Engineering Mesenchymal Stem Cells to Extend Healthspan: Therapeutic Potential, Challenges, and Future Solutions

1. Introduction

Healthspan, defined as the period of life spent in good health without chronic disease or disability, is a central focus of aging research. Mesenchymal stem/stromal cells (MSCs) are multipotent stromal cells with immunomodulatory, regenerative, and trophic properties, making them promising candidates for therapeutic approaches that target age-related pathologies and extend healthspan [1]. These properties have attracted substantial attention in anti-aging research, with evidence showing that MSCs regulate oxidative stress, attenuate endothelial cell senescence, and promote angiogenesis [2].

However, the therapeutic efficacy of wild-type MSCs is limited by poor engraftment, low survival in harsh microenvironments, and reduced functional potency, particularly when cells are derived from older donors [3]. The relevance of healthspan to MSC biology is highlighted owing to shared tissue-level mechanisms underlying many age-associated disorders, including chronic low-grade inflammation, vascular insufficiency, impaired proteostasis, mitochondrial dysfunction, stem/progenitor cell exhaustion, extracellular matrix stiffening, and the accumulation of senescent cells.

Together, these changes impair tissue repair and injury responses by reducing stem cell self-renewal and differentiation while promoting a pro-inflammatory microenvironment through senescence-associated secretory phenotype (SASP) factors. MSC-based interventions are therefore appealing because they may replenish depleted progenitor pools, attenuate inflammaging, and enhance endogenous repair mechanisms in age-related conditions. Rather than functioning solely as replacement cells, MSCs act as dynamic, microenvironment-responsive signaling platforms that modulate multiple interconnected hallmarks of aging by adjusting their paracrine secretome in response to local inflammatory and senescent cues [2,4,5].

Nevertheless, MSCs remain vulnerable to replicative and stress-induced senescence, which often alters their secretory profiles and decreases proliferative capacity [6]. A variety of engineering strategies have been developed to address these limitations and improve MSC survival, proliferation, and pro-regenerative capacity, thereby enhancing clinical performance [7,8]. These approaches include genetic and epigenetic modifications that increase secretion of regenerative factors or strengthen resilience to adverse conditions, as well as preconditioning methods that enhance immunomodulatory activity [9]. For example, preconditioning MSCs with cytokines or hypoxic conditions improves their secretory profile and increases production of anti-inflammatory cytokines [10].

The rationale for engineering MSCs is based on the well-documented age-related decline in stem cell potency, which compromises tissue homeostasis and limits the healing capacity of endogenous cell populations [11]. These limitations are intensified in pathological microenvironments. For example, ischemic tissues are characterized by hypoxia and nutrient deprivation, which reduce MSC survival; diabetic wounds contain high levels of advanced glycation end products, reactive oxygen species, proteases, and inflammatory cytokines, which impair proliferation and differentiation; and fibrotic tissues exhibit abnormal stiffness and disrupted integrin signaling, which promote maladaptive cell behavior [12,13,14,15].

Current strategies, therefore, focus on optimizing MSC viability through targeted priming and genetic enhancement to counteract the senescence-associated secretory phenotype and functional exhaustion observed in vivo [16,17]. Senescent MSCs exhibit altered secretome compositions that can negatively impact tissue repair and homeostasis, necessitating strategies to counteract age-related functional decline [5]. This issue is significant because donor age is inversely correlated with MSC proliferation, differentiation, and immunoregulatory capacity, which may lead to detrimental “inflammaging” outcomes if aged MSCs are used therapeutically [14,18]. Therefore, investigating approaches that improve MSC function and mitigate the negative effects of cellular senescence is essential, especially in therapies targeting older populations [19].

This review examines advanced bioengineering strategies to optimize MSC characteristics for improved outcomes in age-related diseases, with an emphasis on overcoming age-associated functional decline and extending healthspan. Strategies to address donor age-dependent reductions in MSC functionality include deriving MSC-like cells from induced pluripotent stem cells, applying senolytics to remove senescent subpopulations, and enriching juvenile cell fractions through sorting, all of which aim to restore proliferative and immunomodulatory capacities [1,15,20]. In addition, biomaterial-based priming, pharmacological interventions, and customized culture conditions are being investigated to modulate MSC behavior and enhance therapeutic properties [21,22].

This review highlights three major strategies to improve MSC trafficking: increasing chemokine receptor expression, such as CXCR4, through genetic approaches; using cytokine or hypoxia preconditioning to enhance migratory signaling; and engineering the cell surface with biomaterials to facilitate targeted endothelial adhesion [23,24,25]. Understanding these distinctions is essential for selecting the most appropriate strategy to address age-related declines in MSC homing efficiency, including reduced CXCR4 expression and impaired VCAM-1-mediated endothelial tethering in senescent microenvironments [4,15,26]. Careful consideration of each approach’s durability, safety, and compatibility with pathological conditions is necessary for optimal implementation.

2. Unmodified MSCs: Limitations and Challenges

The therapeutic utility of primary mesenchymal stromal cells is often limited by poor engraftment and survival after systemic administration because culture-expanded cells lose critical migratory receptors such as CXCR4 [27]. Native MSCs rely on intrinsic homing receptors, stress-response pathways, and paracrine cues to reach target tissues. However, after transplantation into aged, damaged, or inflamed tissues, these mechanisms may be insufficient. As a result, MSCs often show limited retention, short persistence, and inconsistent functional benefit despite favorable safety profiles [15,17,26]. Furthermore, these cells are rapidly cleared by the mononuclear phagocyte system and frequently become trapped in the pulmonary vasculature, restricting their distribution to distal target organs [28,29].

Disrupted homing mechanisms are especially important for healthspan-focused applications, where therapeutic cells must function within chronically diseased tissues characterized by persistent inflammation, oxidative stress, and fibrotic remodeling that can worsen pulmonary entrapment and phagocyte clearance [15,30]. Donor-to-donor heterogeneity and tissue-source heterogeneity significantly increase variability in MSC potency due to differences in age, genetics, health status, and origin-specific secretory profiles, undermining batch consistency and clinical predictability [9,31,32]. Extensive ex vivo expansion required to achieve therapeutic cell numbers often leads to loss of homing receptor expression and reduced metabolic plasticity [33].

The age of the donor, tissue source, expansion protocols, oxygen tension, and inflammatory stimuli strongly influence MSC potency by altering proliferative capacity, homing receptor expression, and immunomodulatory functions [31,34,35]. These variations complicate the standardization of MSC products and challenge the development of reliable potency tests for preclinical studies [30,31,32]. In older donors, MSCs often experience accelerated senescence during expansion, leading to the loss of functional subpopulations and an increase in clones with reduced regenerative and immunomodulatory potential [4,14,32]. Serial passaging can further select for rapidly dividing but less effective clones, diminishing both heterogeneity and therapeutic versatility.

These age-related changes impair the ability to maintain a pro-regenerative secretome and immunomodulatory activity, as replicative senescence shifts the secretory profile toward proinflammatory SASP components that hinder tissue repair [4,5,36]. Unmodified MSCs exhibit inefficient homing and engraftment after systemic or local administration due to age- and expansion-related downregulation of chemokine receptors such as CXCR4 and impaired VCAM-1-mediated endothelial interactions. These factors lead to pulmonary entrapment and clearance by mononuclear phagocytes [15,23,25,26,30,31].

Following intravenous infusion, MSCs are rapidly sequestered in the pulmonary capillaries due to their size and reduced deformability, triggering an acute inflammatory response and facilitating phagocytic clearance within hours, thereby limiting redistribution to sites of injury or inflammation [24,30,37]. Metabolic stress from pulmonary trapping further impairs the secretory function of aged MSCs, reducing the systemic availability of essential growth factors and cytokines needed for immune modulation [38]. This impairment, combined with replicative senescence, diminishes sustained immunomodulatory interactions and exacerbates inflammaging in chronically inflamed environments [15,39,40].

These challenges hinder clinical translation by preventing standardized manufacturing, regulatory potency validation, and safe systemic delivery in elderly patients. Donor heterogeneity and expansion-induced senescence increase the risk of inconsistent efficacy and may worsen inflammaging through impaired biodistribution and clearance [14,15,17,31]. Rigorous quality control and standardized isolation and cryopreservation protocols are essential to reduce variability [41]. Conventional two-dimensional monolayer expansion exacerbates these issues by accelerating senescence, downregulating homing receptors such as CXCR4, and shifting the secretory profile toward proinflammatory mediators [31,32,36]. Emerging research suggests that preconditioning strategies, including hypoxic exposure or specific growth factor administration, have the ability to enhance CXCR4 expression and improve MSC homing efficiency before clinical use [42].

3. Engineered MSCs: Mechanisms and Therapeutic Potential

Multiple strategies have been designed to improve MSC performance, including genetic modification, microRNA treatment, preconditioning, and epigenetic reprogramming, each offering a distinct route to improved healthspan-related outcomes (Figure 1) [43]. These approaches aim to enhance key MSC characteristics, including homing ability, viability in challenging physiological environments, and targeted paracrine signaling. Genetic modification often uses viral vectors to introduce genes encoding growth factors or anti-apoptotic proteins, thereby increasing MSC survival and regenerative factor production [24,31]. Non-viral methods, such as nucleofection and electroporation, are also being refined to deliver therapeutic genes or regulatory RNAs, offering safer alternatives for clinical applications [44].

Surface modification strategies that use bio-orthogonal chemistry or lipid-based coatings should be matched to the dominant biological challenge in each disease. For example, in ischemic cardiovascular conditions, these modifications can reduce pulmonary entrapment, whereas in inflammatory environments, they can decrease immune clearance. This approach may prolong systemic circulation and improve adhesion and function at target tissues [45,46,47]. In ischemic limb disease and chronic wounds, impaired SDF-1/CXCR4 signaling limits cell recruitment, and poor endothelial adhesion delays angiogenesis and tissue repair. Disease-tailored surface modifications may therefore enhance MSC retention and improve recovery [7,48].

For immunosenescence and inflammatory disorders, the major challenge is rapid immune clearance and inflammation driven by insufficient receptor engagement; in these settings, surface modifications should support durable anti-inflammatory effects and improved communication with immune cells [14,15,39]. For neurodegenerative diseases and frailty syndromes, surface engineering can be designed to deliver neurotrophic factors, support mitochondrial function, and provide anti-senescent vesicles. These examples underscore the need for mechanism-driven, disease-specific MSC platforms rather than a one-size-fits-all approach [43,49,50].

3.1. Genetic, RNA, and Genome-Editing Strategies

In addition to genetic methods, small molecules and growth factors can modulate MSC behavior by activating signaling pathways that support proliferation, differentiation, and stress resistance [51]. Engineering the culture environment with defined physical, chemical, or biological cues can also guide MSC development and enhance therapeutic efficacy [52]. Epigenetic reprogramming, including reversal of age-associated methylation patterns, may rejuvenate aged MSCs and improve their therapeutic utility [4].

CRISPR-Cas9 enables precise genetic modifications that can enhance MSC survival, differentiation, and immunomodulatory function [53]. For example, CRISPR/Cas9-mediated Keap1 knockout in adipose-derived MSCs activates the Nrf2 pathway, strengthening antioxidant defenses and improving cell survival [53,54]. Similarly, insertion of SV40T at the Rosa26 locus can increase the proliferative capacity of bone marrow MSCs without compromising key cellular features [55]. Upregulation of chemokine receptors such as CXCR1, CXCR4, and CXCR7 can improve MSC migration and expansion, thereby enhancing outcomes in disease models [56].

Engineering MSCs to express IL-10, HGF, IDO, Foxp3, or Bcl-2 can strengthen immunomodulation, reduce inflammation, and improve resistance to apoptosis [34,57]. Likewise, increasing expression of CXCR4, IL-10, BDNF, or IFN-beta using viral vectors or CRISPR/Cas9 can enhance MSC immune, antiviral, and neurotrophic functions [50,58]. Finally, post-transcriptional modification strategies may increase long-term MSC resistance to hostile microenvironments [59].

3.2. Preconditioning, Metabolic Reprogramming, and Biomaterial Priming

Pharmacological approaches using small molecules, cytokines, growth factors, hypoxia, extracellular matrix signals, and biomaterials can prime MSCs to maintain stronger secretory, migratory, and immunomodulatory traits under hostile conditions [14,22,60]. For example, diabetes impairs the reparative function of bone marrow–derived stem/progenitor cells, including MSCs (BMD-MSCs). Ex vivo priming with SDF-1α can functionally activate these cells before transplantation. In the referenced study, SDF-1α–primed diabetic BMD-MSCs significantly accelerated wound closure, increased neovascularization, and enhanced endothelial progenitor cell recruitment, while improving cell proliferation without reducing viability [61]. Mechanistically, SDF-1α priming upregulated pro-healing and proangiogenic mediators, including plasminogen and EphB4, and promoted adhesion-related pathways that may support endothelial progenitor cell interaction and vascular engraftment. Importantly, because SDF-1α exposure was performed ex vivo and cells were washed before implantation, this approach improved wound repair without increasing systemic SDF-1α levels [61]. These findings support SDF-1α preconditioning as a practical strategy to enhance the regenerative potency of MSCs/BMD-MSCs for diabetic wound healing.

Exposure to hypoxia or inflammatory cytokines such as IFN-gamma and TNF-alpha can improve the MSC secretome and increase release of cytoprotective and immunomodulatory factors that are often reduced under standard culture conditions [35,62]. In addition, TGF-alpha priming improves MSC survival in ischemic myocardium, demonstrating that targeted biochemical preconditioning can help overcome barriers to engraftment and expansion in vivo [63].

Metabolic reprogramming, such as increasing NAD+ levels or activating AMPK, improves MSC function in aged tissues by restoring mitochondrial health, balancing glycolysis, and increasing resistance to age-related decline [39,64]. These approaches can also precondition MSCs, reducing variability and improving clinical consistency [65,66]. Conversely, poorly controlled culture conditions can increase MSC heterogeneity, reduce immunomodulatory function, and decrease potency consistency [34,51,56]. To ensure reliable clinical outcomes, potency assays for engineered MSCs should include mechanism-specific functional tests, such as immunomodulatory secretome profiling, migration assays, and measurement of critical quality attributes [30,67,68].

3.3. Surface Engineering, Glycoengineering, and HCELL

Cell-surface engineering methods, including lipid-anchored palmitated protein G for antibody or ligand attachment, have been developed to address poor homing and tissue targeting in MSC therapy. These strategies display adhesion molecules that enhance selectin- or integrin-mediated binding, improving MSC trafficking to specific target sites or inflamed tissues [1,24]. Because MSCs commonly lack sufficient selectin ligands for effective capture under physiological flow, surface modifications such as enzymatic fucosylation of cell-surface glycans can increase vascular tethering and tissue entry without altering the cell’s internal program [1,69]. Glycoengineering provides a targeted strategy to increase selectin binding and improve MSC trafficking to inflamed tissues, thereby enhancing delivery efficiency [70].

Specialized scaffolds can also help maintain therapeutic activity and prolong MSC survival in inflamed environments [71]. Fucosylation of MSCs by converting CD44 to the HCELL glycoform has been shown to enhance vascular adhesion and transendothelial migration into damaged tissues by activating integrin alpha4beta1 [72,73]. This modification helps MSCs attach and roll along blood vessels under flow, overcoming their natural lack of E-selectin ligands [74,75]. Because native MSCs lack the sialofucosylated determinants required for E-selectin recognition, HCELL glycoengineering addresses poor bone targeting and limited vascular extravasation [76,77,78].

As a transient, non-genetic modification, this approach improves MSC homing to bone and inflamed sites while preserving cell viability and function and avoiding risks associated with permanent genetic alteration. These features have supported early clinical translation, including phase I trials in osteoporosis [70,72,73]. However, HCELL generation via glycosyltransferase-programmed stereosubstitution is limited to existing glycoproteins, such as CD44, and may unintentionally alter other surface molecules. It may also transiently shift the secretome toward anti-inflammatory effects, which could restrict broader therapeutic applications [1,76].

3.4. MSC-Derived Extracellular Vesicles as Cell-Free Engineering Platforms

MSC-derived extracellular vesicles (EVs) are promising cell-free engineering platforms that may overcome key limitations of cellular therapy, including tumorigenic risk and poor survival in hostile host microenvironments. These vesicles deliver concentrated bioactive proteins, lipids, and nucleic acids [79,80,81]. Their surface can be modified or their cargo can be loaded with specific therapeutic agents to improve targeting and signaling, allowing them to mimic beneficial functions of parent MSCs while reducing risks associated with systemic cell administration [82,83].

In aging studies, vesicles from young or rejuvenated MSCs reduced senescence markers such as p16 and p21, decreased oxidative stress, and increased healthspan in Ercc1-deficient and naturally aged mice by delivering anti-senescence signals that restore stem cell function without the risks of direct cell therapy [84,85,86]. However, clinical translation of engineered EVs remains challenging due to batch variability across different cell sources and culture conditions, the lack of reliable large-scale isolation methods, and the absence of standardized potency assays to ensure consistent biodistribution and safety [79,80,87,88].

Its utility could be advanced through standardized proteomic analysis, functional assays, and quality attributes directly linked to treatment outcomes [80,89]. Preclinical studies provide strong evidence that engineered MSCs, including senescence-resistant cells generated through CRISPR/Cas9 or epigenetic modification, can improve healthspan-related outcomes in aged animal models. These cells reduce cellular senescence, support tissue repair, and slow age-related functional decline. For example, intravenous administration of senescence-resistant mesenchymal progenitor cells improved memory, bone strength, and reproductive health in non-human primates without evident adverse effects [90,91]. Similarly, extracellular vesicles from rejuvenated MSCs increased healthspan in progeroid mice, producing effects comparable to those of young cell infusions [85].

Together, these advanced engineering approaches highlight the potential of MSCs as a flexible therapeutic platform for age-related diseases and healthspan improvement [92]. Because many engineering strategies are available, careful comparisons of efficacy and risk will be essential to identify the most suitable options for clinical use [93].

4. Supercharged MSCs with E-Selectin

Genetic engineering can enhance MSCs by introducing adhesion molecules such as E-selectin, which improves their capacity to home to and persist at target sites. In this approach, adeno-associated virus (AAV)-mediated transduction is used to induce surface expression of E-selectin in murine bone marrow-derived MSCs. E-selectin gene modification markedly enhances both the therapeutic phenotype and function of MSCs. Unmodified MSCs do not express E-selectin; however, after AAV-mediated E-selectin engineering, MSCs acquire a stronger MSC phenotype, including robust upregulation of the key MSC markers CD44 and CD105, while retaining expression of other standard MSC markers such as CD29, CD73, and Sca-1 [94]. Importantly, E-selectin-engineered MSCs preserve their fundamental stem/stromal cell properties, including colony-forming capability, proliferative capacity, and trilineage differentiation potential into chondrogenic, osteogenic, and adipogenic lineages. Thus, E-selectin engineering enhances MSC identity and potency without compromising their native multipotency [93,94]. Figure 2 summarizes this strategy, tracing the transition from conventional MSCs through AAV-mediated E-selectin engineering to the resulting supercharged phenotype and its enhanced therapeutic function in ischemic tissue.

Functionally, E-selectin-engineered MSCs displayed superior regenerative and proangiogenic activity compared with control MSCs. In an ischemic wound model, treatment with E-selectin-engineered MSCs significantly accelerated wound closure, promoted complete re-epithelialization, increased collagen deposition, and enhanced vascular density within ischemic wounds [94] (Figure 2). These cells also showed improved survival and persistence in ischemic tissue, suggesting that E-selectin modification strengthens MSC viability and therapeutic durability in a hostile ischemic microenvironment. Together, these findings indicate that E-selectin engineering transforms conventional MSCs into a more potent regenerative cell product with enhanced tissue repair and angiogenic capacity. Therefore, E-selectin-positive MSCs are appropriately termed “supercharged MSCs” [94,95].

These cells interact strongly with endothelial ligands in ischemic tissues, which improves cell retention, promotes neovascularization, and increases the expression of proangiogenic genes such as Cxcl2 [45,94,95]. Studies show that E-selectin engineering increases expression of nine proangiogenic genes in vitro, including Cxcl2, and reduces muscle atrophy while enhancing neovascularization in limb ischemia models [13,45,94]. In addition to enhancing tissue adhesion, E-selectin also acts as a signaling molecule that increases expression of proangiogenic genes, including Cxcl2, and reduces inflammatory mediators such as TNF. These effects promote release of beneficial paracrine factors, improve cell survival, and enhance neovascularization in ischemic tissues [13,45,94].

These cells interact strongly with endothelial ligands in ischemic tissues, which improves cell retention, promotes neovascularization, and increases the expression of proangiogenic genes such as Cxcl2 [13,45,95]. Studies show that E-selectin engineering increases expression of nine proangiogenic genes in vitro, including Cxcl2, and reduces muscle atrophy while enhancing neovascularization in limb ischemia models [96,97]. In addition to enhancing tissue adhesion, E-selectin also acts as a signaling molecule that increases expression of proangiogenic genes, including Cxcl2, and reduces inflammatory mediators such as TNF. These effects promote the release of beneficial paracrine factors, improve cell survival, and enhance neovascularization in ischemic tissues [13,45,96].

By reshaping the molecular microenvironment, this approach addresses key limitations of standard MSC therapies in severe tissue loss [98]. Future studies should determine whether E-selectin alters the MSC secretome, recruits host endothelial or immune cells, acts primarily through cell retention or paracrine activation, and whether its benefits persist after transgene expression declines. These studies will help determine whether supercharging should focus primarily on adhesion, paracrine activation, or both [13,94,96].

5. Manufacturing, Potency, and Regulatory Considerations for Engineered MSC Products

Clinical translation of engineered MSCs depends as much on manufacturing strategy as on biological rationale. During production, MSCs can undergo replicative aging and clonal selection, reducing multipotency and shifting the secretome toward a pro-inflammatory profile even when canonical surface markers remain stable [32,99]. Each additional engineering step introduces potential variability. Therefore, standardized culture methods and rigorous bioequivalence testing across batches are essential to reduce phenotypic drift and preserve therapeutic potency consistent with the intended mechanism of action [100,101].

Because engineered MSCs may still home inefficiently, effective delivery can require high cell doses. This increases the risk of pulmonary embolism, microthrombi, and vascular occlusion, particularly when infusion parameters are not optimized [30,102,103,104]. Age-related changes in both donors and recipients may further worsen biodistribution and reduce regenerative effects [30]. Because only a small fraction of administered cells typically engraft, repeated treatments may be required; however, repeated expansion and administration can accelerate MSC aging and reduce signaling capacity [105,106].

Hypoxic preconditioning and optimized delivery strategies may help mitigate these limitations [106]. Nevertheless, donor variability and prolonged ex vivo expansion remain major obstacles to producing consistent, high-potency MSC products for reliable clinical outcomes [107]. Potency testing should extend beyond surface-marker analysis and include functional, multi-parameter assays that reflect how engineered cells interact with diseased tissue [108]. Future validation should incorporate real-time monitoring of secretome profiles and cell-surface ligand density to keep batch variability within safe and effective limits [5].

Regulators generally have higher expectations for genetically engineered MSCs than for minimally manipulated products. When viral vectors are used, testing must detect replication-competent viruses and carefully assess the risk of integration to reduce the potential for insertional mutagenesis or tumorigenesis [88,109]. Genome-edited products require thorough off-target analysis, integration-site assessment, and clonal-expansion monitoring to ensure genetic stability and safety [88,110].

For surface-engineered MSCs, the main regulatory focus is confirmation of transient surface changes, such as improved selectin binding, through functional assays demonstrating improved homing. These products generally do not require the same genomic integration or off-target analyses as genome-edited cells [88,111,112]. For MSC-derived EV products, oversight focuses on vesicle identity, purity, sterility after filtration, and potency assays that demonstrate consistent secretome activity [87,113,114]. In all cases, product development should include a clear control strategy with defined acceptance ranges for critical quality attributes.

6. Strategies to Resolve Limitations of Engineered Mesenchymal Stem Cells

Precise delivery of engineered MSCs to target tissues and organs remains a major challenge. Genetic modifications, such as adding chemokine receptors including CCR7, can guide MSCs to lymphoid tissues for improved targeting [115]. Preconditioning MSCs with cytokines such as IFN-gamma can enhance their intrinsic immunosuppressive activity and survival in inflamed environments without requiring extensive genetic manipulation [116]. Using the MSC secretome, particularly extracellular vesicles, as a cell-free therapeutic platform may avoid risks associated with live-cell transplantation, including tumor formation and poor cell survival [87].

It will also be important to determine the optimal method for delivering human E-selectin, whether through genetic modification or protein engineering, to achieve effective surface expression without compromising MSC function or therapeutic potential. Inducible expression systems allow temporal and spatial control of therapeutic protein production. These systems enable precise adjustment of gene expression, reducing off-target effects and limiting long-term immune reactions [117]. They also permit fine-tuning of adhesion molecule expression so that homing can be improved while minimizing immune activation [118]. In addition, inducible systems can adapt protein expression levels to disease-specific conditions, making MSC therapies more precise.

Optimizing MSC culture conditions can help preserve function and therapeutic potency. Appropriate media, scaffold materials, and bioreactor configurations can support immunomodulatory and differentiation capacity, as well as expression of engineered surface molecules [109]. Controlling the timing and duration of engineered molecule expression may also reduce immune responses while preserving native MSC benefits. Transient fucosylation of surface proteins, for example, can improve MSC targeting and retention in inflamed tissues and may increase therapeutic efficacy [73,76].

Biomaterial scaffolds and directed delivery systems can localize MSCs to target tissues, reducing systemic dispersion and improving therapeutic outcomes. For instance, injectable hydrogels and polymer-nanoparticle composites can retain engineered MSCs at local treatment sites. By mimicking aspects of the extracellular matrix, these materials support MSC survival and function while reducing genetic risks and potentially improving scalability and immune compatibility [1,25,119]. They can also be loaded with immunosuppressive agents to shape the local microenvironment and prolong MSC retention and activity [9].

Advanced gene-editing tools such as CRISPR/Cas9 enable precise modifications that can improve MSC therapeutic performance. These tools allow targeted insertion or deletion of genes to enhance immunomodulation, survival, or tissue targeting while reducing limitations associated with older viral approaches [115]. Such modifications can create stable, heritable changes that may improve the safety and efficacy of MSC products for clinical use [120]. In addition, engineering MSCs to produce immunosuppressive factors or molecules that inhibit complement and NK-cell activity may further reduce immune rejection [83].

7. Future Solutions and Clinical Translation for Healthspan Extension

Future MSC engineering approaches for healthspan extension will likely combine strategies to improve targeting, persistence, and resistance to aging. Genetic modifications that increase senescence resistance, combined with biomaterials that support rejuvenation, may prevent cell decline while supporting regeneration and immune function in age-related diseases [41,52,90].

For diabetic wounds, which are characterized by hyperglycemia, oxidative stress, and impaired angiogenesis, MSCs may need to be engineered to release higher levels of angiogenic factors such as VEGF or ANG1. Targeted surface modifications or hydrogel encapsulation can help retain these cells locally and improve vascularization, while transient or cell-free formats may help maintain safety [13,110,121,122]. Another promising direction is the development of cell-free therapies using bioengineered MSC-derived extracellular vesicles (EVs).

These vesicles can be designed for targeted drug delivery or increased stability in the bloodstream, which may improve therapeutic utility [123]. MSC-derived EVs may avoid risks of tumorigenicity and immune reactions associated with live-cell transplantation [9]. For frailty and aging-related conditions, EV-rich products with anti-inflammatory activity may be particularly valuable. MSC-EVs are also attractive because they can be produced at scale and characterized more precisely than parental cell products, helping ensure clinical consistency [124]. To further improve precision, researchers are also using CRISPR-Cas9 to create immortalized cell lines with stable activity and reduced donor-to-donor variability [87,125].

Although these approaches are promising, clinical healthspan extension remains challenging. Long-term monitoring is essential to ensure safety, particularly regarding tumorigenic risk [9,126]. Building on the representative clinical trials and translational experience with MSC-based therapies to date (Table 1), large randomized trials are still needed to define effective dosing and confirm reproducibility. Standardized methods for measuring the secretome and biophysical features of extracellular vesicles are also required for quality control before broad clinical use [31,127]. Finally, regulatory guidelines that account for age-related recipient biology and batch-to-batch variability in stem cell products will be critical for reducing the risk of treatment failure in older patients [35,39].

8. Safety, Study Design, and Clinical Endpoint Considerations

Thorough preclinical testing and standardized manufacturing are necessary to preserve consistency of cell therapy products and reduce risk. New genetic modification tools, including advanced viral vectors and RNA-silencing approaches, are improving MSC engineering and may support the development of broadly available off-the-shelf MSC therapies [138]. The safety of unmodified MSCs has been evaluated in more than 1,200 clinical trials across multiple conditions [30,139,140].

To use engineered MSCs safely, consistent manufacturing and rigorous preclinical validation are required so that enhanced features can be translated into healthspan-focused therapies. Study design should consider that many MSC products have limited persistence in vivo, whereas age-related diseases are typically chronic. Therefore, trials should include surrogate biomarkers of biological aging and tissue recovery rather than relying only on mortality-based endpoints. Functional measures such as the six-minute walk test may be useful in selected contexts [141].

Studies should also assess polypharmacy and possible drug-cell interactions that may influence MSC efficacy in older patients with comorbidities [142]. Finally, data-driven analyses of gene-environment interactions may help identify new therapeutic targets and maintain immune regulation during aging. In healthspan-focused trials, primary outcomes should be both clinically meaningful and disease-specific.

These endpoints should include objective measures of physical function and biological aging, such as reduction of senescence-associated secretory phenotypes or reversal of age-related immune remodeling, to evaluate therapeutic efficacy in older adults [14,15]. Studies should also monitor senescent-cell clearance and systemic consequences, because interactions between MSC therapies and pre-existing secretory phenotypes remain an important determinant of therapeutic response [19,51].

9. Conclusion

Current research and clinical studies suggest that engineered MSCs may benefit conditions such as immune disorders, bone and cartilage injury, and neurological diseases [126,143]. Nevertheless, unmodified MSCs often show poor tissue targeting, limited persistence, donor-to-donor variability, and functional decline in aged or inflamed environments. Engineering approaches can address these limitations by improving survival, homing, paracrine signaling, senescence resistance, and manufacturing consistency.

Important challenges remain in making MSC-based therapies safe, effective, and reproducible. These challenges require strong quality control, standardized manufacturing, and risk-mitigation strategies to improve clinical outcomes [144,145,146]. Additional preclinical and clinical studies are needed to understand long-term effects and optimize delivery of engineered MSCs, particularly for inflammation and age-related metabolic disorders [39].

E-selectin-supercharged MSCs illustrate how modifying cell adhesion can enhance tissue repair, whereas HCELL glycoengineering provides a non-genetic strategy by adding an E-selectin ligand to CD44. Both approaches emphasize the importance of cell trafficking in MSC therapy and can inform the design of more effective regenerative treatments. Progress in this field will require integration of engineering, aging biology, potency testing, scalable manufacturing, and well-designed clinical trials.

Safety features such as inducible suicide systems may improve the safety of genetically engineered MSCs by allowing clinicians to control cell growth and persistence after administration [145]. Despite these advances, improved non-viral gene delivery methods, including lipid nanoparticles and polymer polyplexes, are still needed. These methods avoid genomic integration and may be safer than viral vectors, but they must remain efficient in MSCs, which are difficult to transfect [44,147].

Engineered MSCs and MSC-derived EVs may support tissue repair and healthspan extension, but their success will depend on well-defined mechanisms of action, reliable manufacturing, rigorous safety evaluation, and clinically meaningful endpoints. Inflammation-responsive or doxycycline-inducible promoters may allow cells to release immunomodulatory factors only when needed, reducing off-target effects, immune reactions, and toxicity [148,149]. With improved plasmid designs and cell preparation methods, these advances can make engineered MSC therapies more precise and clinically effective [150,151].