INTRODUCTION
Telomeres are repetitive nucleotide sequences (TTAGGG) located at the ends of chromosomes that protect genomic DNA from degradation, end-to-end fusion, and abnormal recombination. With each cell division, telomeres progressively shorten due to end-to-end replication issues, and this wear and tear can be accelerated by oxidative stress, inflammation, and lifestyle factors (Blackburn et al., 2015). When telomere length reaches a critical threshold, cells enter replicative senescence or undergo apoptosis, which contributes to tissue aging and functional decline (López-Otín et al., 2023). Telomere length measured in peripheral blood leukocytes is widely used as a surrogate for biological age and is associated with mortality, cardiovascular disease, the risk of cancer development, and various age-related diseases (Goglin et al., 2016; Haycock et al., 2014; Vaiserman and Krasnienkov, 2021). Therapeutics that alleviate telomere shortening or promote telomere elongation are receiving significant attention in regenerative medicine and longevity research. Given that telomere biology plays a crucial role in the aging process, researchers are focusing on developing methods to delay or reduce the onset of age-related diseases by inhibiting telomere wear (Schellnegger et al., 2024).
Mesenchymal stem cells (MSCs) were officially named over 25 years ago and refer to cells derived from the bone marrow and periosteum of humans and mammals. These cells can be proliferated through isolation and culture, and they maintain various mesodermal phenotypes and the ability to form tissues in vitro. MSCs are pluripotent stromal cells possessing immunomodulatory, anti-inflammatory, and secretory signaling properties. In fact, stem cells present in specific sites and tissues of a patient form new tissues upon stimulation by bioactive factors secreted by externally supplied MSCs (Caplan, 2017). MSCs are attracting attention for their tremendous potential in regenerative medicine. Possessing immunomodulatory and regenerative properties, MSCs have diverse potential applications in the clinical field. MSCs are key candidates applicable to various organ systems because they exhibit characteristics such as multilineage differentiation and paracrine signaling, and can be isolated from diverse tissues. After intravenous injection, MSCs migrate to the site of inflammation and regulate the cellular microenvironment by secreting growth factors, extracellular vesicles, and cytokines (Maldonado et al., 2023). Preclinical studies have shown that MSCs-derived extracellular vesicles carry human telomerase reverse transcriptase (hTERT) messenger ribonucleic acid (mRNA) and regulatory microRNAs capable of stimulating endogenous telomerase activity in recipient cells (Likonen et al., 2022; Tan et al., 2024; Wu et al., 2022).
Despite the increasing clinical interest in MSCs therapy for antiaging purposes, human data quantifying changes in telomere length after MSCs infusion are still lacking. This case study is one of several studies that systematically investigated telomere length before and after intravenous MSCs administration in a real clinical setting.
MATERIALS AND METHODS
Study design
This study is a retrospective observational case study conducted at the Stem Cell Laboratory of Gangnam Seran Clinic (Korea). All subjects received intravenous MSCs therapy as part of their routine clinical practice. Telomere length measurements were taken at the baseline time (before treatment) and at the follow-up visit (after treatment). This study was conducted as part of the Advanced Regenerative Medicine Project (RM-1-006) in compliance with the Declaration of Helsinki (2013 Revision). Written informed consent was obtained from all subjects prior to treatment and data collection.
Subjects
A total of 19 cases from 18 subjects were included (Table 1). The ages of the subjects ranged from 34 to 78 years (median, 55 years), with 9 males and 9 females (1 male subject, CBA, was measured at 2 follow-up times). Clinical indications included antiaging, chronic fatigue, musculoskeletal pain, diabetes, and postoperative recovery. Subjects were anonymized using initialized identifiers.
MSCs preparation and administration
MSCs were derived from allogeneic umbilical cord tissue. The cells were prepared in compliance with Good Manufacturing Practice and quality-checked for viability (>90%), sterility, and identity. Specified dose (X×106 cells) was slowly infused intravenously. Some patients received additional local injections (intra-articular or subcutaneous) for concomitant musculoskeletal conditions. All treatments were performed directly by the medical staff at Gangnam Seran Clinic.
Telomere length measurement
DNA was extracted from peripheral blood collected at baseline and follow-up. Telomere length was quantified in kilobases per cell (kb/cell) using specific methods (quantitative polymerase chain reaction telomere/single copy gene (T/S) ratio, flow cytometry-based fluorescence in situ hybridization, Southern blot terminal restriction fragment) (Cawthon, 2002). All measurements were performed at designated accredited laboratories. Two cases (KBH and KIS) were reported in log-scale format by the reference laboratory and are presented separately.
Statistical analysis
Due to the nature of the retrospective case study design, formal hypothesis testing was a secondary task compared to descriptive analysis. Descriptive statistics (median, interquartile range, range) were used to characterize telomere length at each time point. Group-level changes were reported as mean±standard error of the mean.
RESULTS
Overall pattern of change
An increase in the median telomere length was observed after intravenous MSCs treatment in all 19 evaluable cases, with only one KDH patient (−0.3 kb/cell, −5.2%) being an exception. The magnitude of the change varied from −0.3 kb/cell to +10.1 kb/cell (mean change after excluding cases with log scale=+2.47 kb/cell, n=17), and a measurable increase was observed in 94% of cases (Fig. 1). This directional change was consistently observed across subjects with various variables, including age (34–78 years), sex, duration of treatment (25 days–14 months), and baseline telomere length, suggesting a reproducible biological effect associated with MSC administration. The sign test for the probability of observing an increase in 17 or more of the 17 cases yielded P<0.001. Significant variability in response magnitude between individuals is discussed below.
Subjects with very short basal telomere lengths
Basal telomere lengths were found to be significantly short in several subjects, including CWS, LJE, and CBA (primary measure) (median ≤2 kb/cell). After MSCs treatment, the median telomere length of these subjects increased significantly, and follow-up showed that the distribution changed from ≤2 kb/cell to the range of 2.5–11 kb/cell (Fig. 1; cases 1, 2, 7, 8, 12). This pattern is consistent with telomerase acting preferentially on very short telomeres (Hockemeyer and Collins, 2015; Mir et al., 2020).
Continuous measurements
Telomere length was measured in one subject (CBA) over 3 time points (baseline, 4 months after treatment, and 8 months after treatment). The median telomere length gradually increased from approximately 1.0 kb/cell at baseline to approximately 2.7 kb/cell after 4 months, and to approximately 11.0 kb/cell after 8 months. These in-subject responses over time support the occurrence of cumulative or auto-amplifying biological processes following a single MSCs infusion.
Older subjects
All six subjects aged 60 years or older showed an increase in telomere length during the follow-up period (Fig. 1; cases 3, 6, 9, 11, 15, 19). Given that telomere wear leads to the most severe consequences in the elderly population (Blackburn et al., 2015; López-Otín et al., 2023), the consistent occurrence of these results in the oldest group is of particular clinical significance.
Short-term measurement responders
Measurable telomere growth was observed even in subjects with short measurement intervals (Fig. 1; cases 13, 14, 15, 17, 18). These results suggest that MSCs-mediated telomere homeostasis regulatory effects may appear within weeks, which is consistent with the rapid response rate of extracellular vesicle-mediated gene expression changes (Tan et al., 2024).
DISCUSSION
The principal finding of this case series is that intravenous MSCs therapy was consistently associated with increases in peripheral blood telomere length across a heterogeneous cohort of subjects. This observation aligns with several proposed mechanisms of MSCs-mediated cellular rejuvenation, centered on paracrine signaling via extracellular vesicles carrying hTERT mRNA, regulatory microRNAs, and growth factors (Likonen et al., 2022; Tan et al., 2024; Wu et al., 2022). MSCs-derived extracellular vesicles have been shown to carry hTERT mRNA that, upon uptake by recipient cells, is translated into functionally active telomerase enzyme, conferring a replicative advantage and reducing cellular senescence (Likonen et al., 2022). Additionally, the potent anti-inflammatory and antioxidant paracrine effects of MSCs reduce the reactive oxygen species (ROS) burden that accelerates telomere erosion in vivo (Armstrong and Boonekamp, 2023; Barnes et al., 2022; De Rosa et al., 2021).
Basal telomere length predicts response size
In this dataset, the most powerful predictor of treatment response is baseline telomere length. Subjects with the shortest baseline telomere lengths (≤2 kb/cell) showed the largest absolute increase, whereas subjects with longer baseline telomere lengths showed minimal change. This pattern can be explained by a protein counting model of telomere length homeostasis. Shorter telomeres activate the shelterin complex (telomeric repeat factor 1/telomeric repeat factor 2) less, reducing the inhibition of the “open” state accessible to telomerase and consequently enabling preferential elongation of critically short ends by telomerase (Hockemeyer and Collins, 2015; Mir et al., 2020).
Continuous measurement and cumulative effect
The progressive increase in telomere length observed in CBA over 8 months is consistent with a self-amplifying biological process rather than a transient or artifactual change. Extracellular vesicles-mediated transcriptional reprogramming can sustain altered gene expression for weeks to months following a single infusion (Tan et al., 2024). This suggests that the full biological effect of a single MSCs administration may not be captured by early follow-up measurements, and that serial telomere monitoring at 3- to 6-month intervals may be optimal in clinical practice.
Older subjects and antiaging effects
The sustained increase in telomere length observed in subjects aged 60 to 78 is particularly noteworthy. Telomere wear plays both a causative role and an aggravating factor in human diseases (Blackburn et al., 2015). Telomere reduction is the most serious issue in the aging population, and excessively short telomere lengths lead to tissue dysfunction, immunosenescence, and increased disease susceptibility (Blackburn et al., 2015; López-Otín et al., 2023). The results of this study are consistent with previous case reports of a reduction in biological age through MSCs-based combined protocols (Lee and Burns, 2024).
Causes of interindividual variability in telomere response
The most prominent feature of this case study is the interindividual variability in telomere length changes after treatment (range, −0.3 to +10.1 kb/cell). Understanding this variability is essential for interpreting results and optimizing future treatment protocols. Seven key determinants are presented.
Reference telomere length
As explained earlier, the preferential elongation of critically short telomeres by telomerase creates an inverse relationship between reference telomere length and response size. This is supported by protein counting models and direct experimental evidence (Hockemeyer and Collins, 2015; Mir et al., 2020).
Genetic polymorphisms of telomere maintenance genes
Polymorphisms in hTERT (particularly rs2736100 on chromosome 5p15.33), telomerase RNA component, dyskerin pseudouridine synthase 1, and telomeric repeat-binding factor 1-interacting nuclear factor 2 can cause several-fold differences in basal telomerase activity between individuals (Choi et al., 2015; Snetselaar et al., 2018). The C allele of rs2736100 is associated with increased leukocyte telomere length, whereas the A allele is associated with decreased telomere length (Snetselaar et al., 2018). Epigenetic silencing of the hTERT promoter, which is common in aged cells, can further reduce responsiveness to paracrine telomerase activation.
Differences in extracellular vesicle uptake efficiency
Uptake of MSCs-derived extracellular vesicle by receptor leukocytes is regulated by tetraspanin expression (CD9, CD63, CD81) on the receptor cell membrane, the systemic inflammatory environment, and the redox state of the circulatory environment. Severe systemic inflammation may impair extracellular vesicle internalization and downstream telomerase activation (Tan et al., 2024; Wu et al., 2022).
Oxidative stress
Telomere DNA, which is rich in GGG trinucleotides, is the genomic region most vulnerable to 8-oxoguanine (8-hydroxy-2′-deoxyguanosine) oxidative damage (Barnes et al., 2022; De Rosa et al., 2021). In individuals with high baseline ROS levels (e.g., diabetes, metabolic syndrome), a significant portion of the antioxidant capacity of MSCs-derived extracellular vesicles is consumed in inhibiting sustained oxidative damage rather than in telomere lengthening. This “competitive consumption” effect implies that telomere responses may vary depending on an individual’s oxidative status, even with the same MSCs dosage (Armstrong and Boonekamp, 2023).
Treatment duration and cumulative dose
Although no monotonic relationship between treatment duration and response was observed among subjects, intrasubject longitudinal data obtained from CBA suggest a gradual cumulative effect over 8 months. Subjects who received multiple MSCs infusions (KKY, 4 infusions; HHJ, 5 infusions) may experience an additive effect.
Concomitant antiaging therapy
Some subjects received recombinant growth hormone, chelation therapy, or photobiomodulation therapy in conjunction with MSCs infusion. Growth hormone activates the insulin-like growth factor-1/phosphatidylinositol 3-kinase/protein kinase B signaling axis, which independently upregulates hTERT expression and enhances telomerase activity by phosphorylating the Ser-824 site of hTERT (Aulinas et al., 2013; Kaplan et al., 2009; Méndez-Pertuz et al., 2017). The telomere changes observed in subjects with confirmed increases in insulin-like growth factor-1 (IJY, KDH) may represent a complex effect rather than an MSCs-specific effect (Cawthon, 2002).
Heterogeneity of analytical methods
Two cases were reported regarding log kb/cell values, indicating that different measurement platforms were used across the entire cohort. Quantitative polymerase chain reaction-based T/S methods, flow fluorescence in situ hybridization telomere length measurements, and Southern blot terminal restriction fragment analysis can yield estimates showing a 1.5- to 3.0-fold difference for the same sample, and the coefficient of variation between analyses can reach 5%–10% even within a single platform (Cawthon, 2002). Standardization of analytical methods is essential for quantitative comparison in future studies.
In summary, the interindividual variability observed in this case study reflects multifactorial biological factors regulating telomere homeostasis rather than a lack of consistency in MSCs treatment. The distinct inverse correlation between baseline telomere length and the degree of treatment response has clinical significance. This helps identify patients with accelerated biological aging as optimal candidates for MSCs-based telomere restoration therapy and supports the use of baseline telomere measurements as patient selection criteria and key efficacy biomarkers in future clinical trials.
Several limitations must be considered. First, due to the absence of an untreated control group, a clear causal relationship regarding whether telomere elongation is caused by MSCs treatment cannot be established. Second, statistical inference is limited due to the small sample size and clinical heterogeneity. Third, quantitative comparisons are difficult due to differences in telomere measurement methods among subjects. Fourth, combination therapies (growth hormone, chelation, photobiomodulation) may be potential confounding variables. Fifth, in most cases, the follow-up period was limited to a single time point after treatment, making it impossible to evaluate the long-term persistence of the observed changes. Despite these limitations, the consistent results showing improvement of measurable cases are unlikely to be due to chance, and future prospective verification is required.
This retrospective case study presents preliminary clinical evidence that intravenous MSCs treatment is associated with a consistent and measurable increase in peripheral blood telomere length across diverse cohorts spanning various age groups, baseline telomere status, and treatment durations. These results support the hypothesis that MSCs administration exerts telomere-protective or telomere-prolonging effects, which are likely mediated through extracellular signaling, including hTERT mRNA delivery and ROS reduction.
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