Botanical Nanovesicles Boost Mesenchymal Stem Cell Therapy: Next-Gen Advanced Therapy Medicinal Products for Spinal Cord Injury

Manufacturing Challenges: Achieving Good Manufacturing Practice-compliant MSC Production

Maintaining optimal cellular activity and quality is critical to the efficacy of cell-based therapies. The production of Good Manufacturing Practice (GMP)-compliant MSCs is an intricate and highly regulated process encompassing various stages of cell handling, characterization, and rigorous quality control. ²⁴ To safeguard the quality, safety, and efficacy of such innovative cell and gene therapies, dedicated regulatory frameworks were established. While initial European Union medicines legislation (Directive 2003/63/EC, Regulation 726/2004/EC) addressed the potential of cell therapies as medicines, the specific ATMP framework arrived later with Regulation 1394/2007/EC in 2007. Subsequent directives have further defined scientific and technical requirements for ATMPs, notably Directive 2009/120/EC and the EU GMP-ATMP guidelines. ²⁵ The FDA also uses a risk-based approach, categorizing cell-based products into two groups as follows: minimally manipulated (cryopreservation, thawing, density gradient isolation, washing, and dilution) and more than minimally manipulated (expansion and genetic modification). ²⁵ Consequently, MSC cultures derived from diverse tissue sources must adhere to GMP standards, with rigorous monitoring to ensure sterility, safety, potency, and compliance with predefined specifications.

A central challenge arises from the tension between clinical demand and manufacturing constraints. Achieving therapeutic doses (typically 1–2 million cells per kilogram of body weight) necessitates extensive passaging during in vitro expansion. ²⁶ However, prolonged passaging risks compromising cell quality and functionality, directly conflicting with GMP requirements for maintaining batch consistency and safety. This scalability-efficacy paradox represents a primary barrier to the widespread clinical adoption of MSC therapies.

Cellular senescence: Functional decline of aged MSCs

Cellular senescence, a stress-triggered irreversible proliferative arrest, manifests through persistent metabolic activity and senescence-associated secretory phenotype (SASP)-mediated hypersecretion of pro-inflammatory/proteolytic factors.^27–29 As soon as the number of cell divisions reaches Hayflick’s limit, cellular senescence promotes the aging and dysfunction of organisms. ³⁰ These senescence-associated processes also substantially impair the regenerative capacity and therapeutic efficacy of MSCs.

Autologous MSC transplantation has emerged as a clinically recommended therapeutic strategy, offering distinct advantages in personalized regenerative medicine for sufficient quantities directly from patients. However, the problem we may face is that the quality and lifespan of MSCs are influenced strongly by the chronological age of a donor. ³¹ In donors, intrinsic aging (also termed in vivo aging) directly triggers profound functional impairments in MSCs, evidenced by decreased cell population, compromised migratory potential, and attenuated differentiation capacity.^32–35 Particularly concerning is the emergence of SASP, which transforms these cells into pro-inflammatory mediators through paracrine signaling (Fig. 3). ³⁶ The impaired functionality of MSCs derived from elderly donors may notably contribute to the marked disparities in clinical therapeutic outcomes.

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FIG. 3.

Alterations in cellular functions during MSC senescence. MSC, mesenchymal stem cell.

Umbilical cord mesenchymal stem cells (UC-MSCs) provided an alternative strategy due to their derivation from young donors, circumventing age-related cellular senescence inherent in adult-derived stem cells. However, their clinical application is limited by the substantial costs associated with long-term cell banking for maintaining cell inventories. Critically, the challenge of replicative senescence during extended in vitro culture periods remains a persistent concern in cellular applications.^37,38 Researchers demonstrated that even cells with initially robust proliferative potential eventually undergo senescence due to telomere shortening and accumulated DNA damage during repeated divisions. ³⁹ In vivo aging pertains to the chronological age of MSC donors, which directly impacts cellular longevity, whereas in vitro aging manifests as the progressive loss of stem cell properties due to senescence-associated phenotypic alterations during culture expansion. ⁴⁰ Analogous to in vivo aging, in vitro aging induces marked molecular divergence that profoundly impacts cellular architecture, mitochondrial dynamics, and age-related stress responses, accompanied by characteristic cytomorphological transformations, including nuclear enlargement and chromatin structural reorganization. ⁴¹ The distinction lies in that in vitro aging of MSCs appears to result in more severe consequences, including a complete loss of their progenitor cell signature. ⁴² Long-term in vitro culture may affect MSCs more than the natural aging process of their host. ⁴³ The artificial environment fails to replicate native niche conditions, with suboptimal nutrient gradients and mechanical stimuli precipitating premature cell cycle arrest (G1 phase accumulation) and senescence-associated β-galactosidase overexpression. Some external exposures, such as reactive oxygen species (ROS), significantly accelerate the functional degradation of MSCs. ⁴⁴ These physical and chemical cues indicate that senescence resulting from in vitro replicative expansion definitively leads to the functional deterioration of MSCs.

The senescence of MSCs, referred to as in vitro or in vivo, compromises their functional capacity and poses a significant barrier to clinical translation. Future efforts focused on combating the MSC senescence will be essential in the advancement of ATMPs.

Suboptimal treatment responses: A consequence of physiologically limited regeneration

Although MSC-based therapies have demonstrated therapeutic efficacy in SCI, current treatment regimens remain insufficient to reverse the pathological sequelae of neural damage fully. MSC-mediated therapeutic outcomes for SCI stem fundamentally from intrinsic biological constraints on their regenerative potential. Understanding the intrinsic biological determinants governing MSCs constitutes a pivotal prerequisite for advancing their therapeutic applications.

The intrinsic biological abilities of MSCs directly correlate with their therapeutic efficacy across pathological conditions. MSCs exhibit distinct regenerative capacities dictated by their embryonic germ layer origins and distribution position.^45,46 For instance, ectodermal mesenchymal stem cells (EMSCs) exhibit fundamental functional divergence from their mesoderm-derived counterparts, particularly in neuroregenerative applications. Originating from neuroectodermal progenitors sharing ontogenetic lineage with neural progenitor cells, they demonstrate enhanced neurogenic differentiation capacity and superior neurotrophic factor secretion efficacy. ⁴⁷ Other studies highlighted that UC-MSCs exhibit superior immune regulatory capabilities compared with other stem cells.^48–50 Despite the advantages of MSCs derived from various tissue sources in specific fields, their current efficacy remains insufficient to reverse SCI completely. Achieving complete functional recovery necessitates a multifaceted enhancement of MSC performance (referred to as MSC v2.0) across various therapeutic dimensions.

Hostile microenvironment: Poor MSC survival post-transplantation

The mechanism underlying the efficacy of MSCs in treating diseases remains controversial within the scientific community. Researchers have proposed divergent mechanisms to explain the therapeutic effects of MSCs: some emphasize that the paracrine activity of surviving MSCs at the injury site plays a predominant role. In contrast, others contend that the regenerative function is mediated through MSC apoptosis in vivo.^51–53 Notably, post-transplantation apoptosis patterns alone cannot fully account for the observed clinical efficacy variations. A plausible hypothesis is that the survival rate of MSCs determines the duration of their paracrine effects, thereby influencing the overall recovery outcome. ⁵⁴

The engraftment efficiency of transplanted MSCs at lesion sites is critically modulated by two distinct yet interconnected phases as follows: (i) preimplantation parameters encompassing administration protocol design (including delivery route optimization and dosage calibration), cellular preparation techniques (involving cell source selection, viability assessment, and tissue engineering), and (2) postimplantation dynamics governed by the pathophysiological microenvironment (characterized by inflammatory cytokine profiles and hypoxic stress gradients). The postimplantation phase of MSCs constitutes a critical survival rate determinant, in which dynamic microenvironmental interactions mediate cellular survival kinetics. This pivotal biological process warrants in-depth discussion due to its fundamental role in modulating engraftment success rates and long-term tissue regeneration outcomes.

SCI triggers a self-perpetuating pathological cascade that catastrophically disrupts the homeostatic equilibrium of the spinal cord microenvironment, establishing a pro-inflammatory microenvironment. The pathological overproduction of ROS at the lesion epicenter has been identified as a principal pathogenic driver, which amplifies neuroinflammatory cascades and dysregulates multiple regulated cell death pathways. ⁵⁵ In the hostile microenvironment of the injury site, transplanted MSCs must initially confront intrinsic viability challenges before executing their therapeutic potential. Chronic ROS accumulation and sustained pro-inflammatory signaling at the injury site accelerate MSC senescence and/or programmed necrosis. ⁵⁶ Meanwhile, these cells are subjected to macrophage-mediated phagocytosis, further contributing to MSC loss.^57,58 The ultimate result is that only a minimal fraction of the transplanted MSCs can be detected at the injury site post-transplantation.^59–61 The strategic modulation of MSCs to resist inflammatory damage from hostile microenvironments may bring new hope for enhancing MSC viability and functional persistence post-transplantation.

Rejuvenating MSCs: BNs counteract MSC senescence

MSC senescence is a cellular response to intrinsic and extrinsic pressures, constituting a significant barrier to the GMP-compliant MSCs and markedly diminishing their therapeutic potential. ⁷¹ Contemporary research reveals that the aging state of MSCs could be reversible through targeted external interventions. ⁷² BNs, which contain intrinsic drug compositions, lipids, proteins, and mRNAs, act as carriers for bioactive molecules. ⁷³ They play a crucial role in advancing therapeutic applications and enhancing biological interactions by facilitating intercellular communication. Mechanistically, these BNs are readily taken up and integrated by target cells, leading to significant biological effects (Fig. 5).^74,75

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FIG. 5.

BN-mediated cellular rejuvenation strategy for aging MSCs. BN, botanical nanovesicle; MSCs, mesenchymal stem cells.

Oxidative damage to cellular components has gained widespread recognition as a key pathophysiological event that contributes to the onset and progression of various diseases as well as the aging process. ⁷⁶ It was also recognized as a major driver of MSC senescence. ⁷⁷ Plant fruits are one of the most popular and widely consumed healthy diet sources today for antiaging purposes.^78,79 Their popularity is primarily due to their high concentration of vitamin C. However, vitamin C has a short half-life, requiring continuous infusion to maintain sufficient in vivo levels. Its pharmacokinetics are complex and influenced by numerous environmental and lifestyle factors, complicating its effective delivery. ⁸⁰ Recent study has highlighted the ability of BNs to improve the bioavailability and efficacy of vitamin C, thereby augmenting its antioxidant properties. ⁸¹ When combined with MSCs, these plant-derived compounds may help prevent or reverse oxidative damage, thereby delaying the onset of MSC senescence and preserving their regenerative capacity.^82–84 Another potential reason BNs regulate the youthful state of MSCs is in the drug components they encapsulate. The active ingredients extracted from natural plants have shown superior antiaging properties, involving polysaccharide, polyphenol, saponin, and so on.^85–88 These pharmacological effects also apply to the regulation of MSC function. For instance, fucoxanthin derived from brown seaweed could mitigate the age-related decline in vitality and function as well as the detrimental effects of prolonged in vitro culture by modulating the PI3K/Akt/Nrf-2 signaling pathway. ⁸⁹ The natural bioactive lipid named oleoylethanolamide (OEA) also has been demonstrated to enhance the activity of antioxidant enzymes, including superoxide dismutase and glutathione peroxidase (GSH-px), in MSCs. Pretreatment with OEA improves MSC viability and overall bioperformance. ⁹⁰ Recent research demonstrated that curcumin liposomes alleviate senescence of bone marrow mesenchymal stem cells by activating mitophagy, which provides new evidence for the antiaging effect of drugs on MSCs. ⁹¹ Our recent findings also demonstrated that Lycium barbarum oligosaccharides (LBOs) promote GSH synthesis in EMSCs, reshaping EMSC functional condition via facilitating the removal of ROS. ⁹²

The large-scale production of MSCs is inherently stringent and lengthy. While current methods enhance MSC quality through direct addition of antioxidants like vitamin C and bioactive compounds such as fucoxanthin, these exogenous additives face critical limitations: characteristically short half-lives, unpredictable metabolic kinetics, and inherently low water solubility—all of which drastically constrain bioavailability and consistent efficacy. These fundamental challenges of transient activity and solubility-driven delivery inefficiency underscore the compelling advantages of BNs in MSC production. BNs provide an ideal nano-platform for efficient encapsulation, protection, and targeted delivery of diverse actives. This capability significantly extends compound bioavailability and presents a transformative solution to core biomanufacturing challenges.

MSC functional improvement: BNs augment MSC therapeutic efficiency in SCI

The active ingredients and efficacy profiles of different drugs are distinct, resulting in marked differences in their biological effects. The pathogenesis and progression of certain diseases often guide the selection of BN-based strategies for MSCs, which can be pivotal in influencing cell-based therapies. The severe local pathological cascade and limited nerve regeneration following SCI are primary obstacles to effective treatment. ⁸⁴ Thereby, the key challenge in MSC therapy lies in enhancing MSC immune regulation and nerve regeneration.

In our previous work, we reported that LBO confers an inflammatory license to EMSCs, enhancing their paracrine effects and ultimately augmenting the therapeutic efficacy of EMSCs in treating SCI. ⁹³ Recently, we have successfully isolated BNs derived from L. barbarum. They serve as an efficient natural carrier, contain various active ingredients, including LBO, and have demonstrated a therapeutic role. ⁹⁴ Collective clues provide valuable insights for future research to explore the promotive effects of L. barbarum-derived BNs on MSCs for treating SCI. Due to their superior aqueous solubility, polysaccharides can rapidly confer an inflammatory “license” on MSCs. This pharmacological characteristic has motivated our thinking about whether insoluble therapeutic agents could similarly augment the regenerative potential of MSCs.

Numerous insoluble drugs exist, with curcumin being the primary focus of this discussion. Curcumin, a polyphenolic compound, is renowned for its broad spectrum of biological activities, including anti-inflammatory and neuroprotective properties. ⁹⁵ Curcumin-based therapeutic strategies have achieved significant success in treating SCI.^96,97 More intriguingly, some scholars have proposed the application of curcumin to “domesticate” MSCs, thereby equipping them with enhanced immune-regulatory effects. In their study, they evaluated the impact of curcumin on a peripheral blood mononuclear cell/MSC coculture model. They found that low concentrations of curcumin reduced the levels of inflammatory cytokines (IL-17, IL-6, IL-1β, and IFN-γ) while increasing the levels of anti-inflammatory cytokines (TGF-β and IL-10). ⁹⁸ In addition, other studies have demonstrated that curcumin enhances the immunomodulatory function of MSCs by upregulating the expression of Indoleamine 2,3-dioxygenase 1, which in turn inhibits allogeneic T cell responses.^99,100 The above clues also highlight that the effects of curcumin on MSCs are markedly concentration dependent. High concentrations of curcumin promote apoptosis and are detrimental to MSCs, whereas low concentrations inhibit apoptosis, promote cell proliferation, and facilitate the acquisition of an inflammatory “license.” This suggests that the process of “domesticating” MSCs with curcumin is a gradual, concentration-dependent process. However, applying these strategies generally requires methods to enhance the solubility and stability of curcumin to overcome its inherent limitations in these aspects.^101–103 Natural BNs exhibit unique advantages for addressing drug delivery challenges without undesirable side effects caused by chemical or biological carriers. ¹⁰⁴ Liu et al. isolated and purified nanovesicles from turmeric by ultracentrifugation and sucrose gradient centrifugation. They identified bioactive constituents termed curcumin in nanovesicles with the support of mass spectrometry and HPLC analysis. ¹⁰⁵ This discovery positions BNs as efficient natural carriers for curcumin, suggesting a novel delivery paradigm for insoluble pharmaceuticals. Even more promising is the possibility that these BNs, combined with MSCs, could bring novel breakthroughs. BNs may solve this challenge in future research, as they facilitate a durable and more controlled endocytosis process for cellular uptake. ¹⁰⁶ As mentioned above, concentration gradients of curcumin have diametrically opposed effects: high concentrations induce MSC apoptosis, whereas low doses promote proliferation and confer inflammatory licensing capacity. This biphasic response underscores the need for precise control in the “domestication” of MSCs, highlighting it as a delicate and finely tuned process.

The preceding examples highlight the superior mechanistic profile of BN-mediated curcumin delivery systems for targeting MSC conditioning relative to conventional strategies. BNs offer dual advantages: (1) overcoming the critical solubility/stability barrier via a safer, more biocompatible solution than synthetic carriers, and (2) circumventing curcumin’s detrimental concentration dependence through controlled intracellular release and sustained low-dose exposure in MSCs. BN-mediated preconditioning licenses MSCs for enhanced therapeutic efficacy in SCI by strategically modulating their paracrine secretion profile. Critically, this functional improvement occurs without compromising cellular viability, thereby amplifying MSC-mediated repair mechanisms.

Prolong MSC survival status: BN-mediated biological effect

While aiming to improve therapeutic outcomes, the arbitrary escalation of dosage or frequency in MSC-based therapies may compromise therapeutic rationality due to the elevated risks of severe clinical complications (e.g., pulmonary embolism) and the disproportionate increase in treatment costs relative to efficacy gains. Optimizing the potency of MSCs to transform them into enhanced ATMPs capable of thriving in hostile biological environments is a top priority.

To date, the low survival rate of transplanted cells remains a significant factor affecting treatment efficacy, especially in SCI, where cell survival is often limited. This highlights the need to explore combined strategies to enhance cell survival. ¹⁰⁷ These strategies are tailored to the mechanism of injury and focus on protecting MSCs against detrimental microenvironments characterized by hypoxia, oxidative stress, inflammatory cytokines, and metabolic disturbances.

Long-term severe hypoxia exposure induces metabolic dysregulation and cellular stress responses that significantly impair the survival of transplanted MSCs, as hypoxic microenvironments disrupt their physiological homeostasis and regenerative functions. ¹⁰⁸ Concurrently, oxidative stress exacerbates this damage by promoting ROS accumulation, directly disrupting redox balance and triggering inflammatory cascades. ¹⁰⁹ In SCI, ROS imbalance perpetuates neuronal damage and correlates with poor clinical outcomes, suggesting that oxidative stress management is critical for modulating post-SCI tissue repair processes.^110,111

Enhancing MSC survival under oxidative stress at injury sites involves three primary strategies as follows: tissue engineering, gene modification, and pharmaceutical interventions. (1) Tissue Engineering: This approach requires meticulous optimization of biocompatibility, material toxicity, and MSC mobility within scaffolds to ensure functional integration and regenerative efficacy. However, the complexity of balancing these factors often limits its practicality. (2) Gene modification: While genetically modified MSCs show promise for regenerative therapies, their engineering processes carry risks such as off-target effects and unintended mutations, potentially compromising genomic stability.^112,113 These technical and safety challenges collectively underscore the necessity for stringent regulatory oversight in the clinical use of genetically modified MSCs. (3) Pharmaceutical Empowerment: A more streamlined strategy involves pretreating MSCs with exogenous pharmacological agents. Controlled drug treatment could improve the ability of MSCs to resist excessive oxidative stress following transplantation.^89,114,115 Several studies have created cellular oxidative stress damage models by exposing MSCs to hydrogen peroxide (H₂O₂). The results from these studies consistently suggest that pretreating MSCs with specific drugs, such as antioxidants or other protective agents, can significantly enhance their survival under oxidative stress conditions.^116,117 This strategy reduces cellular damage caused by ROS and preserves MSC functionality, improving their ability to proliferate, migrate, and differentiate. This pharmaceutical approach demonstrates superior operational precision and process controllability in MSC improvement compared with conventional genetic or physical modification techniques, establishing a paradigm-shifting methodology in cellular stress management.

Studies demonstrate that sustained low-dose exposure to lipopolysaccharide (LPS), a bacterial exotoxin, induces immune tolerance in mice and confers protection against subsequent severe inflammatory diseases.^118,119 Extending these discoveries, scholars exploring LPS stimulation of MSCs at varying concentrations and durations confirmed that prolonged, low-dose LPS exposure effectively promotes an inflammatory tolerance state, significantly enhancing MSC survival. ¹²⁰ This success has spurred broader interest in MSC preconditioning strategies, particularly within plant-derived therapeutics. Although established as a potent antitumor agent inducing dose-dependent cancer cell apoptosis and oxidative stress, andrographolide demonstrates unexplored potential in MSC research. ¹²¹ A recent study revealed that andrographolide preconditioning equips MSCs to withstand environmental stressors such as H₂O₂ and oxygen-glucose deprivation. ¹²² A key divergence lies in the effective concentration: andrographolide necessitates 10–30 μM for tumor applications, but triggers effective MSC preconditioning at just 1–10 μM. To leverage this difference, concentrations must be carefully controlled, enabling MSCs to acquire inflammatory tolerance while avoiding cytotoxicity. Consequently, we speculate that all antitumor natural compounds that kill tumors via oxidative stress could potentially enhance MSC tolerance to inflammatory damage. Novel drug delivery systems such as BNs, with their capacity for controlled, gentle release, emerge as compelling candidates for future MSC “training” protocols to induce functional tolerance.

Enhancing the inflammatory tolerance threshold of MSCs constitutes a slow, gentle, long-term process, making sustained drug release and precise concentration control essential for generating high-tolerance MSCs. BNs address this need through a controlled, sustained drug delivery approach that circumvents cellular damage from high drug concentrations and leverages gradual release kinetics to elevate the tolerance threshold efficiently. From this perspective, BNs offer distinct advantages for enhancing MSC survival in inflammatory environments.