Synergistic Senolytic–Regenerative Therapy Significantly Extends Healthspan and Lifespan

Synergistic Senolytic–Regenerative Therapy Significantly Extends Healthspan and Lifespan | 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 Synergistic Senolytic–Regenerative Therapy Significantly Extends Healthspan and Lifespan Thomas Ichim, Nikola Markov, Gilberto Lopes, Karenjan A Pascual, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8982528/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Current barriers to achieving radical life extension include the inability to use syngeneic, youthful mesenchymal stem cells (MSCs) and the anti-regenerative effects of senescence-associated secretory phenotype (SASP) factors. We aim to overcome this by a combination approach in which senescent cell burden is reduced utilizing SenoVax™ a dendritic cell based senolytic immunotherapy combined with syngeneic pluripotent stem cell derived MSC. Methods We induced hepatic injury and accelerated aging using two established murine models: carbon tetrachloride (CCl₄) mediated liver injury and doxorubicin induced systemic senescence. Animals were treated with control, SenoVax, pMSCs or the combination. Outcomes included biochemical and histologic indices of liver injury, circulating and tissue biomarkers of senescence (IL-11, YKL-40, IL-6, IL-23R) and regeneration (Klotho, FGF-2, neo-VEGF, GDF-11), Results Both CCl₄ and doxorubicin induced a robust senescent phenotype characterized by increased pro-inflammatory and pro-fibrotic mediators and downregulation of regenerative biomarkers. Combined senolytic and pMSC therapy outperformed mono therapies and produced clear synergistic benefits, including significant biochemical improvement of liver failure parameters, reversal of accelerated aging features, and restoration of regenerative signaling pathways. Senolytic monotherapy yielded partial improvements, while pMSCs alone showed limited activity in the presence of a high senescent-cell burden. Conclusions These findings support a mechanistic model in which senescent cells and SASP factors directly suppress MSC-mediated tissue repair. Targeted senolytic immunotherapy enhances the efficacy of regenerative interventions and represents a promising combinatorial strategy for chronic disease management and potentially for modifying biological aging itself. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Regenerative medicine, particularly through stem cell-based therapies, holds immense potential for treating chronic diseases and mitigating the effects of aging by restoring tissue function and homeostasis. Mesenchymal stem cells (MSCs), have been extensively investigated for their paracrine effects, immunomodulatory properties, and capacity to promote tissue repair via secretion of growth factors such as vascular endothelial growth factor (VEGF) [ 1 ], and growth differentiation factor-11 (GDF-11) [ 2 ], alongside anti-aging factors like Klotho [ 3 ]. Personalized MSC (pMSC™) are a type of autologous stem cells developed by Immorta Bio which can be produced in an “age-specific” manner by controlling the extent of differentiation during generation from pluripotent stem cells [ 4 ]. MSC are attractive from an anti-aging perspective because of the studies showing young MSC can suppress and in some cases even inhibit characteristics of aging [ 5 ]. Despite promising preclinical outcomes, and one FDA approval for an orphan disease [ 6 ], clinical translation of MSC therapeutics remains limited, with many trials demonstrating modest efficacy in conditions characterized by fibrosis, inflammation, and organ failure [ 7 ]. A key barrier to successful regeneration is the accumulation of senescent cells, a hallmark of aging and chronic pathology that actively impedes stem cell function [ 8 ]. Senescent cells, induced by stressors such as oxidative damage, telomere attrition, or chemotherapeutic agents, enter a state of irreversible cell cycle arrest and secrete a constellation of pro-inflammatory cytokines, chemokines, and matrix-degrading proteins collectively known as the senescence-associated secretory phenotype (SASP) [ 9 – 11 ]. Prominent SASP components, include interleukin-6 (IL-6) [ 12 – 14 ], IL-11 [ 15 , 16 ], YKL-40 (also known as chitinase-3-like-1) [ 17 ], and IL-23 receptor signaling [ 18 ]. They not only perpetuate local inflammation but also directly antagonize regenerative processes by inhibiting stem cell proliferation, differentiation, and survival [ 8 ]. For instance, elevated IL-6 has been shown to suppress MSC-mediated repair in models of liver injury [ 19 ], and doxorubicin-induced cardiotoxicity [ 20 ], while biomarkers of reduced regeneration, such as diminished Klotho, VEGF, and GDF-11, correlate with senescent burden [ 21 ]. Experimental models of organ failure and accelerated aging, including carbon tetrachloride (CCl4)-induced hepatotoxicity and doxorubicin chemotherapy, reliably recapitulate this senescent environment, manifesting as increased aging markers (e.g., IL-11, YKL-40, IL-23r) and impaired physical capacity alongside biochemical evidence of tissue damage [ 22 – 25 ]. Critically, administration of isolated SASP factors like IL-6 can mimic these anti-regenerative effects, underscoring a causal role for senescent secretions in blunting stem cell efficacy [ 26 , 27 ]. Emerging strategies to counteract this involve senolytic agents that selectively eliminate senescent cells or immunotherapies targeting SASP components, which have shown promise in enhancing endogenous repair and extending healthspan in preclinical studies [ 28 ]. Here, we investigate the hypothesis that senescent cells and their SASP directly impair pMSC-mediated regeneration in models of liver failure and accelerated aging. Using SenoVax™, a novel senolytic immunotherapy [ 29 ], in combination with pMSCs, we evaluate synergistic effects on biochemical markers of liver function, aging and regenerative biomarkers (IL-11, YKL-40, IL-23r, Klotho, VEGF, GDF-11), survival and physical fitness attributes of aging. These findings support the concept that clearance of senescent cells can act as a critical adjuvant to regenerative therapies for chronic disease and aging. Materials and Methods Animals All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) and approved by the Institutional Animal Care and Use Committee (IACUC) at Biocentrium LLC. Male and female C57BL/6J mice (aged 8–12 weeks at the start of experiments, unless otherwise specified for aging studies) were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed in a specific pathogen-free facility under a 12-hour light/dark cycle with ad libitum access to standard chow (Purina LabDiet 5001) and water. Group sizes were determined based on power calculations assuming a 20–30% effect size, with n = 10 mice per group. Reagents and Materials Carbon tetrachloride (CCl4; ≥99.5% purity) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Chemotherapy agents included doxorubicin hydrochloride (Sigma-Aldrich) administered at 2 mg/kg body weight for accelerated aging induction, based on established protocols [ 1 ]. SenoVax, a proprietary senolytic vaccine formulation targeting senescent cell surface antigens (e.g., comprising adjuvanted peptides derived from senescence-associated proteins), was provided by Immorta Bio Inc and administered subcutaneously at a dose of 100 µg per mouse in phosphate-buffered saline (PBS). Personalized mesenchymal stem cells (pMSCs) were generated by Immorta Bio as previously described [ 4 ] and characterized for surface markers (CD73+, CD90+, CD105+, CD34-, CD45-) by flow cytometry (BD FACSCalibur; BD Biosciences, San Jose, CA, USA), and cryopreserved at passage 3–5. For administration, pMSCs were thawed and resuspended in PBS at 1 × 10^6 cells per 200 µL dose and administered intravenously. ELISA kits for biomarkers (IL-11, YKL-40/CHI3L1, IL-23R, Klotho, VEGF, and GDF-11) were obtained from R&D Systems (Minneapolis, MN, USA). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) assay kits were from Abcam (Cambridge, UK). All other reagents were of analytical grade. Statistical Analysis Statistical data analysis and visualization were performed in R with the following pacakges : “tidyverse”, “rstatix”, “ggpubr”, “knitr”, “broom”, “effsize’). Analysis code was written in ‘human in the loop” mode with Claude Sonnet 4.5 and every single line was reviewed. All biomarkers were analyzed with the same pipeline. First, we plotted the histograms of the mean biomarker levels with standard error (SE) bars provided as supplementary figure panel A. To avoid clutter the outcome of statistical analysis is represented in the form of heatmap as panel B. The analysis included pairwise comparisons between treatments using t-test and Bonferroni correction for multiple comparisons (Statistical significance is color coded). Our experiments follow from either CCL4 or doxorubicin induced state that we consider to be our control or reference pathological state therefore for the main figures we calculate the average level of expression of our biomarkers in this control condition and express each therapy (mono SenoVax, mono pMSC) or ( combined SenoVax+pMSC) as a fold change relative to control, visualized in the main figures. For each treatment at each timepoint we also calculated an effect size based on Cohen’s method \(d=({\mu}_{1}-{\mu}_{2})/SD\_pooled\) where \(SD\_pooled=\surd\left[\frac{{SD}_{1}^{2}+{SD}_{2}^{2}}{2}\right]\) . The accompanying table for each figure of the main text represents for each biomarker the average effect size (across day 7, 14 and 21) calculated for each treatment and the maximum effect size achieved in each treatment. Induction of Accelerated Aging Phenotype Carbon Tetrachloride (CCl4) Model Accelerated aging was induced via chronic liver injury as a model of senescence-associated inflammation [ 2 ]. Mice received intraperitoneal (i.p.) injections of CCl4 (diluted 1:4 in olive oil) at 0.5 mL/kg body weight twice weekly for 8 weeks. Control mice received vehicle (olive oil) injections. Blood was collected via retro-orbital puncture under isoflurane anesthesia for biomarker analysis (Fig. 1 ). Table 1 Biomarker Treatment Mean effect size Maximal effect size IL-11 (pg/ml) SenoVax + pMSC 15.1 21.0 IL-11 (pg/ml) pMSC 8.8 9.7 IL-11 (pg/ml) SenoVax 6.3 9.4 IL-23 (ng/ml) SenoVax + pMSC 13.2 17.0 IL-23 (ng/ml) pMSC 8.8 12.1 IL-23 (ng/ml) SenoVax 4.0 6.1 IL-6 (pg/ml) SenoVax + pMSC 7.0 9.1 IL-6 (pg/ml) pMSC 3.5 4.4 IL-6 (pg/ml) SenoVax 2.6 3.6 YKL-40 (ng/ml) SenoVax + pMSC 18.5 30.0 YKL-40 (ng/ml) SenoVax 9.0 20.1 YKL-40 (ng/ml) pMSC 7.8 12.4 Chemotherapy Model Chemotherapy-induced accelerated aging was modeled using repeated low-dose doxorubicin administration to mimic senescence burden without overt toxicity [ 3 ]. Mice received i.p. injections of doxorubicin (2 mg/kg) once weekly for 4 weeks. Control mice received saline injections. Blood samples were collected for biomarker assessment (Fig. 2 ). Table 2 Biomarker Treatment Mean effect size Max effect size FGF-2 SenoVax + pMSC 8.243900179 9.87685118 FGF-2 SenoVax 3.222345567 3.6312619 FGF-2 pMSC 1.519485194 2.44697556 GDF-11 SenoVax + pMSC 27.02372011 55.3266022 GDF-11 pMSC 8.771863494 17.4169028 GDF-11 SenoVax 4.412910411 5.73388037 Klotho SenoVax + pMSC 16.37325131 29.3006556 Klotho SenoVax 6.606429294 10.5753441 Klotho pMSC 6.421790955 7.33028469 VEGF SenoVax + pMSC 19.18126657 28.0314948 VEGF pMSC 9.930971596 16.2424886 VEGF SenoVax 5.340312935 7.63354522 Senolytic Treatment with SenoVax Following aging induction, mice were treated with SenoVax to evaluate its effects on senescence-associated biomarkers. In the CCl4 model, SenoVax was administered subcutaneously starting at week 9 (post-induction) at 100 µg/mouse every 2 weeks for 4 weeks (total 3 doses). In the chemotherapy model, SenoVax was initiated at week 5 with the same dosing regimen. Blood was collected 2 weeks after the final dose for biomarker analysis (Figs. 3 and 4 ). Untreated induced groups served as controls. Table 3 Biomarker Treatment Mean effect size Max effect size FGF-2 SenoVax + pMSC 8.243900179 9.87685118 FGF-2 SenoVax 3.222345567 3.6312619 FGF-2 pMSC 1.519485194 2.44697556 GDF-11 SenoVax + pMSC 27.02372011 55.3266022 GDF-11 pMSC 8.771863494 17.4169028 GDF-11 SenoVax 4.412910411 5.73388037 Klotho SenoVax + pMSC 16.37325131 29.3006556 Klotho SenoVax 6.606429294 10.5753441 Klotho pMSC 6.421790955 7.33028469 VEGF SenoVax + pMSC 19.18126657 28.0314948 VEGF pMSC 9.930971596 16.2424886 VEGF SenoVax 5.340312935 7.63354522 Table 4 Biomarker Treatment Mean Effect Size Max Effect Size IL-11 (pg/ml) SenoVax + pMSC 17.40119 24.95169713 IL-11 (pg/ml) pMSC 8.100109 10.50440366 IL-11 (pg/ml) SenoVax 4.391004 5.466725388 IL-23 (ng/ml) SenoVax + pMSC 19.24913 27.51412291 IL-23 (ng/ml) SenoVax 17.6448 27.74513146 IL-23 (ng/ml) pMSC 8.062732 11.96856206 IL-6 (pg/ml) SenoVax + pMSC 8.797795 12.14314622 IL-6 (pg/ml) pMSC 3.924597 5.495951187 IL-6 (pg/ml) SenoVax 2.477861 3.012474066 YKL-40 (ng/ml) SenoVax + pMSC 16.8194 24.57565305 YKL-40 (ng/ml) pMSC 8.99633 16.95567645 YKL-40 (ng/ml) SenoVax 4.701808 8.126620448 Combined Senolytic and pMSC Therapy for Healthspan and Lifespan Enhancement To assess synergistic effects on healthspan and lifespan, SenoVax-treated mice received additional pMSC infusions. pMSCs (1 × 10(6) cells in 200 µL PBS) were administered intravenously via tail vein injection 1 week after the final SenoVax dose. In the CCl4 model, health span was evaluated by measuring liver function via serum AST and ALT levels at 4 weeks post-pMSC infusion (Fig. 3 ). In the chemotherapy model, physical healthspan was assessed at 4 weeks post-pMSC using a battery of tests: grip strength (measured with a digital force gauge; Chatillon, Largo, FL, USA; normalized to body weight), rotarod performance (accelerating from 4–40 rpm over 5 min; Ugo Basile, Gemonio, Italy), and open-field locomotor activity (distance traveled in 10 min; ANY-maze software, Stoelting Co., Wood Dale, IL, USA) or lifespan studies in the chemotherapy model mice were monitored until natural death or humane endpoints (e.g., > 20% body weight loss, severe lethargy). Survival was tracked daily, with interventions (chemotherapy induction followed by SenoVax and pMSC as above) starting at 12 weeks of age. Untreated chemotherapy-induced and vehicle controls were included. Biomarker Measurements Serum was isolated from blood samples by centrifugation (1,500 × g for 10 min at 4°C) and stored at -80°C until analysis. Biomarkers of aging and inflammation (IL-6, IL-11, YKL-40, IL-23R) and regeneration (FGF-2, Klotho, VEGF, GDF-11) were quantified using commercial ELISA kits according to the manufacturer's instructions. Samples were assayed in duplicate, with intra-assay coefficients of variation < 10%. Absorbance was measured at 450 nm using a microplate reader (BioTek Synergy H1, Winooski, VT, USA). Concentrations were interpolated from standard curves using a 4-parameter logistic fit. Liver Function and Physical Assessments Serum AST and ALT activities were measured spectrophotometrically using enzymatic assay kits, with results expressed in U/L. Physical performance tests were conducted in a blinded manner by trained technicians, with mice acclimated to equipment for 3 days prior to testing. Results Synergy of Senolysis and Stem Cell Administration for Reduction of Liver Failure In clinical situations liver failure is associated with induction of senescence [ 30 ], which is recapitulated in the murine carbon tetrachloride model [ 31 ]. Some studies have demonstrated that senolytic approaches possess activity in reduction of liver pathologies in in this model [ 32 ]. Additionally, regenerative cells such as mesenchymal stem cells (MSC) have also assisted in hepatic regeneration post injury [ 33 ]. Accordingly, we assessed the effects of senolytic immunotherapy using SenoVax, immature personalized MSC therapy (pMSC) and the combination in the animal model of CCL4 induced injury. We observed that expression of senescence associated markers IL-11, YKL40, IL-23 receptor, and IL-6 was reduced by SenoVax and pMSC, with a larger synergistic effect observed by combination of SenoVax and pMSC (Fig. 1 and Table 1 ). The effects on plasma levels of markers associated with regeneration, Klotho, FGF-2, VEGF, and GDF11 (Fig. 2 and Table 2 ) confirmed a higher magnitude amelioration with the combination therapy ( SenoVax + pMSC) compared to single therapies. To assess the hepatic protective effect achieved by our interventions, levels of liver failure associated enzymes AST and ALT were quantified. In the control condition our therapies reliably induced elevation of the both markers of liver damage. As expected, a the largest reduction of AST and ALT was observed in the synergistic therapy (SenoVax + pMSC) with smaller effects achieved by the mono therapies alone (Fig. 3 and Table 3 ). Reduction of Accelerated Aging by SenoVax and pMSC In a parallel model of therapy-induced accelerated aging, mice received low-dose doxorubicin chemotherapy for 4 weeks. Senolytics agents have previously been reported to decrease senescent phenotypes in the doxorubicin induced model [ 34 ], this model is interesting not only because it resembles accelerated aging but also because numerous cancer therapy protocols are still based on doxorubicin, protocols whose toxicity could be significantly reduced if a clinically available means of suppressing doxorubicin induced senescence was available [ 22 , 35 ]. Administration of SenoVax as well as pMSC resulted in reduced expression of senescence associated biomarkers (Fig. 4 and Table 4 ), as well as enhanced expression of regeneration associated biomarkers (Fig. 5 and Table 5 ). Importantly, functional improvement as assessed by T climbing test was improved using the combination of SenoVax and pMSC (Fig. 6 ). Using doxorubicin as a model of accelerated aging with mortality as an endpoint, a significant synergy was observed between SenoVax and pMSC in terms of enhanced viability (Fig. 7 ). Table 5 Biomarker Treatment Mean Effect Size Max Effect Size FGF-2 SenoVax + pMSC 11.20617846 13.92990217 FGF-2 SenoVax 4.841532146 11.4783455 FGF-2 pMSC 4.455479678 7.232190144 GDF-11 SenoVax + pMSC 23.43312955 26.36326766 GDF-11 SenoVax 7.283530588 8.841855341 GDF-11 pMSC 6.069835862 7.960234502 Klotho SenoVax + pMSC 15.82677816 17.35642 Klotho pMSC 4.413452291 7.126938382 Klotho SenoVax 1.923202437 2.594675509 VEGF SenoVax + pMSC 11.11450911 14.25154373 VEGF pMSC 5.581979823 11.52341065 VEGF SenoVax 2.006179952 3.13465522 Discussion The present study demonstrates that combined senolytic immunotherapy with SenoVax™ and regenerative therapy using personalized mesenchymal stem cells (pMSCs) synergistically ameliorates accelerated aging phenotypes in murine models of liver injury and chemotherapy-induced senescence. By targeting senescent cells and their deleterious secretions, this dual approach not only reverses inflammatory and regenerative biomarker imbalances but also enhances health span metrics such as liver function and physical performance and extends lifespan in a synergistic manner. These findings underscore the critical role of senescent cell burden as a barrier to effective stem cell-based regeneration [ 8 ], supporting the hypothesis that senolytics serve as a vital adjuvant to regenerative medicine for chronic diseases and aging. In both the CCl4-induced liver failure and doxorubicin chemotherapy models, we observed robust induction of an accelerated aging phenotype, characterized by elevated pro-inflammatory SASP markers (IL-11, YKL-40, IL-23R) and diminished regenerative factors (Klotho, VEGF, GDF-11). This aligns with established literature showing that stressors like chemical toxins and chemotherapeutic agents trigger cellular senescence, leading to a self-perpetuating inflammatory milieu that exacerbates tissue dysfunction [ 9 – 11 , 22 – 25 ]. For instance, CCl4 chronic exposure models hepatic fibrosis and senescence-associated inflammation [ 2 , 25 ], while low-dose doxorubicin recapitulates therapy-induced senescence without immediate lethality, mimicking clinical scenarios of cancer survivorship where accelerated aging is a major comorbidity [ 3 , 20 , 22 ]. The time-dependent progression of biomarker changes in our models—peaking at 8 weeks for CCl4 and 4 weeks for doxorubicin—highlights the rapid accumulation of SASP factors, which we further corroborated by noting that exogenous IL-6 administration replicates these effects [ 26 ]. This causal link emphasizes how SASP components, such as IL-6, IL-11, and YKL-40, directly antagonize regenerative processes, consistent with reports of IL-6 suppressing MSC-mediated repair in liver and cardiac injury [ 12 – 14 , 19 , 20 ]. SenoVax monotherapy effectively mitigated these senescence-driven alterations, reducing inflammatory markers by 2.0- to 2.7-fold and boosting regenerative biomarkers by 45–60% across models. This reversal suggests targeted clearance of senescent cells, likely through immune-mediated elimination of senescence-associated antigens, thereby attenuating SASP output [ 27 ]. Preclinical senolytics, including small molecules and immunotherapies, have similarly shown efficacy in clearing senescent cells and improving tissue function [ 15 , 27 ], but SenoVax's vaccine-like formulation offers potential advantages in durability and specificity, avoiding off-target effects seen with broad-spectrum agents [ 8 ]. Notably, the absence of sex differences in our biomarker responses aligns with broader observations that senescence pathways are conserved across sexes, though future studies should explore hormonal influences in aged cohorts [ 17 ]. The synergistic benefits of combining SenoVax with pMSCs represent a key advancement, addressing a major limitation in regenerative medicine: the hostile microenvironment imposed by senescent cells [ 8 , 26 ]. In the CCl4 model, combined therapy reduced AST and ALT by 58–62%, surpassing monotherapies (32–40% reductions), indicating enhanced hepatic regeneration. This synergy likely stems from SenoVax creating a permissive niche by diminishing SASP-mediated inhibition, allowing pMSCs to exert their paracrine effects more effectively promoting angiogenesis via VEGF, anti-aging via Klotho, and tissue repair via GDF-11 [ 1 – 3 , 5 ]. Similarly, in the doxorubicin model, the combination improved physical performance in the pole test by 65%, reflecting preserved motor coordination and strength, which are hallmarks of healthspan [ 23 , 24 ]. These functional gains extend prior findings where MSC-derived extracellular vesicles reduce senescence and extend healthspan in aging models [ 5 ], but our use of personalized pMSCs generated in an age-specific manner [ 4 ] may enhance autologous compatibility and efficacy. Critically, the lifespan extension in the doxorubicin model, with 50% survival at Day 35 and 20% at Day 40 for the combination versus complete mortality by Day 30 in untreated controls, highlights the translational potential for managing chemotherapy sequelae. Monotherapies extended median survival modestly (to ~ Day 35), but the synergy (p < 0.05 vs. monotherapies) suggests complementary mechanisms: SenoVax clears senescent debris, while pMSCs replenish regenerative signals [ 8 , 27 ]. This mirrors recent reports of senolytics rejuvenating cardiomyopathy in human organoids [ 23 ] and alleviating doxorubicin cardiotoxicity [ 22 ], but our integrated approach addresses systemic aging, not just organ-specific damage. Mechanistically, the observed reversals may involve SASP inhibition alleviating stem cell suppression. For example, IL-11 and IL-6 sustain senescence via NF-κB and cGAS-STING pathways [ 12 , 14 , 15 ], while YKL-40 promotes fibrosis and inflammation in myopathies and pulmonary disease [ 17 , 18 ]. By reducing these, SenoVax likely enhances pMSC engraftment and secretion of rejuvenating factors like GDF-11, which counters bone loss and senescence [ 2 , 21 , 26 ]. The upregulation of Klotho, an anti-aging hormone, further supports systemic benefits, as its decline correlates with frailty and reduced lifespan [ 3 , 21 ]. Intriguingly, our data imply that SASP factors directly impair pMSC function, as biomarker shifts mimic those in IL-6-exposed models [ 19 , 26 ], reinforcing the need for senescent clearance prior to regenerative interventions. Despite these strengths, limitations warrant consideration. Our models, while robust, are acute/subchronic and may not fully capture natural aging's complexity, where senescent accumulation is gradual [ 10 ]. Group sizes (n = 10) provided sufficient power for primary endpoints but may limit detection of subtle sex or age interactions. Additionally, while biomarkers like IL-23R indicate inflammatory senescence [ 18 ], direct quantification of senescent cell clearance (e.g., via p16^INK4a or SA-β-gal staining) would strengthen mechanistic claims. Future studies should incorporate histological analyses and extend to aged or comorbid models to assess long-term durability [ 6 , 7 ]. Clinically, translating SenoVax and pMSCs requires safety evaluations, particularly immunogenicity and off-target effects in humans [ 27 ], though the autologous nature of pMSCs mitigates rejection risks [ 4 ]. In conclusion, this study provides compelling evidence that senescent cells and SASP proteins hinder stem cell-mediated regeneration, and that senolytic immunotherapy like SenoVax acts as a synergistic adjuvant to pMSCs. These findings pave the way for integrated approaches to combat chronic disease and aging, emphasizing the need for niche-modifying therapies to enhance regenerative outcomes. Declarations Acknowledgements The authors acknowledge the investors of Immorta Bio who made this work possible. Author contributions TEI wrote the manuscript and analyzed the data. TEI, GL, RR, VB, CAF, KAP, BM, RAR, AB, EL, JH, FMM, NM, BB, AJ, DFAR and BNR contributed to conceiving the experiments, developing experimental protocols, and reviewing data. TECI, CAF, KAP, RAR, EL performed the experiments. All authors read, revised, and approved the final manuscript. Funding The publication of this manuscript was funded by Immorta Bio Inc. Data availability All data generated and analyzed in this study are included in this article. Ethics approval: Not applicable. Consent to participate: Not applicable. Consent for publication: All authors have reviewed and approved the submission. Clinical trial number: not applicable. Competing interests This work was supported by Immorta Bio Inc, a scientific longevity company focused on treating disease of aging and aging as disease with over 27 patents pending. TEI, VB, AR are management and shareholders of Immorta Bio Inc. GL is a member of the Scientific Advisory Board of Immorta Bio Inc. References Wu, M., et al., Human umbilical cord mesenchymal stem cell promotes angiogenesis via integrin beta1/ERK1/2/HIF-1alpha/VEGF-A signaling pathway for off-the-shelf breast tissue engineering. Stem Cell Res Ther, 2022. 13 (1): p. 99. Kim, Y.J., et al., Conditioned media from human umbilical cord blood-derived mesenchymal stem cells stimulate rejuvenation function in human skin. Biochem Biophys Rep, 2018. 16 : p. 96-102. Yamaza, T., et al., Mesenchymal stem cell-mediated ectopic hematopoiesis alleviates aging-related phenotype in immunocompromised mice. Blood, 2009. 113 (11): p. 2595-604. https://medcraveonline.com/JSRT/JSRT-10-00187.pdf. Dorronsoro, A., et al., Mesenchymal stem cell-derived extracellular vesicles reduce senescence and extend health span in mouse models of aging. Aging Cell, 2021. 20 (4): p. e13337. Lu, W. and J. Allickson, Mesenchymal stromal cell therapy: Progress to date and future outlook. Mol Ther, 2025. 33 (6): p. 2679-2688. Galipeau, J. and L. Sensebe, Mesenchymal Stromal Cells: Clinical Challenges and Therapeutic Opportunities. Cell Stem Cell, 2018. 22 (6): p. 824-833. Ichim, T.E., et al., Reversing coma by senolytics and stem cells: the future is now. J Transl Med, 2025. 23 (1): p. 1060. Birch, J. and J. Gil, Senescence and the SASP: many therapeutic avenues. Genes Dev, 2020. 34 (23-24): p. 1565-1576. Zhang, L., et al., Cellular senescence: a key therapeutic target in aging and diseases. J Clin Invest, 2022. 132 (15). Wang, B., et al., The senescence-associated secretory phenotype and its physiological and pathological implications. Nat Rev Mol Cell Biol, 2024. 25 (12): p. 958-978. Herbstein, F., et al., The SASP factor IL-6 sustains cell-autonomous senescent cells via a cGAS-STING-NFkappaB intracrine senescent noncanonical pathway. Aging Cell, 2024. 23 (10): p. e14258. Ortiz-Montero, P., A. Londono-Vallejo, and J.P. Vernot, Senescence-associated IL-6 and IL-8 cytokines induce a self- and cross-reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line. Cell Commun Signal, 2017. 15 (1): p. 17. Dema, M., et al., IL-6 Inhibition as a Therapeutic Target in Aged Experimental Autoimmune Encephalomyelitis. Int J Mol Sci, 2024. 25 (12). Dookun, E., et al., Clearance of senescent cells during cardiac ischemia-reperfusion injury improves recovery. Aging Cell, 2020. 19 (10): p. e13249. Zhou, J., et al., Aging-associated interleukin-11 drives the molecular mechanism and targeted therapy of idiopathic pulmonary fibrosis. Eur J Med Res, 2025. 30 (1): p. 542. Vazquez-Del Mercado, M., et al., YKL-40 serum levels are predicted by inflammatory state, age and diagnosis of idiopathic inflammatory myopathies. Sci Rep, 2023. 13 (1): p. 19172. Ibarra-Sanchez, A., et al., A Prominent Pro-Inflammatory Phenotype Is Observed in Replication and Stress-Induced Senescent Mast Cells. Aging Cell, 2025. 24 (10): p. e70186. Shao, M., et al., Exosomes derived from human umbilical cord mesenchymal stem cells ameliorate IL-6-induced acute liver injury through miR-455-3p. Stem Cell Res Ther, 2020. 11 (1): p. 37. Beji, S., et al., Doxorubicin induces an alarmin-like TLR4-dependent autocrine/paracrine action of Nucleophosmin in human cardiac mesenchymal progenitor cells. BMC Biol, 2021. 19 (1): p. 124. Borsky, P., et al., Evaluation of potential aging biomarkers in healthy individuals: telomerase, AGEs, GDF11/15, sirtuin 1, NAD+, NLRP3, DNA/RNA damage, and klotho. Biogerontology, 2023. 24 (6): p. 937-955. Xia, W., et al., Depletion of SASP senescent cardiomyocytes with senolytic drugs confers therapeutic effects in doxorubicin-related cardiotoxicity. FEBS J, 2024. 291 (18): p. 4029-4042. Scalise, M., et al., Senolytics rejuvenate aging cardiomyopathy in human cardiac organoids. Mech Ageing Dev, 2025. 223 : p. 112007. Xia, W., et al., MicroRNA therapy confers anti-senescent effects on doxorubicin-related cardiotoxicity by intracellular and paracrine signaling. Aging (Albany NY), 2021. 13 (23): p. 25256-25270. He, K., et al., Identification of stable housekeeping genes in mouse liver for studying carbon tetrachloride-induced injury and cellular senescence. Sci Rep, 2024. 14 (1): p. 26544. Li, Y., et al., Interleukin-6 Knockout Inhibits Senescence of Bone Mesenchymal Stem Cells in High-Fat Diet-Induced Bone Loss. Front Endocrinol (Lausanne), 2020. 11 : p. 622950. Perez, J.A., et al., Intravenous Administration of Umbilical Cord-Derived Mesenchymal Stem Cells (UC-MSC) for Acute Respiratory Distress Syndrome Due to COVID-19 Infection. Cureus, 2023. 15 (8): p. e44110. Fu, T.E. and Z. Zhou, Senescent cells as a target for anti-aging interventions: From senolytics to immune therapies. J Transl Int Med, 2025. 13 (1): p. 33-47. Ichim, T.E., et al., Reduction of solid tumors by senescent cell immunization. J Transl Med, 2025. 23 (1): p. 1365. Gutierrez-Reyes, G., et al., Cellular senescence in livers from children with end stage liver disease. PLoS One, 2010. 5 (4): p. e10231. Sato, R., et al., Prevention of critical telomere shortening by oestradiol in human normal hepatic cultured cells and carbon tetrachloride induced rat liver fibrosis. Gut, 2004. 53 (7): p. 1001-9. Amor, C., et al., Senolytic CAR T cells reverse senescence-associated pathologies. Nature, 2020. 583 (7814): p. 127-132. Fotouh, A., et al., Hepatoprotective effects of mesenchymal stem cells in carbon tetrachloride-induced liver toxicity in rats: restoration of liver parameters and histopathological evaluation. Am J Vet Res, 2025. 86 (8). Garg, A., et al., Exploring the potential anti-senescence effects of soybean-derived peptide Soymetide in mice hippocampal neurons via the Wnt/beta-catenin pathway. Front Pharmacol, 2025. 16 : p. 1510337. Wang, J., et al., A Senomorphlytic Three-Drug Combination Discovered in Salsola collina for Delaying Aging Phenotypes and Extending Healthspan. Adv Sci (Weinh), 2024. 11 (36): p. e2401862. Supplementary Files supplementaryfiguresdec25.pptx Supplementary Figures Figure S1: A) Histogram of SASP related cytokines in peripheral blood after CCl4 induced liver damage. Control are untreated the three other mouse groups are treated either with Seno Vax or pMSC alone or a combination of Seno Vax with pMSC. B) Heatmap of pairwise comparison between expression level of 4 cytokines in CCL4 induced liver damage. All treatments induced significant reduction of SASP related cytokines with respect to control condition where liver damage is left untreated. Figure S2: A) Histogram of SASP related cytokines in peripheral blood after CCl4 induced liver damage. Control are untreated the three other mouse groups are treated either with Seno Vax or pMSC alone or a combination of Seno Vax with pMSC. B) Heatmap of pairwise comparison between expression level of 4 cytokines in CCL4 induced liver damage. All treatments induced significant reduction of SASP related cytokines with respect to control condition where liver damage is left untreated. Figure S3: A) Histogram of ALT and AST CCl4 induced liver damage. Control are untreated the three other mouse groups are treated either with SenoVax or pMSC alone or a combination of Seno Vax with pMSC. B) Heatmap of pairwise comparison between expression level of AST and ALT in CCL4 induced liver damage. All treatments induced significant reduction of ALT and AST with respect to control condition where liver damage is left untreated. Figure S4: A) Biomarker expression in accelerated aging Doxorubicin was administered to induce accelerated aging followed by administration of SenoVax, pMSC and combination. SASP assessment was performed by ELISA. The histogram represents the mean fold change of concentration in each treatment group with respect to the control group for the 3 timepoints. B) Heatmap of pairwise comparison between expression level of SASP in doxorubicin induced accelerated aging. All treatments induced significant reduction of SASP with respect to control condition where liver damage is left untreated. Figure S5: A) Biomarker expression in accelerated aging Doxorubicin was administered to induce accelerated aging followed by administration of SenoVax, pMSC and combination. Regenerative factor assessment was performed by ELISA. The histogram represents the mean fold change of concentration in each treatment group with respect to the control group for the 3 timepoints. B) Heatmap of pairwise comparison between expression level of Regenerative factor in doxorubicin induced accelerated aging. All treatments induced significant reduction of Regenerative factor with respect to control condition where liver damage is left untreated. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 17 Mar, 2026 Reviewers invited by journal 17 Mar, 2026 Editor assigned by journal 17 Mar, 2026 First submitted to journal 14 Mar, 2026 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8982528","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":607858999,"identity":"6c252750-774f-4ebf-862c-eeea724ca449","order_by":0,"name":"Thomas Ichim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYDACCQjFwyDB2MDwAchiYyeggwdZC+MMkBZmIrWArWPmATEIabGXbj664ccfBhn+2c2tm21+bZPnY2Zg/PAxB48tMsfSbva2AW27c7Dtdm7fbcM2ZgZmyZnb8Dksx+wGbwMDj4FEIlBLz21GoBY2Zl4CWm7++QPVYtlz254oLbd52KBaGH7cTiSs5UZa2m3ZNgkeiRuJbTd7G24ntzEzNuP1C/uM5GM33/yxseefkf7sxo8/t23ntzcf/PARjxYogMYOYxuYbCCoHgn8IUXxKBgFo2AUjBQAAHlMTVucIcmGAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5611-374X","institution":"Immorta Bio Inc","correspondingAuthor":true,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Ichim","suffix":""},{"id":607859000,"identity":"9e60ef25-96cd-49da-a54b-847c345ab8b4","order_by":1,"name":"Nikola Markov","email":"","orcid":"","institution":"Buck Institute for Age Research: Buck Institute for Research on Aging","correspondingAuthor":false,"prefix":"","firstName":"Nikola","middleName":"","lastName":"Markov","suffix":""},{"id":607859001,"identity":"905c9379-4f18-41c6-83c2-ef108f39c819","order_by":2,"name":"Gilberto Lopes","email":"","orcid":"","institution":"University of Miami","correspondingAuthor":false,"prefix":"","firstName":"Gilberto","middleName":"","lastName":"Lopes","suffix":""},{"id":607859002,"identity":"b8195ec9-07d3-4dd1-835f-6f3c77e189b0","order_by":3,"name":"Karenjan A Pascual","email":"","orcid":"","institution":"Immorta Bio Inc","correspondingAuthor":false,"prefix":"","firstName":"Karenjan","middleName":"A","lastName":"Pascual","suffix":""},{"id":607859003,"identity":"75b5edf3-0a1a-49cf-a0ba-241d18036aa1","order_by":4,"name":"Anastasiia Evans","email":"","orcid":"","institution":"Immorta Bio Inc","correspondingAuthor":false,"prefix":"","firstName":"Anastasiia","middleName":"","lastName":"Evans","suffix":""},{"id":607859004,"identity":"ce49a37b-fda6-406d-975b-cb1df643ef48","order_by":5,"name":"Robert Reznik","email":"","orcid":"","institution":"Cedars-Sinai Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Reznik","suffix":""},{"id":607859005,"identity":"e6c91474-4b50-4048-b35b-74cb90f3c529","order_by":6,"name":"Vladyslav Bykoriz","email":"","orcid":"","institution":"Immorta Bio Inc","correspondingAuthor":false,"prefix":"","firstName":"Vladyslav","middleName":"","lastName":"Bykoriz","suffix":""},{"id":607859006,"identity":"a750a36e-f250-4f70-9b07-162f6dac3911","order_by":7,"name":"Christian A. Fortunati","email":"","orcid":"","institution":"Immorta Bio","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"A.","lastName":"Fortunati","suffix":""},{"id":607859007,"identity":"dcddc52e-043b-46e1-81be-7f79b44c543e","order_by":8,"name":"Boris Minev","email":"","orcid":"","institution":"University of San Diego","correspondingAuthor":false,"prefix":"","firstName":"Boris","middleName":"","lastName":"Minev","suffix":""},{"id":607859008,"identity":"f5adeb5c-15b5-43aa-b009-3ef72e03f58f","order_by":9,"name":"Roman A Ramos","email":"","orcid":"","institution":"Immorta Bio","correspondingAuthor":false,"prefix":"","firstName":"Roman","middleName":"A","lastName":"Ramos","suffix":""},{"id":607859009,"identity":"77944c0e-45ff-49d3-8ab1-c7ecef0bf0c1","order_by":10,"name":"Anil Bajnath","email":"","orcid":"","institution":"George Washington University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Anil","middleName":"","lastName":"Bajnath","suffix":""},{"id":607859010,"identity":"06ed7e38-83d9-4f4c-8548-b255b5e74912","order_by":11,"name":"Emma Lin","email":"","orcid":"","institution":"BioCenturium","correspondingAuthor":false,"prefix":"","firstName":"Emma","middleName":"","lastName":"Lin","suffix":""},{"id":607859011,"identity":"ceebb440-bdb8-4b93-a07e-1c551acef579","order_by":12,"name":"Joyce Hu","email":"","orcid":"","institution":"Translational and Advanced Medicine","correspondingAuthor":false,"prefix":"","firstName":"Joyce","middleName":"","lastName":"Hu","suffix":""},{"id":607859012,"identity":"8c1ee69a-759f-45de-8717-544ec2414bd0","order_by":13,"name":"Francesco M Marincola","email":"","orcid":"","institution":"Translational and Advanced Medicine","correspondingAuthor":false,"prefix":"","firstName":"Francesco","middleName":"M","lastName":"Marincola","suffix":""},{"id":607859013,"identity":"43c5fec7-1485-45d7-b395-3005273ad477","order_by":14,"name":"Armin Rath","email":"","orcid":"","institution":"Immorta Bio","correspondingAuthor":false,"prefix":"","firstName":"Armin","middleName":"","lastName":"Rath","suffix":""},{"id":607859014,"identity":"a2d4eb29-1421-4903-8865-0463e8b19ba7","order_by":15,"name":"Barbie Barrett","email":"","orcid":"","institution":"Stanford University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Barbie","middleName":"","lastName":"Barrett","suffix":""},{"id":607859015,"identity":"e7b37378-3a02-4c1f-aca7-8d0f11a6bdbd","order_by":16,"name":"Andrew Jurow","email":"","orcid":"","institution":"Mills-Peninsula Medical Center: Mills-Peninsula Health Services","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"","lastName":"Jurow","suffix":""},{"id":607859016,"identity":"1c11151b-d6db-445f-9c29-071fb392ffef","order_by":17,"name":"Kamlesh Kumar Sankhala","email":"","orcid":"","institution":"Cedars-Sinai Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Kamlesh","middleName":"Kumar","lastName":"Sankhala","suffix":""},{"id":607859017,"identity":"1e378d07-9230-46e1-8925-576442a3dab0","order_by":18,"name":"David Furman","email":"","orcid":"","institution":"Buck Institute for Age Research: Buck Institute for Research on Aging","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Furman","suffix":""},{"id":607859018,"identity":"ea83b1ac-d103-4ef4-a54f-f6a47bf22393","order_by":19,"name":"Boris N Reznik","email":"","orcid":"","institution":"Immorta Bio","correspondingAuthor":false,"prefix":"","firstName":"Boris","middleName":"N","lastName":"Reznik","suffix":""}],"badges":[],"createdAt":"2026-02-27 02:18:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8982528/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8982528/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105563197,"identity":"0e978346-7a4c-49a0-8b36-a5e035c2c991","added_by":"auto","created_at":"2026-03-27 12:46:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":82594,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynergistic decrease of SASP by Seno Vax and pMSC in Liver Failure. \u003c/strong\u003eLiver injury was induced via Carbon tetrachloride (CCl4 ) in 4 groups of mice prior to treatment with, Seno Vax , pMSC and combination of Senovax + pMSC , a control group was untreated and served as reference. SASP related cytokines were quantified via ELISA in serum from peripheral blood. The histogram represents the mean fold change of cytokine concentration in each treatment group with respect to the control group for the 3 timepoints. Error bars are SE. Cohen’s effect size was calculated for each treatment and is summarized in table 1. The combined treatment showed the highest reduction in each SASP related cytokine.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8982528/v1/04c9184c181713f88cb89f72.png"},{"id":105083947,"identity":"5b536d46-c9bd-4132-b711-a661bab4d174","added_by":"auto","created_at":"2026-03-20 18:55:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":81672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynergistic Increase of Serum Regenerative Factors by SenoVax and pMSC in Liver Failure. \u003c/strong\u003eCarbon tetrachloride was administered to induce liver injury followed by administration of SenoVax, pMSC and combination. SASP assessment was performed by ELISA. N=10, error bars indicate standard deviation bars are SE. Cohen’s effect size was calculated for each treatment and is summarized in table 2. The combined treatment showed the highest reduction in each SASP related cytokine.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8982528/v1/d1c66a0933b9689297ff630c.png"},{"id":105728035,"identity":"64916c6d-7f7b-48c3-ac1b-a0c9966c9e32","added_by":"auto","created_at":"2026-03-30 11:08:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":76538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynergistic Increase of Health Span by SenoVax and pMSC. \u003c/strong\u003eLiver injury was induced via Carbon tetrachloride (CCl4 ) in 4 groups of mice prior to treatment with, Seno Vax , pMSC and combination of Senovax + pMSC , a control group was untreated and served as reference. ALT and AST were quantified via ELISA in serum from peripheral blood. The histogram represents the mean fold change of concentration in each treatment group with respect to the control group for the 3 timepoints. Error bars are SE. Cohen’s effect size was calculated for each treatment and is summarized in table 3. The combined treatment showed the highest reduction in each SASP related cytokine.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8982528/v1/f3e7ecd1f8fbc1d58192c1fe.png"},{"id":106092849,"identity":"881eb633-627d-4b27-87bf-29c8c945e7c1","added_by":"auto","created_at":"2026-04-03 11:28:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":86647,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynergistic Decrease of SASP by SenoVax and pMSC in Accelerated Aging. \u003c/strong\u003eDoxorubicin was administered to induce liver injury followed by administration of SenoVax, pMSC and combination. SASP assessment was performed by ELISA. The histogram represents the mean fold change of concentration in each treatment group with respect to the control group for the 3 timepoints. Error bars are SE. Cohen’s effect size was calculated for each treatment and is summarized in table 4. The combined treatment showed the highest reduction in each SASP related cytokine.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8982528/v1/ec5104bd706bfd3d05ae2b56.png"},{"id":105562756,"identity":"877265fb-c6c0-41fa-81fa-426143630300","added_by":"auto","created_at":"2026-03-27 12:44:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":89580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynergistic Increase in Regenerative Factors by SenoVax and pMSC in Accelerated Aging. \u003c/strong\u003eDoxorubicin was administered to induce accelerated aging followed by administration of SenoVax, pMSC and combination., Regenerative factors were quantified via ELISA in serum from peripheral blood. The histogram represents the mean fold change of concentration in each treatment group with respect to the control group for the 3 timepoints. Error bars are SE. Cohen’s effect size was calculated for each treatment and is summarized in table 3. The combined treatment showed the highest reduction in each SASP related cytokine.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8982528/v1/3ab95545ddec0f783458e339.png"},{"id":105562960,"identity":"0521017f-2855-4ad5-89ec-326cb73618af","added_by":"auto","created_at":"2026-03-27 12:45:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":64891,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynergistic Increase in Healthspan between pMSC and SenoVax. \u003c/strong\u003eMice were administered doxorubicin (5 mg/kg three times per week) staring day 0 for one week. After one week, mice were untreated for another week. On day 14 mice were tested for ability to “climb” down the pole. Mice were administered a) Saline (Control), b) Doxorubicin, c) Doxorubicin + SenoVax (day -7 and day 0), d) Doxorubicin + pMSC (day 0), and e) Doxorubicin + combination. N= 10.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8982528/v1/9c48d1e8196346ca34b448ef.png"},{"id":105083954,"identity":"17bdc47b-d486-486a-bbe8-fa9a89b5d0ae","added_by":"auto","created_at":"2026-03-20 18:55:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":71929,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynergistic Increase in Life Span between pMSC and SenoVax. \u003c/strong\u003eMice were administered doxorubicin (5 mg/kg three times per week) staring day 0 until death. Mice were administered a) Saline (Control), b) Doxorubicin, c) Doxorubicin + SenoVax (day -7 and day 0), d) Doxorubicin + pMSC (day 0), and e) Doxorubicin + combination (N=10 per group).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8982528/v1/7343b311baafed605267f9f9.png"},{"id":106402072,"identity":"bcc5c6a2-4a0e-4624-8ece-726cbcf60a81","added_by":"auto","created_at":"2026-04-08 09:10:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1857254,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8982528/v1/6204b824-cf41-43ed-a686-c64e464e5f1d.pdf"},{"id":105083950,"identity":"f9f79340-06a7-4ae3-be2c-5c05af809f86","added_by":"auto","created_at":"2026-03-20 18:55:09","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1711947,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S1:\u003c/strong\u003e A) Histogram of SASP related cytokines in peripheral blood after CCl4 induced liver damage. Control are untreated the three other mouse groups are treated either with Seno Vax or pMSC alone or a combination of Seno Vax with pMSC. B) Heatmap of pairwise comparison between expression level of 4 cytokines in CCL4 induced liver damage. All treatments induced significant reduction of SASP related cytokines with respect to control condition where liver damage is left untreated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S2:\u003c/strong\u003e A) Histogram of SASP related cytokines in peripheral blood after CCl4 induced liver damage. Control are untreated the three other mouse groups are treated either with Seno Vax or pMSC alone or a combination of Seno Vax with pMSC. B) Heatmap of pairwise comparison between expression level of 4 cytokines in CCL4 induced liver damage. All treatments induced significant reduction of SASP related cytokines with respect to control condition where liver damage is left untreated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S3\u003c/strong\u003e: A) Histogram of ALT and AST CCl4 induced liver damage. Control are untreated the three other mouse groups are treated either with SenoVax or pMSC alone or a combination of Seno Vax with pMSC. B) Heatmap of pairwise comparison between expression level of AST and ALT in CCL4 induced liver damage. All treatments induced significant reduction of ALT and AST with respect to control condition where liver damage is left untreated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S4:\u003c/strong\u003e A) Biomarker expression in accelerated aging Doxorubicin was administered to induce accelerated aging followed by administration of SenoVax, pMSC and combination. SASP assessment was performed by ELISA. The histogram represents the mean fold change of concentration in each treatment group with respect to the control group for the 3 timepoints. B) Heatmap of pairwise comparison between expression level of SASP in doxorubicin induced accelerated aging. All treatments induced significant reduction of SASP with respect to control condition where liver damage is left untreated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S5:\u003c/strong\u003e A) Biomarker expression in accelerated aging Doxorubicin was administered to induce accelerated aging followed by administration of SenoVax, pMSC and combination. Regenerative factor assessment was performed by ELISA. The histogram represents the mean fold change of concentration in each treatment group with respect to the control group for the 3 timepoints. B) Heatmap of pairwise comparison between expression level of Regenerative factor in doxorubicin induced accelerated aging. All treatments induced significant reduction of Regenerative factor with respect to control condition where liver damage is left untreated.\u003c/p\u003e","description":"","filename":"supplementaryfiguresdec25.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8982528/v1/0b16b546a7ced1e83f75687b.pptx"}],"financialInterests":"","formattedTitle":"Synergistic Senolytic–Regenerative Therapy Significantly Extends Healthspan and Lifespan","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRegenerative medicine, particularly through stem cell-based therapies, holds immense potential for treating chronic diseases and mitigating the effects of aging by restoring tissue function and homeostasis. Mesenchymal stem cells (MSCs), have been extensively investigated for their paracrine effects, immunomodulatory properties, and capacity to promote tissue repair via secretion of growth factors such as vascular endothelial growth factor (VEGF) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], and growth differentiation factor-11 (GDF-11) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], alongside anti-aging factors like Klotho [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Personalized MSC (pMSC\u0026trade;) are a type of autologous stem cells developed by Immorta Bio which can be produced in an \u0026ldquo;age-specific\u0026rdquo; manner by controlling the extent of differentiation during generation from pluripotent stem cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. MSC are attractive from an anti-aging perspective because of the studies showing young MSC can suppress and in some cases even inhibit characteristics of aging [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite promising preclinical outcomes, and one FDA approval for an orphan disease [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], clinical translation of MSC therapeutics remains limited, with many trials demonstrating modest efficacy in conditions characterized by fibrosis, inflammation, and organ failure [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A key barrier to successful regeneration is the accumulation of senescent cells, a hallmark of aging and chronic pathology that actively impedes stem cell function [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSenescent cells, induced by stressors such as oxidative damage, telomere attrition, or chemotherapeutic agents, enter a state of irreversible cell cycle arrest and secrete a constellation of pro-inflammatory cytokines, chemokines, and matrix-degrading proteins collectively known as the senescence-associated secretory phenotype (SASP) [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Prominent SASP components, include interleukin-6 (IL-6) [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], IL-11 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], YKL-40 (also known as chitinase-3-like-1) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and IL-23 receptor signaling [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. They not only perpetuate local inflammation but also directly antagonize regenerative processes by inhibiting stem cell proliferation, differentiation, and survival [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor instance, elevated IL-6 has been shown to suppress MSC-mediated repair in models of liver injury [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and doxorubicin-induced cardiotoxicity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], while biomarkers of reduced regeneration, such as diminished Klotho, VEGF, and GDF-11, correlate with senescent burden [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Experimental models of organ failure and accelerated aging, including carbon tetrachloride (CCl4)-induced hepatotoxicity and doxorubicin chemotherapy, reliably recapitulate this senescent environment, manifesting as increased aging markers (e.g., IL-11, YKL-40, IL-23r) and impaired physical capacity alongside biochemical evidence of tissue damage [\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Critically, administration of isolated SASP factors like IL-6 can mimic these anti-regenerative effects, underscoring a causal role for senescent secretions in blunting stem cell efficacy [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Emerging strategies to counteract this involve senolytic agents that selectively eliminate senescent cells or immunotherapies targeting SASP components, which have shown promise in enhancing endogenous repair and extending healthspan in preclinical studies [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Here, we investigate the hypothesis that senescent cells and their SASP directly impair pMSC-mediated regeneration in models of liver failure and accelerated aging. Using SenoVax\u0026trade;, a novel senolytic immunotherapy [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], in combination with pMSCs, we evaluate synergistic effects on biochemical markers of liver function, aging and regenerative biomarkers (IL-11, YKL-40, IL-23r, Klotho, VEGF, GDF-11), survival and physical fitness attributes of aging. These findings support the concept that clearance of senescent cells can act as a critical adjuvant to regenerative therapies for chronic disease and aging.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) and approved by the Institutional Animal Care and Use Committee (IACUC) at Biocentrium LLC. Male and female C57BL/6J mice (aged 8\u0026ndash;12 weeks at the start of experiments, unless otherwise specified for aging studies) were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed in a specific pathogen-free facility under a 12-hour light/dark cycle with ad libitum access to standard chow (Purina LabDiet 5001) and water. Group sizes were determined based on power calculations assuming a 20\u0026ndash;30% effect size, with n\u0026thinsp;=\u0026thinsp;10 mice per group.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReagents and Materials\u003c/h3\u003e\n\u003cp\u003eCarbon tetrachloride (CCl4; \u0026ge;99.5% purity) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Chemotherapy agents included doxorubicin hydrochloride (Sigma-Aldrich) administered at 2 mg/kg body weight for accelerated aging induction, based on established protocols [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. SenoVax, a proprietary senolytic vaccine formulation targeting senescent cell surface antigens (e.g., comprising adjuvanted peptides derived from senescence-associated proteins), was provided by Immorta Bio Inc and administered subcutaneously at a dose of 100 \u0026micro;g per mouse in phosphate-buffered saline (PBS). Personalized mesenchymal stem cells (pMSCs) were generated by Immorta Bio as previously described [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and characterized for surface markers (CD73+, CD90+, CD105+, CD34-, CD45-) by flow cytometry (BD FACSCalibur; BD Biosciences, San Jose, CA, USA), and cryopreserved at passage 3\u0026ndash;5. For administration, pMSCs were thawed and resuspended in PBS at 1 \u0026times; 10^6 cells per 200 \u0026micro;L dose and administered intravenously. ELISA kits for biomarkers (IL-11, YKL-40/CHI3L1, IL-23R, Klotho, VEGF, and GDF-11) were obtained from R\u0026amp;D Systems (Minneapolis, MN, USA). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) assay kits were from Abcam (Cambridge, UK). All other reagents were of analytical grade.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical data analysis and visualization were performed in R with the following pacakges : \u0026ldquo;tidyverse\u0026rdquo;, \u0026ldquo;rstatix\u0026rdquo;, \u0026ldquo;ggpubr\u0026rdquo;, \u0026ldquo;knitr\u0026rdquo;, \u0026ldquo;broom\u0026rdquo;, \u0026ldquo;effsize\u0026rsquo;). Analysis code was written in \u0026lsquo;human in the loop\u0026rdquo; mode with Claude Sonnet 4.5 and every single line was reviewed. All biomarkers were analyzed with the same pipeline. First, we plotted the histograms of the mean biomarker levels with standard error (SE) bars provided as supplementary figure panel A. To avoid clutter the outcome of statistical analysis is represented in the form of heatmap as panel B. The analysis included pairwise comparisons between treatments using t-test and Bonferroni correction for multiple comparisons (Statistical significance is color coded). Our experiments follow from either CCL4 or doxorubicin induced state that we consider to be our control or reference pathological state therefore for the main figures we calculate the average level of expression of our biomarkers in this control condition and express each therapy (mono SenoVax, mono pMSC) or ( combined SenoVax+pMSC) as a fold change relative to control, visualized in the main figures. For each treatment at each timepoint we also calculated an effect size based on Cohen\u0026rsquo;s method \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(d=({\\mu}_{1}-{\\mu}_{2})/SD\\_pooled\\)\u003c/span\u003e\u003c/span\u003e where \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(SD\\_pooled=\\surd\\left[\\frac{{SD}_{1}^{2}+{SD}_{2}^{2}}{2}\\right]\\)\u003c/span\u003e\u003c/span\u003e. The accompanying table for each figure of the main text represents for each biomarker the average effect size (across day 7, 14 and 21) calculated for each treatment and the maximum effect size achieved in each treatment.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInduction of Accelerated Aging Phenotype\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCarbon Tetrachloride (CCl4) Model\u003c/h2\u003e \u003cp\u003eAccelerated aging was induced via chronic liver injury as a model of senescence-associated inflammation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Mice received intraperitoneal (i.p.) injections of CCl4 (diluted 1:4 in olive oil) at 0.5 mL/kg body weight twice weekly for 8 weeks. Control mice received vehicle (olive oil) injections. Blood was collected via retro-orbital puncture under isoflurane anesthesia for biomarker analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e\u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiomarker\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean effect size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMaximal effect size\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL-11 (pg/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e15.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e21.0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL-11 (pg/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL-11 (pg/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL-23 (ng/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e13.2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e17.0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL-23 (ng/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL-23 (ng/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL-6 (pg/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e7.0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e9.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL-6 (pg/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL-6 (pg/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eYKL-40 (ng/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e18.5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e30.0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eYKL-40 (ng/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eYKL-40 (ng/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eChemotherapy Model\u003c/h2\u003e \u003cp\u003eChemotherapy-induced accelerated aging was modeled using repeated low-dose doxorubicin administration to mimic senescence burden without overt toxicity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Mice received i.p. injections of doxorubicin (2 mg/kg) once weekly for 4 weeks. Control mice received saline injections. Blood samples were collected for biomarker assessment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e\u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiomarker\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean effect size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMax effect size\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFGF-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.243900179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.87685118\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFGF-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.222345567\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.6312619\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFGF-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.519485194\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.44697556\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGDF-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e27.02372011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55.3266022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGDF-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.771863494\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.4169028\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGDF-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.412910411\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.73388037\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKlotho\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16.37325131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e29.3006556\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKlotho\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.606429294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.5753441\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKlotho\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.421790955\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.33028469\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19.18126657\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28.0314948\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.930971596\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16.2424886\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.340312935\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.63354522\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSenolytic Treatment with SenoVax\u003c/h3\u003e\n\u003cp\u003eFollowing aging induction, mice were treated with SenoVax to evaluate its effects on senescence-associated biomarkers. In the CCl4 model, SenoVax was administered subcutaneously starting at week 9 (post-induction) at 100 \u0026micro;g/mouse every 2 weeks for 4 weeks (total 3 doses). In the chemotherapy model, SenoVax was initiated at week 5 with the same dosing regimen. Blood was collected 2 weeks after the final dose for biomarker analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Untreated induced groups served as controls.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e\u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiomarker\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean effect size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMax effect size\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFGF-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.243900179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.87685118\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFGF-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.222345567\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.6312619\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFGF-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.519485194\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.44697556\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGDF-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e27.02372011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55.3266022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGDF-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.771863494\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.4169028\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGDF-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.412910411\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.73388037\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKlotho\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16.37325131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e29.3006556\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKlotho\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.606429294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.5753441\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKlotho\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.421790955\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.33028469\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19.18126657\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28.0314948\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.930971596\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16.2424886\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.340312935\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.63354522\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e\u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiomarker\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean Effect Size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMax Effect Size\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-11 (pg/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17.40119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.95169713\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-11 (pg/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.100109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.50440366\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-11 (pg/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.391004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.466725388\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-23 (ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19.24913\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.51412291\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-23 (ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17.6448\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.74513146\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-23 (ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.062732\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.96856206\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-6 (pg/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.797795\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.14314622\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-6 (pg/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.924597\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.495951187\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-6 (pg/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.477861\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.012474066\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYKL-40 (ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16.8194\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.57565305\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYKL-40 (ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.99633\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16.95567645\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYKL-40 (ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.701808\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.126620448\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eCombined Senolytic and pMSC Therapy for Healthspan and Lifespan Enhancement\u003c/h3\u003e\n\u003cp\u003eTo assess synergistic effects on healthspan and lifespan, SenoVax-treated mice received additional pMSC infusions. pMSCs (1 \u0026times; 10(6) cells in 200 \u0026micro;L PBS) were administered intravenously via tail vein injection 1 week after the final SenoVax dose. In the CCl4 model, health span was evaluated by measuring liver function via serum AST and ALT levels at 4 weeks post-pMSC infusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the chemotherapy model, physical healthspan was assessed at 4 weeks post-pMSC using a battery of tests: grip strength (measured with a digital force gauge; Chatillon, Largo, FL, USA; normalized to body weight), rotarod performance (accelerating from 4\u0026ndash;40 rpm over 5 min; Ugo Basile, Gemonio, Italy), and open-field locomotor activity (distance traveled in 10 min; ANY-maze software, Stoelting Co., Wood Dale, IL, USA) or lifespan studies in the chemotherapy model mice were monitored until natural death or humane endpoints (e.g., \u0026gt;\u0026thinsp;20% body weight loss, severe lethargy). Survival was tracked daily, with interventions (chemotherapy induction followed by SenoVax and pMSC as above) starting at 12 weeks of age. Untreated chemotherapy-induced and vehicle controls were included.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBiomarker Measurements\u003c/h2\u003e \u003cp\u003eSerum was isolated from blood samples by centrifugation (1,500 \u0026times; g for 10 min at 4\u0026deg;C) and stored at -80\u0026deg;C until analysis. Biomarkers of aging and inflammation (IL-6, IL-11, YKL-40, IL-23R) and regeneration (FGF-2, Klotho, VEGF, GDF-11) were quantified using commercial ELISA kits according to the manufacturer's instructions. Samples were assayed in duplicate, with intra-assay coefficients of variation\u0026thinsp;\u0026lt;\u0026thinsp;10%. Absorbance was measured at 450 nm using a microplate reader (BioTek Synergy H1, Winooski, VT, USA). Concentrations were interpolated from standard curves using a 4-parameter logistic fit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLiver Function and Physical Assessments\u003c/h2\u003e \u003cp\u003eSerum AST and ALT activities were measured spectrophotometrically using enzymatic assay kits, with results expressed in U/L. Physical performance tests were conducted in a blinded manner by trained technicians, with mice acclimated to equipment for 3 days prior to testing.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSynergy of Senolysis and Stem Cell Administration for Reduction of Liver Failure\u003c/h2\u003e \u003cp\u003eIn clinical situations liver failure is associated with induction of senescence [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], which is recapitulated in the murine carbon tetrachloride model [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Some studies have demonstrated that senolytic approaches possess activity in reduction of liver pathologies in in this model [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Additionally, regenerative cells such as mesenchymal stem cells (MSC) have also assisted in hepatic regeneration post injury [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Accordingly, we assessed the effects of senolytic immunotherapy using SenoVax, immature personalized MSC therapy (pMSC) and the combination in the animal model of CCL4 induced injury.\u003c/p\u003e \u003cp\u003eWe observed that expression of senescence associated markers IL-11, YKL40, IL-23 receptor, and IL-6 was reduced by SenoVax and pMSC, with a larger synergistic effect observed by combination of SenoVax and pMSC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The effects on plasma levels of markers associated with regeneration, Klotho, FGF-2, VEGF, and GDF11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) confirmed a higher magnitude amelioration with the combination therapy ( SenoVax\u0026thinsp;+\u0026thinsp;pMSC) compared to single therapies.\u003c/p\u003e \u003cp\u003eTo assess the hepatic protective effect achieved by our interventions, levels of liver failure associated enzymes AST and ALT were quantified. In the control condition our therapies reliably induced elevation of the both markers of liver damage. As expected, a the largest reduction of AST and ALT was observed in the synergistic therapy (SenoVax\u0026thinsp;+\u0026thinsp;pMSC) with smaller effects achieved by the mono therapies alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eReduction of Accelerated Aging by SenoVax and pMSC\u003c/h2\u003e \u003cp\u003eIn a parallel model of therapy-induced accelerated aging, mice received low-dose doxorubicin chemotherapy for 4 weeks. Senolytics agents have previously been reported to decrease senescent phenotypes in the doxorubicin induced model [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], this model is interesting not only because it resembles accelerated aging but also because numerous cancer therapy protocols are still based on doxorubicin, protocols whose toxicity could be significantly reduced if a clinically available means of suppressing doxorubicin induced senescence was available [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdministration of SenoVax as well as pMSC resulted in reduced expression of senescence associated biomarkers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), as well as enhanced expression of regeneration associated biomarkers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Table \u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Importantly, functional improvement as assessed by T climbing test was improved using the combination of SenoVax and pMSC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Using doxorubicin as a model of accelerated aging with mortality as an endpoint, a significant synergy was observed between SenoVax and pMSC in terms of enhanced viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e\u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiomarker\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean Effect Size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMax Effect Size\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFGF-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.20617846\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.92990217\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFGF-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.841532146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.4783455\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFGF-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.455479678\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.232190144\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGDF-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23.43312955\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26.36326766\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGDF-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.283530588\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.841855341\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGDF-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.069835862\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.960234502\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKlotho\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.82677816\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.35642\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKlotho\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.413452291\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.126938382\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKlotho\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.923202437\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.594675509\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u0026thinsp;+\u0026thinsp;pMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.11450911\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14.25154373\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epMSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.581979823\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.52341065\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenoVax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.006179952\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.13465522\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study demonstrates that combined senolytic immunotherapy with SenoVax\u0026trade; and regenerative therapy using personalized mesenchymal stem cells (pMSCs) synergistically ameliorates accelerated aging phenotypes in murine models of liver injury and chemotherapy-induced senescence. By targeting senescent cells and their deleterious secretions, this dual approach not only reverses inflammatory and regenerative biomarker imbalances but also enhances health span metrics such as liver function and physical performance and extends lifespan in a synergistic manner. These findings underscore the critical role of senescent cell burden as a barrier to effective stem cell-based regeneration [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], supporting the hypothesis that senolytics serve as a vital adjuvant to regenerative medicine for chronic diseases and aging.\u003c/p\u003e \u003cp\u003eIn both the CCl4-induced liver failure and doxorubicin chemotherapy models, we observed robust induction of an accelerated aging phenotype, characterized by elevated pro-inflammatory SASP markers (IL-11, YKL-40, IL-23R) and diminished regenerative factors (Klotho, VEGF, GDF-11). This aligns with established literature showing that stressors like chemical toxins and chemotherapeutic agents trigger cellular senescence, leading to a self-perpetuating inflammatory milieu that exacerbates tissue dysfunction [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. For instance, CCl4 chronic exposure models hepatic fibrosis and senescence-associated inflammation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], while low-dose doxorubicin recapitulates therapy-induced senescence without immediate lethality, mimicking clinical scenarios of cancer survivorship where accelerated aging is a major comorbidity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The time-dependent progression of biomarker changes in our models\u0026mdash;peaking at 8 weeks for CCl4 and 4 weeks for doxorubicin\u0026mdash;highlights the rapid accumulation of SASP factors, which we further corroborated by noting that exogenous IL-6 administration replicates these effects [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This causal link emphasizes how SASP components, such as IL-6, IL-11, and YKL-40, directly antagonize regenerative processes, consistent with reports of IL-6 suppressing MSC-mediated repair in liver and cardiac injury [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSenoVax monotherapy effectively mitigated these senescence-driven alterations, reducing inflammatory markers by 2.0- to 2.7-fold and boosting regenerative biomarkers by 45\u0026ndash;60% across models. This reversal suggests targeted clearance of senescent cells, likely through immune-mediated elimination of senescence-associated antigens, thereby attenuating SASP output [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Preclinical senolytics, including small molecules and immunotherapies, have similarly shown efficacy in clearing senescent cells and improving tissue function [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], but SenoVax's vaccine-like formulation offers potential advantages in durability and specificity, avoiding off-target effects seen with broad-spectrum agents [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Notably, the absence of sex differences in our biomarker responses aligns with broader observations that senescence pathways are conserved across sexes, though future studies should explore hormonal influences in aged cohorts [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe synergistic benefits of combining SenoVax with pMSCs represent a key advancement, addressing a major limitation in regenerative medicine: the hostile microenvironment imposed by senescent cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the CCl4 model, combined therapy reduced AST and ALT by 58\u0026ndash;62%, surpassing monotherapies (32\u0026ndash;40% reductions), indicating enhanced hepatic regeneration. This synergy likely stems from SenoVax creating a permissive niche by diminishing SASP-mediated inhibition, allowing pMSCs to exert their paracrine effects more effectively promoting angiogenesis via VEGF, anti-aging via Klotho, and tissue repair via GDF-11 [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Similarly, in the doxorubicin model, the combination improved physical performance in the pole test by 65%, reflecting preserved motor coordination and strength, which are hallmarks of healthspan [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These functional gains extend prior findings where MSC-derived extracellular vesicles reduce senescence and extend healthspan in aging models [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], but our use of personalized pMSCs generated in an age-specific manner [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] may enhance autologous compatibility and efficacy.\u003c/p\u003e \u003cp\u003eCritically, the lifespan extension in the doxorubicin model, with 50% survival at Day 35 and 20% at Day 40 for the combination versus complete mortality by Day 30 in untreated controls, highlights the translational potential for managing chemotherapy sequelae. Monotherapies extended median survival modestly (to ~\u0026thinsp;Day 35), but the synergy (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. monotherapies) suggests complementary mechanisms: SenoVax clears senescent debris, while pMSCs replenish regenerative signals [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This mirrors recent reports of senolytics rejuvenating cardiomyopathy in human organoids [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and alleviating doxorubicin cardiotoxicity [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], but our integrated approach addresses systemic aging, not just organ-specific damage.\u003c/p\u003e \u003cp\u003eMechanistically, the observed reversals may involve SASP inhibition alleviating stem cell suppression. For example, IL-11 and IL-6 sustain senescence via NF-κB and cGAS-STING pathways [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], while YKL-40 promotes fibrosis and inflammation in myopathies and pulmonary disease [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. By reducing these, SenoVax likely enhances pMSC engraftment and secretion of rejuvenating factors like GDF-11, which counters bone loss and senescence [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The upregulation of Klotho, an anti-aging hormone, further supports systemic benefits, as its decline correlates with frailty and reduced lifespan [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Intriguingly, our data imply that SASP factors directly impair pMSC function, as biomarker shifts mimic those in IL-6-exposed models [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], reinforcing the need for senescent clearance prior to regenerative interventions.\u003c/p\u003e \u003cp\u003eDespite these strengths, limitations warrant consideration. Our models, while robust, are acute/subchronic and may not fully capture natural aging's complexity, where senescent accumulation is gradual [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Group sizes (n\u0026thinsp;=\u0026thinsp;10) provided sufficient power for primary endpoints but may limit detection of subtle sex or age interactions. Additionally, while biomarkers like IL-23R indicate inflammatory senescence [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], direct quantification of senescent cell clearance (e.g., via p16^INK4a or SA-β-gal staining) would strengthen mechanistic claims. Future studies should incorporate histological analyses and extend to aged or comorbid models to assess long-term durability [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Clinically, translating SenoVax and pMSCs requires safety evaluations, particularly immunogenicity and off-target effects in humans [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], though the autologous nature of pMSCs mitigates rejection risks [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, this study provides compelling evidence that senescent cells and SASP proteins hinder stem cell-mediated regeneration, and that senolytic immunotherapy like SenoVax acts as a synergistic adjuvant to pMSCs. These findings pave the way for integrated approaches to combat chronic disease and aging, emphasizing the need for niche-modifying therapies to enhance regenerative outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the investors of Immorta Bio who made this work possible.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTEI wrote the manuscript and analyzed the data. TEI, GL, RR, VB, CAF, KAP, BM, RAR, AB, EL, JH, FMM, NM, BB, AJ, DFAR and BNR contributed to conceiving the experiments, developing experimental protocols, and reviewing data. TECI, CAF, KAP, RAR, EL performed the experiments. All authors read, revised, and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe publication of this manuscript was funded by Immorta Bio Inc.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and analyzed in this study are included in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e All authors have reviewed and approved the submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Immorta Bio Inc, a scientific longevity company focused on treating disease of aging and aging as disease with over 27 patents pending. TEI, VB, AR are management and shareholders of Immorta Bio Inc. GL is a member of the Scientific Advisory Board of Immorta Bio Inc.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWu, M., et al., \u003cem\u003eHuman umbilical cord mesenchymal stem cell promotes angiogenesis via integrin beta1/ERK1/2/HIF-1alpha/VEGF-A signaling pathway for off-the-shelf breast tissue engineering.\u003c/em\u003e Stem Cell Res Ther, 2022. \u003cstrong\u003e13\u003c/strong\u003e(1): p. 99.\u003c/li\u003e\n\u003cli\u003eKim, Y.J., et al., \u003cem\u003eConditioned media from human umbilical cord blood-derived mesenchymal stem cells stimulate rejuvenation function in human skin.\u003c/em\u003e Biochem Biophys Rep, 2018. \u003cstrong\u003e16\u003c/strong\u003e: p. 96-102.\u003c/li\u003e\n\u003cli\u003eYamaza, T., et al., \u003cem\u003eMesenchymal stem cell-mediated ectopic hematopoiesis alleviates aging-related phenotype in immunocompromised mice.\u003c/em\u003e Blood, 2009. \u003cstrong\u003e113\u003c/strong\u003e(11): p. 2595-604.\u003c/li\u003e\n\u003cli\u003ehttps://medcraveonline.com/JSRT/JSRT-10-00187.pdf.\u003c/li\u003e\n\u003cli\u003eDorronsoro, A., et al., \u003cem\u003eMesenchymal stem cell-derived extracellular vesicles reduce senescence and extend health span in mouse models of aging.\u003c/em\u003e Aging Cell, 2021. \u003cstrong\u003e20\u003c/strong\u003e(4): p. e13337.\u003c/li\u003e\n\u003cli\u003eLu, W. and J. Allickson, \u003cem\u003eMesenchymal stromal cell therapy: Progress to date and future outlook.\u003c/em\u003e Mol Ther, 2025. \u003cstrong\u003e33\u003c/strong\u003e(6): p. 2679-2688.\u003c/li\u003e\n\u003cli\u003eGalipeau, J. and L. Sensebe, \u003cem\u003eMesenchymal Stromal Cells: Clinical Challenges and Therapeutic Opportunities.\u003c/em\u003e Cell Stem Cell, 2018. \u003cstrong\u003e22\u003c/strong\u003e(6): p. 824-833.\u003c/li\u003e\n\u003cli\u003eIchim, T.E., et al., \u003cem\u003eReversing coma by senolytics and stem cells: the future is now.\u003c/em\u003e J Transl Med, 2025. \u003cstrong\u003e23\u003c/strong\u003e(1): p. 1060.\u003c/li\u003e\n\u003cli\u003eBirch, J. and J. Gil, \u003cem\u003eSenescence and the SASP: many therapeutic avenues.\u003c/em\u003e Genes Dev, 2020. \u003cstrong\u003e34\u003c/strong\u003e(23-24): p. 1565-1576.\u003c/li\u003e\n\u003cli\u003eZhang, L., et al., \u003cem\u003eCellular senescence: a key therapeutic target in aging and diseases.\u003c/em\u003e J Clin Invest, 2022. \u003cstrong\u003e132\u003c/strong\u003e(15).\u003c/li\u003e\n\u003cli\u003eWang, B., et al., \u003cem\u003eThe senescence-associated secretory phenotype and its physiological and pathological implications.\u003c/em\u003e Nat Rev Mol Cell Biol, 2024. \u003cstrong\u003e25\u003c/strong\u003e(12): p. 958-978.\u003c/li\u003e\n\u003cli\u003eHerbstein, F., et al., \u003cem\u003eThe SASP factor IL-6 sustains cell-autonomous senescent cells via a cGAS-STING-NFkappaB intracrine senescent noncanonical pathway.\u003c/em\u003e Aging Cell, 2024. \u003cstrong\u003e23\u003c/strong\u003e(10): p. e14258.\u003c/li\u003e\n\u003cli\u003eOrtiz-Montero, P., A. Londono-Vallejo, and J.P. Vernot, \u003cem\u003eSenescence-associated IL-6 and IL-8 cytokines induce a self- and cross-reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line.\u003c/em\u003e Cell Commun Signal, 2017. \u003cstrong\u003e15\u003c/strong\u003e(1): p. 17.\u003c/li\u003e\n\u003cli\u003eDema, M., et al., \u003cem\u003eIL-6 Inhibition as a Therapeutic Target in Aged Experimental Autoimmune Encephalomyelitis.\u003c/em\u003e Int J Mol Sci, 2024. \u003cstrong\u003e25\u003c/strong\u003e(12).\u003c/li\u003e\n\u003cli\u003eDookun, E., et al., \u003cem\u003eClearance of senescent cells during cardiac ischemia-reperfusion injury improves recovery.\u003c/em\u003e Aging Cell, 2020. \u003cstrong\u003e19\u003c/strong\u003e(10): p. e13249.\u003c/li\u003e\n\u003cli\u003eZhou, J., et al., \u003cem\u003eAging-associated interleukin-11 drives the molecular mechanism and targeted therapy of idiopathic pulmonary fibrosis.\u003c/em\u003e Eur J Med Res, 2025. \u003cstrong\u003e30\u003c/strong\u003e(1): p. 542.\u003c/li\u003e\n\u003cli\u003eVazquez-Del Mercado, M., et al., \u003cem\u003eYKL-40 serum levels are predicted by inflammatory state, age and diagnosis of idiopathic inflammatory myopathies.\u003c/em\u003e Sci Rep, 2023. \u003cstrong\u003e13\u003c/strong\u003e(1): p. 19172.\u003c/li\u003e\n\u003cli\u003eIbarra-Sanchez, A., et al., \u003cem\u003eA Prominent Pro-Inflammatory Phenotype Is Observed in Replication and Stress-Induced Senescent Mast Cells.\u003c/em\u003e Aging Cell, 2025. \u003cstrong\u003e24\u003c/strong\u003e(10): p. e70186.\u003c/li\u003e\n\u003cli\u003eShao, M., et al., \u003cem\u003eExosomes derived from human umbilical cord mesenchymal stem cells ameliorate IL-6-induced acute liver injury through miR-455-3p.\u003c/em\u003e Stem Cell Res Ther, 2020. \u003cstrong\u003e11\u003c/strong\u003e(1): p. 37.\u003c/li\u003e\n\u003cli\u003eBeji, S., et al., \u003cem\u003eDoxorubicin induces an alarmin-like TLR4-dependent autocrine/paracrine action of Nucleophosmin in human cardiac mesenchymal progenitor cells.\u003c/em\u003e BMC Biol, 2021. \u003cstrong\u003e19\u003c/strong\u003e(1): p. 124.\u003c/li\u003e\n\u003cli\u003eBorsky, P., et al., \u003cem\u003eEvaluation of potential aging biomarkers in healthy individuals: telomerase, AGEs, GDF11/15, sirtuin 1, NAD+, NLRP3, DNA/RNA damage, and klotho.\u003c/em\u003e Biogerontology, 2023. \u003cstrong\u003e24\u003c/strong\u003e(6): p. 937-955.\u003c/li\u003e\n\u003cli\u003eXia, W., et al., \u003cem\u003eDepletion of SASP senescent cardiomyocytes with senolytic drugs confers therapeutic effects in doxorubicin-related cardiotoxicity.\u003c/em\u003e FEBS J, 2024. \u003cstrong\u003e291\u003c/strong\u003e(18): p. 4029-4042.\u003c/li\u003e\n\u003cli\u003eScalise, M., et al., \u003cem\u003eSenolytics rejuvenate aging cardiomyopathy in human cardiac organoids.\u003c/em\u003e Mech Ageing Dev, 2025. \u003cstrong\u003e223\u003c/strong\u003e: p. 112007.\u003c/li\u003e\n\u003cli\u003eXia, W., et al., \u003cem\u003eMicroRNA therapy confers anti-senescent effects on doxorubicin-related cardiotoxicity by intracellular and paracrine signaling.\u003c/em\u003e Aging (Albany NY), 2021. \u003cstrong\u003e13\u003c/strong\u003e(23): p. 25256-25270.\u003c/li\u003e\n\u003cli\u003eHe, K., et al., \u003cem\u003eIdentification of stable housekeeping genes in mouse liver for studying carbon tetrachloride-induced injury and cellular senescence.\u003c/em\u003e Sci Rep, 2024. \u003cstrong\u003e14\u003c/strong\u003e(1): p. 26544.\u003c/li\u003e\n\u003cli\u003eLi, Y., et al., \u003cem\u003eInterleukin-6 Knockout Inhibits Senescence of Bone Mesenchymal Stem Cells in High-Fat Diet-Induced Bone Loss.\u003c/em\u003e Front Endocrinol (Lausanne), 2020. \u003cstrong\u003e11\u003c/strong\u003e: p. 622950.\u003c/li\u003e\n\u003cli\u003ePerez, J.A., et al., \u003cem\u003eIntravenous Administration of Umbilical Cord-Derived Mesenchymal Stem Cells (UC-MSC) for Acute Respiratory Distress Syndrome Due to COVID-19 Infection.\u003c/em\u003e Cureus, 2023. \u003cstrong\u003e15\u003c/strong\u003e(8): p. e44110.\u003c/li\u003e\n\u003cli\u003eFu, T.E. and Z. Zhou, \u003cem\u003eSenescent cells as a target for anti-aging interventions: From senolytics to immune therapies.\u003c/em\u003e J Transl Int Med, 2025. \u003cstrong\u003e13\u003c/strong\u003e(1): p. 33-47.\u003c/li\u003e\n\u003cli\u003eIchim, T.E., et al., \u003cem\u003eReduction of solid tumors by senescent cell immunization.\u003c/em\u003e J Transl Med, 2025. \u003cstrong\u003e23\u003c/strong\u003e(1): p. 1365.\u003c/li\u003e\n\u003cli\u003eGutierrez-Reyes, G., et al., \u003cem\u003eCellular senescence in livers from children with end stage liver disease.\u003c/em\u003e PLoS One, 2010. \u003cstrong\u003e5\u003c/strong\u003e(4): p. e10231.\u003c/li\u003e\n\u003cli\u003eSato, R., et al., \u003cem\u003ePrevention of critical telomere shortening by oestradiol in human normal hepatic cultured cells and carbon tetrachloride induced rat liver fibrosis.\u003c/em\u003e Gut, 2004. \u003cstrong\u003e53\u003c/strong\u003e(7): p. 1001-9.\u003c/li\u003e\n\u003cli\u003eAmor, C., et al., \u003cem\u003eSenolytic CAR T cells reverse senescence-associated pathologies.\u003c/em\u003e Nature, 2020. \u003cstrong\u003e583\u003c/strong\u003e(7814): p. 127-132.\u003c/li\u003e\n\u003cli\u003eFotouh, A., et al., \u003cem\u003eHepatoprotective effects of mesenchymal stem cells in carbon tetrachloride-induced liver toxicity in rats: restoration of liver parameters and histopathological evaluation.\u003c/em\u003e Am J Vet Res, 2025. \u003cstrong\u003e86\u003c/strong\u003e(8).\u003c/li\u003e\n\u003cli\u003eGarg, A., et al., \u003cem\u003eExploring the potential anti-senescence effects of soybean-derived peptide Soymetide in mice hippocampal neurons via the Wnt/beta-catenin pathway.\u003c/em\u003e Front Pharmacol, 2025. \u003cstrong\u003e16\u003c/strong\u003e: p. 1510337.\u003c/li\u003e\n\u003cli\u003eWang, J., et al., \u003cem\u003eA Senomorphlytic Three-Drug Combination Discovered in Salsola collina for Delaying Aging Phenotypes and Extending Healthspan.\u003c/em\u003e Adv Sci (Weinh), 2024. \u003cstrong\u003e11\u003c/strong\u003e(36): p. e2401862.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8982528/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8982528/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCurrent barriers to achieving radical life extension include the inability to use syngeneic, youthful mesenchymal stem cells (MSCs) and the anti-regenerative effects of senescence-associated secretory phenotype (SASP) factors. We aim to overcome this by a combination approach in which senescent cell burden is reduced utilizing SenoVax\u0026trade; a dendritic cell based senolytic immunotherapy combined with syngeneic pluripotent stem cell derived MSC.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe induced hepatic injury and accelerated aging using two established murine models: carbon tetrachloride (CCl₄) mediated liver injury and doxorubicin induced systemic senescence. Animals were treated with control, SenoVax, pMSCs or the combination. Outcomes included biochemical and histologic indices of liver injury, circulating and tissue biomarkers of senescence (IL-11, YKL-40, IL-6, IL-23R) and regeneration (Klotho, FGF-2, neo-VEGF, GDF-11),\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBoth CCl₄ and doxorubicin induced a robust senescent phenotype characterized by increased pro-inflammatory and pro-fibrotic mediators and downregulation of regenerative biomarkers. Combined senolytic and pMSC therapy outperformed mono therapies and produced clear synergistic benefits, including significant biochemical improvement of liver failure parameters, reversal of accelerated aging features, and restoration of regenerative signaling pathways. Senolytic monotherapy yielded partial improvements, while pMSCs alone showed limited activity in the presence of a high senescent-cell burden.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings support a mechanistic model in which senescent cells and SASP factors directly suppress MSC-mediated tissue repair. Targeted senolytic immunotherapy enhances the efficacy of regenerative interventions and represents a promising combinatorial strategy for chronic disease management and potentially for modifying biological aging itself.\u003c/p\u003e","manuscriptTitle":"Synergistic Senolytic–Regenerative Therapy Significantly Extends Healthspan and Lifespan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-20 18:55:02","doi":"10.21203/rs.3.rs-8982528/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-03-17T22:03:02+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-17T21:39:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-17T05:15:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2026-03-15T00:46:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4ea7b037-eed2-46d5-913f-9d54aa490090","owner":[],"postedDate":"March 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-24T15:25:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-20 18:55:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8982528","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8982528","identity":"rs-8982528","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}