advancing Frameworks[J]. Cells, 2025, 14(21): 1660.
[15] FÄHNRICH S, WEDEMANN A, STEEBPASS L, et al. Optimized for Routine: Highly Sensitive Fluorescent Telomeric Repeat Ampli-fication Protocol (f-TRAP)[J]. Biotechniques, 2024, 76(10): 517-522.
[16] WU X, WU J, DAI J, et al. Aggregation-Induced Emission Luminogens Reveal Cell Cycle-Dependent Telomerase Activity in Cancer Cells[J]. National Science Review, 2021, 8(6): nwaa306.
[17] GONZÁLEZ-GUALDA E, BAKER AG, FRUK L, et al. A Guide to Assessing Cellular Senescence in vitro and in vivo[J]. FEBS J, 2021, 288(1): 56-80.
[18] ZHANG S, XIONG W, XU S, et al.Recent Advances in DNA Systems for In Situ Telomerase Activity Detection and Imaging[J]. Chemosensors, 2025, 13(1): 17.
[19] KARAGYAUR M, PRIMAK A, BASALOVA N, et al.Safety and Regenerative Properties of Immortalized Human Mesenchymal Stromal Cell Secretome[J]. International Journal of Molecular Sciences, 2025, 26(19): 9322.
[20] SCHELLNEGGER M, HOFMANN E, CARNIELETTO M, et al.Unlocking Longevity: the Role of Telomeres and Its Targeting Interventions[J]. Front Aging, 2024, 5: 1339317.
[21] LI Y, ZHANG F, QINZ, et al. Development of 3D Cell-Based Fluorescent Reporter Assay for Screening of Drugs Downregulating Telomerase Reverse Transcriptase[J]. Bioengineering, 2025, 12(4): 335.
[22] MATVEEVA D, KASHIRINA D, EZDAKOVA M, et al.Senescence-Associated Alterations in Matrisome of Mesenchymal Stem Cells[J]. International Journal of Molecular Sciences, 2024, 25(10): 5332.