Mesenchymal stem cell secretome and exosomes as potential advanced therapy medicinal products for treating a failing heart

Abstract

Heart failure results in impaired cardiac systolic and/or diastolic functions. Traditional pharmacological and non-pharmacological approaches provide, at best, only short-term symptomatic relief, and the long-term prognosis remains poor. Regenerative medicine, particularly mesenchymal stem cells (MSCs), presents a promising alternative for cardiac repair. Ye and Liu published a study in the recent issue of World Journal of Stem Cell highlights the cell-free therapy approach, focusing on the therapeutic potential of MSC-derived paracrine secretions, which exert crucial pleiotropic effects on cardiac repair and regeneration. Their mechanisms involve the seven primary modules that promote angiogenesis, inhibit apoptosis, reduce oxidative stress, modulate inflammatory responses, facilitate cardiomyogenesis, and reduce myocardial fibrosis. Furthermore, their complex repair mechanisms, regenerative pathways, and emerging strategies, which bring us a step closer to their clinical translation, have been discussed as part of the future perspective. While MSC-derived secretomes and exosomes show immense promise for myocardial reparability and improving cardiac function, further research is warranted to fully understand their roles, optimize techniques, and establish their safety and effectiveness for clinical use.

Key Words: Cell-free therapy; Conditioned medium; Exosomes; Heart failure; Mesenchymal stem cells; Myocardial infarction; Secretome

Core Tip: This paper reviews advancements in the treatment and management of heart failure through cell-free therapies, including mesenchymal stem cell-derived secretomes and exosomes that deliver an array of bioactive molecules and participate in the repair process. The seven primary modules, which explain the reparative and regenerative potential of secretome and exosomes, have been discussed in the context of mechanistic understanding, drawing on published data from experimental and clinical settings that act on the myocardium to achieve the therapeutic benefits of the treatment.

This editorial refers to “Engineering the future of nanomedicine: Strategic approaches to extracellular vesicle-based drug administration regimens” by Ye and Liu, 2025; https://doi.org/10.4252/wjsc.v17.i7.107080.

---

INTRODUCTION

Heart failure (HF) is a complex, progressive, clinical syndrome with the underlying pathophysiology involving an intricate interplay between multiple neurohormonal pathways. They also contribute to disease progression, including water retention, vasoconstriction, myocardial hypertrophy, fibrosis, and remodeling[1-3]. The contemporary pharmacotherapy approach for HF is primarily symptomatic and carries its limitations[4]. At the same time, non-pharmacological treatment strategies, such as implantable cardioverter-defibrillators and cardiac resynchronization therapy, can only delay the inevitable, as they fail to address the underlying cause of the problem[5,6]. Heart transplantation is the gold standard treatment, but suffers from high medical costs, serious postoperative complications, need for immunosuppression, and, most importantly, limited donor availability[7]. In sum, none of the contemporary surgical or non-surgical treatment options can reverse myocardial degeneration or necrosis or fundamentally improve cardiac pump function. With recent advances in regenerative medicine, stem cell-based therapies hold promise, as they can support myocardial regeneration through multifactorial mechanisms[8]. As part of the fast-emerging cell-free therapy approach, the secretome provides a potential biological milieu of active factors that can aid in the theragnostic of HF[9]. It contains a heterogeneous profile of growth factors, autocrine and paracrine hormones, cytokines, proteases, chemokines, exosomes, macrovesicles, and secretory pathway constituents, depending on the parent cells[9,10].

The characteristic features of the most well-studied mesenchymal stem cells (MSCs), stipulated by SCRT for their identification, are noteworthy and are used to ensure that a uniform population of cells is used in both experimental and clinical settings[11]. The diverse tissue sources for MSCs can be categorized into two groups: Adult tissues, such as bone marrow, peripheral blood, and adipose tissue, and neonatal birth-associated tissues, including the placenta, umbilical cord, and cord blood[12]. After extensive characterization, MSCs have progressed to the late phases of clinical assessment and have also been launched as living biopharmaceuticals for routine clinical applications in specific disease conditions[13]. Moving forward, the use of their paracrine secretions, including secretomes and exosomes, provides a rapidly emerging cell-free therapy approach to promote tissue regeneration and modulate disease[9]. In this editorial, we extend the discussion from the recent issue of World Journal of Stem Cells published by Ye and Liu[14] entitled “Engineering the future of nanomedicine: Strategic approaches to extracellular vesicle-based drug administration regimens”.

OVERVIEW OF MSC-DERIVED SECRETOME AND EXOSOMES

MSC-derived secretome comprises a spectrum of soluble paracrine factors, including proteins, nucleic acids, and lipids, each with distinct regulatory and trophic functions in intercellular communication and therapeutic processes. For example, among secreted proteins, growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) play a crucial role in angiogenesis. At the same time, interleukin (IL)-10 reduces inflammation in the injured heart, while pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, play a dual role in both damaging and repairing tissue, alongside enzymes like matrix metalloproteinases (MMPs). Nucleic acids, particularly mRNAs and microRNAs (miRNAs), play a crucial role in the genetic regulation of recipient cells. Lipids, such as prostaglandins and leukotrienes, also participate in cell signaling and coordinate various cellular activities[15]. Together, the stem cell-derived secretome constituents orchestrate a reparative and regenerative microenvironment by modulating immune responses, enhancing cell survival, promoting angiogenesis, and facilitating tissue repair with reduced fibrosis[16].

On the contrary, exosomes are nanosized, insoluble factors that carry specific cargo with a heterogeneous profile representative of the parent cells. The composition of the cargo is orchestrated by highly regulated sorting mechanisms that selectively incorporate specific proteins, lipids, and nucleic acids into exosomes, which are tailored to mediate intercellular communication for distinct biological functions[17]. They are released by almost every cell type in the body. Their cargo profile may reveal essential information about the cells and tissues of origin, making them useful as minimally invasive diagnostic and predictive biomarkers, in addition to their therapeutic potential. Exosomes and cardiokines released by cardiomyocytes and endothelial cells, the two dominant cell types in the myocardial architecture, are also being investigated as potential biomarkers for diagnosis and prognosis, in addition to the existing, commonly used cardiac biomarkers, such as cardiac troponins[18,19]. Together, cell secretome and exosomes are transitioning from mere cellular byproducts to established key mediators of intercellular communication, significantly influencing all aspects of diagnosis and therapy.

CARDIAC REPAIR AND REGENERATION MECHANISTIC PATHWAYS

The adult stem cell-derived secretome has demonstrated anti-apoptotic, cytoprotective, reparative, and regenerative effects in the treatment of ischemic heart disease[20]. Figure 1 provides a schematic illustration of the multifaceted role of MSC-derived secretome (represented by rectangular boxes) and exosomal cargos (represented in circles) in promoting cardiac repair following HF. The injured myocardium at the center is surrounded by seven key protective, immunomodulatory, reparative, and regenerative mechanisms (modules), each involving distinct molecular components and biological pathways discussed below.

Figure 1 Mesenchymal stem cell secretome and exosomal cargo-mediated mechanisms of cardiac repair. The figure created by BioRender (Supplementary material). This schematic illustrates the multifaceted roles of mesenchymal stem cell-derived secretome (represented by rectangular boxes) and exosome cargo (represented by circular elements) in promoting cardiac repair following heart failure. The injured myocardium at the center is surrounded by seven key regenerative mechanisms, each involving distinct molecular components and biological pathways as discussed in the text. VEGF: Vascular endothelial growth factor; FGF2: Fibroblast growth factor 2; PDGF: Platelet-derived growth factor; TGF-β: Transforming growth factor beta; HGF: Hepatocyte growth factor; IGF-1: Insulin-like growth factor 1; Bcl-2: B-cell lymphoma 2; PDCD4: Programmed cell death protein 4; Bim: B-cell lymphoma 2-interacting mediator of cell death; IL-10: Interleukin-10; MMP-2/9: Matrix metalloproteinases 2/9; TIMP-1: Tissue inhibitor of metalloproteinases-1; CTGF: Connective tissue growth factor; SOD: Superoxide dismutase; PTEN: Phosphatase and tensin homolog; NF-κB: Nuclear factor kappa B; PGE2: Prostaglandin E2; TSG-6: Tumor necrosis factor-alpha-stimulated gene-6; IDO: Indoleamine 2,3-dioxygenase; HLA-G: Human leukocyte antigen G; SDF-1: Stromal cell-derived factor 1; MCP-1: Monocyte chemoattractant protein 1; G-CSF: Granulocyte colony-stimulating factor; ECM: Extracellular matrix; FSTL1: Follistatin-like 1.

The anti-apoptotic module

In the anti-apoptotic module, MSC-derived paracrine factors in the secretome, such as hepatocyte growth factor (HGF), stromal cell-derived factor-1α (SDF-1α), nerve growth factor, VEGF, and insulin-like growth factor 1 (IGF-1), promote endothelial and cardiomyocyte survival through activation of diverse anti-apoptotic signaling pathways. Gnecchi et al[21] were the first to report that treatment with conditioned medium (CM) from genetically modified bone marrow-derived MSCs (BM-MSCs) overexpressing protein kinase B (Akt) significantly reduced cardiomyocyte apoptosis and caspase-3 activity. Elucidating the mechanism using a comprehensive functional genomic strategy, they reported a pivotal role for secreted frizzled-related protein 2, a modulator of Wnt signaling, in CM cytoprotection[22]. Building on these data, Jiang et al[23] reported that supportive interactions between Akt and proangiogenic factors can further enhance the anti-apoptotic effects of the treatment. Other commonly reported signaling pathways include protein kinase C, phosphoinositide 3-kinase/Akt, nuclear factor kappa B (NF-κB), and signal transducer and activator of transcription 3 (STAT3)[24]. Besides the stem cell secretome, the mechanistic role of exosomes and their payload of miRNAs, i.e., miR-21, miR-22, miR-24, and miR-125b, reportedly inhibits pro-apoptotic targets (programmed cell death protein 4, B-cell lymphoma 2-interacting mediator of cell death, etc.), resulting in reduced caspase-3 activity and activation of the phosphoinositide 3-kinase/Akt and extracellular regulated kinase 1/2 pathways[25]. On the same note, miR-301a in stem cells attenuates cardiomyocyte apoptosis by abrogating apoptosis signal-regulating kinase 1, which regulates the downstream pathways of p38/c-Jun N-terminal kinase/NF-κB signaling[26].

The angiogenesis module

This module is integral to both reparative and regenerative processes, as it plays a crucial role in restoring regional blood flow to the ischemic myocardium. The development of neovascular structures increases blood vessel density in the area, allowing “biological bypassing” to address the root cause of nutritional and blood flow impairment. Simply put, the angiogenesis module is driven by pro-angiogenic factors in the secretome, including VEGF, angiopoietin-1, angiopoietin-2, FGF2, platelet-derived growth factor (PDGF), and transforming growth factor (TGF)-β. Together with the critical participation of an exosomal array of miR-30b, miR-21, miR-126, miR-132, miR-181b, miR-210, and miR-296, they act on endothelial progenitor cells to trigger their migration, homing in, and proliferation via the AKT/endothelial nitric oxide synthase signaling to participate in the angiogenesis[27]. For example, hypoxamiR-210 represses Efna in endothelial cells to promote angiogenesis, while miR-125a plays a dual role, promoting angiogenesis and inhibiting apoptosis by repressing Notch ligand delta-like-4 expression. Yu et al[28] reported that exosomes derived from GATA-binding protein 4-expressing MSCs, upon internalization by cardiomyocytes, improved cardiomyocyte survival, reduced infarct size, and preserved global cardiac function in an experimental animal model. Molecular studies revealed elevated levels of miR-19a in cardiomyocytes and myocardium. Abrogating miR-19a also abolished the therapeutic benefits of GATA-binding protein-expressing MSC-derived exosomes. Recent research has highlighted the role of CD44, which is expressed on the surface of diverse population of cells (i.e., stem cells, monocytes, vascular endothelial cells, and also involved in the membrane organization of exosomes), binds and activates pro-angiogenic factors like FGF2, PDGF, secreted by the infiltrating monocytes to induce the development of collateral circulation in the infarcted myocardium[29]. The role of bone morphogenetic protein-9, which binds to the high-affinity activin-A receptor-like type 1, has been reported to signal via SMAD1/5 and to regulate both paracrine/autocrine signaling and angiogenesis[30].

The oxidative stress reduction module

This module is significant because it addresses the imbalance between oxygen radical production and scavenging, a hallmark of the failing heart that involves diverse signaling pathways[31]. One of the leading causes of oxidative stress in a failing heart is mitochondrial dysfunction, which not only leads to an oxidative imbalance but also impairs ATP production and initiates apoptotic and necrotic signaling pathways[32]. Hence, the oxidative stress reduction module primarily revolves around antioxidant enzymes (superoxide dismutase, catalase, peroxiredoxins) and miR-21 and miR-19a, which suppress phosphatase and tensin homolog, enhance antioxidant defenses, and decrease reactive oxygen species levels[33,34]. Besides antioxidants such as CoQ10 and MitoQ, emerging treatment strategies also include metabolic modulators and gene therapy[32].

The immunomodulatory and anti-inflammatory module

This module ensures the rejuvenation of the failing heart, as the inflammatory response and oxidative stress are closely linked and often proceed in tandem[35]. The immunomodulatory effects of soluble secretomes and exosomes, primarily derived from MSCs, have been extensively studied because they mimic the functional potential of MSC-based therapy to modulate immune responses[36]. Aside from entering cell-to-cell contact, MSCs also interact with immune cells by secreting paracrine signals that regulate immune cell function, proliferation, and differentiation[37]. For example, MSCs inhibit T-helper 17 (Th17) cell differentiation by releasing IL-10 and prostaglandin E2 (PGE2), thereby suppressing IL-17, IL-22, and interferon-γ production and inducing a regulatory T phenotype[38]. Similarly, MSCs block IL-2-induced natural killer cell proliferation during the innate immune response[37]. Wang et al[39] reported IL25-mediated transcriptional regulation of programmed death ligand-1 via STAT3 in human MSCs to suppress the Th17 response, and ablation of IL25 in MSCs abrogated Th17 suppression. Some of the other immunomodulatory molecules include PGE2, TNF-α-stimulated gene-6, IL-10, indoleamine 2,3-dioxygenase, HLA-G, and galectin-1, along with an array of exosomal miRNAs[40], including miR-146a and miR-21-5p, which inhibit NF-κB, thereby reducing TNF-α, IL-6, and IL-1β, and promoting the polarization of M2 macrophages. More recently, engineering or preconditioning approaches for MSCs have been developed to overexpress immunomodulatory molecules, thereby enhancing their therapeutic immunomodulatory activity[41,42].

The module of extracellular matrix remodeling and recruitment of resident stem/progenitor cells

This module is critical for halting HF progression. Cardiac fibroblasts produce constituent proteins of the extracellular matrix (ECM), including collagens type I and III, as well as collagenases, fibronectin, and vitronectin[43]. A recent study comparing a de-cellularized normal heart with a failing heart has revealed that the ECM in a failing heart is more aligned, flatter, and exhibits compact fiber bundles with reduced elasticity and organizational complexity, features that remain the hallmark of standard myocardial ECM[44]. Molecular analysis revealed an overrepresentation of dysregulated genes involved in ECM organization in cardiac fibroblasts of the failing heart, as well as connections to TGFβ1, IL-1, TNF-α, and brain-derived neurotrophic factor signaling pathways. Yes-associated protein was a key player in ECM remodeling in the diseased heart. Treatment with secretome and exosomes accelerates wound healing. Given the structural dynamism in ECM, the roles of MMPs and their tissue inhibitors are crucial for its maintenance, enabling it to perform physiological functions beyond structural support alone[45]. These two enzymes also play a critical role in structural remodeling and homeostasis, thereby balancing ECM turnover. One of the essential functions of ECM is to serve as a reservoir of hormones, growth factors, cytokines, and other factors that enable resident stem/progenitor cells to migrate and remain at the site of injury, thereby participating in the repair process. This is facilitated by bioactive molecules such as SDF-1, monocyte chemoattractant protein 1, and granulocyte colony-stimulating factor[46]. It remains an essential player in the stem cell niche, determining their functionality through processes such as proliferation, self-renewal, and differentiation. A recent study has identified proprotein convertase subtilisin/kexin type 6 as an essential player in cardiac remodeling and fibrosis[47]. Besides, a comprehensive analysis of the exosome-derived potential miRNA candidates with a role in healing and remodeling of the myocardium having ischemia-reperfusion injury and shortlisted 4 of them, including miR-149-5p, miR-29b-3p, let-7i-5p, and miR-7a-5p for their regulatory role[48].

The cardiomyocyte regeneration module

Cardiomyocyte regeneration is at the heart of mechanisms that help the damaged myocardium recover from the functional deficit caused by ischemic episodes. Although the mechanisms of self-renewal in cardiomyocytes remain inconclusive, myocardial regeneration primarily revolves around the possibility of cardiomyocytes re-entering the cell cycle or of resident progenitor cells undergoing cardiomyogenic differentiation. Both of these processes require chemical cues from tissue factors[49]. A study by Kastner et al[50] reported that treatment with secretome from hypoxia-preconditioned MSCs induced cardiomyocyte proliferation. Dynamic changes in different ischemia and regeneration-related genes, including hypoxia-inducible factor-1 alpha, Ras homolog family member A, and IL-18, were observed. Kukumberg et al[51] reported cardiomyocyte proliferation in response to treatment with the amniotic fluid stem cell secretome. Analysis of the secretome revealed its richness in proangiogenic and anti-inflammatory factors, including HGF, leukemia inhibitory factor, placental growth factor, stem cell factor, VEGF-A, IL-8, and SDF-1α, as well as an array of ILs and cytokines. A recent study has demonstrated that treatment with an engineered secretome from preconditioned adipose tissue-derived stem cells is therapeutically effective[52]. However, the nanoparticle-released secretome was rich in IGF-1, FGF2, PDGF, and VEGF, promoting angiogenesis and cardiomyocyte hypertrophy without evidence of cardiomyocyte proliferation, but with increased proliferation of non-cardiomyocyte cells. Besides the secretome, exosomal miRNAs play a significant role in myocardial repair and regeneration, as summarized by Bhaskara et al[53]. The role of neuregulin-1, VEGF, IGF-1, and follistatin-like 1, in concert with exosomal miR-199a, miR-17-92 cluster, and miR-590, which activate the Hippo/Yes-associated protein and Wnt/β-catenin pathways to induce cardiomyocyte proliferation, has been reported.

The anti-fibrotic module

This module interferes with the heart’s intrinsic repair mechanism, which replaces damaged myocardium with fibrotic tissue. Fibrotic changes in the myocardium are associated with most cardiac pathologies[54]. It generally involves the activation of cardiac fibroblasts, which is accompanied by chronic activation of inflammatory pathways and mechanical stress, leading to excessive ECM production, specifically collagen, in the myocardium[55]. The fibrotic changes lead to structural and functional alterations in the myocardium, as fibroblasts transition into myofibroblasts, which are primarily regulated by p38 mitogen-activated protein kinase, a component of the noncanonical TGFβ pathway activated by both chemical and mechanical stress. Although the process can be slowed by pharmacological interventions with RAAS inhibitors and beta-blockers, these fail to reverse the already established fibrotic tissue. The use of MSCs and their paracrine secretions has shown promise in inhibiting fibrosis, primarily by preventing the transformation of fibroblasts into myofibroblasts[56]. At the molecular level, the use of secretome and exosomes revealed distinct mechanisms. Treatment with secretome upregulated NF-κB target genes (COX2, IL-6, etc.) in fibroblasts, as the secretome was found to be rich in NF-κB activators[57]. On the contrary, the exosomal fraction was interestingly enriched in TGF-β, Notch, IGF, and Wnt pathway activators[56]. The antifibrotic effects that are reportedly mediated by but not restricted to the secreted VEGF, HGF, FGF2, IL-10, HGF, monocyte chemoattractant protein 1, MMP-2/9, and tissue inhibitor of metalloproteinases-1, alongside exosomal let7, miR-21, miR-26, miR-29, miR-122, miR-125b, miR-133, miR-146, miR-148, and miR-150, which downregulate collagen I/III, TGF-β, and connective tissue growth factor, inhibiting the TGF-β/SMAD pathway and ECM deposition and should be investigated further for their anti-fibrotic effects[58].

Collectively, these synergistic mechanisms contribute to myocardial protection, regeneration, angiogenesis, and immunomodulation, highlighting the therapeutic potential of MSC-derived secretome and exosomes in HF. A less established mechanism involves the recruitment of endogenous stem cells by specific paracrine factors, such as SDF-1, VEGF, FGF-2, HGF, and C-X-C chemokine ligand 12, as well as microvesicles released by stem cells. This mechanism enables resident cardiac and cardiovascular lineage cells to re-enter the cell cycle, compensating for the loss of functioning cardiomyocytes.

SECRETOME EFFICACY EVIDENCE IN PRECLINICAL STUDIES

Preclinical studies using MSC-derived secretomes have consistently demonstrated cardioprotective effects in animal models of myocardial infarction (MI) and HF. Starting with a simple infusion of MSC-conditioned media, which led to nearly a 60% reduction in infarct size, improved stroke volume and left ventricular ejection fraction (LVEF), and enhanced diastolic function, treatment using secretome has come a long way[59]. Kompa et al[60] implanted rats with a TheraCyte device containing W8B2+ cardiac stem cells; the device continuously released its secretome into the systemic circulation, thereby preserving LVEF, attenuating scar area, reducing interstitial fibrosis, and inhibiting cardiomyocyte hypertrophy. Two independent studies reported that systemic infusion of cell-mimicking microparticles and synthetic MSC microparticles carrying a concentrated secretome improved LVEF and reduced infarct size, while increasing infarct wall thickness without eliciting an immune response[61,62]. Intramyocardial injection under direct vision of a nanocomposite secretome-loaded hydrogel (nSi Gel) improved LVEF, left ventricular fractional shortening, and cardiac output, and significantly attenuated infarct size[63]. At the molecular level, the intravenous delivery of secretome in experimental rodent models downregulated profibrotic genes (Col1α1, MMP-2, MMP-9) and the pro-inflammatory cytokine IL-12p70, resulting in a concomitant improvement in LVEF[64]. Translational studies in a porcine model of experimental MI treated with APOSEC (a secretome from apoptotic blood mononuclear cells) preserved LVEF, reduced transmural infarct size, increased blood vessel density, and repressed pro-inflammatory genes, i.e., TNF-α, IL-1β, and CCL2[65]. Using a combinatorial approach with adipose-derived MSC secretome, modest improvements in vessel density and upregulation of angiogenic genes were observed compared with cells alone[66]. Current research focuses on exploring the most effective delivery methods and utilizing various devices and hydrogels that provide sustained release, thereby enhancing retention and prolonging therapeutic benefits. A summary of preclinical studies using secretome-based therapy in animal models is presented in Table 1[59,64-69].

Table 1 Pre-clinical studies using mesenchymal stem cell-derived secretome in small and large experimental animal models for cardiac regeneration and repair.

Ref.	Model	Source	Isolation methods	Conditioned medium	ROA	Dose and groups	Further details
Timmers et al[59], 2011	Dalland landrace pigs (LCX ligation MI)	hESC-derived MSCs	Centrifugation and 220 nm filtration	Not explicitly mentioned	ICI	MSC-CM, non-CM	CM treatment enhanced cardiac function after MI and suggested potential paracrine-mediated cardioprotection
Hynes et al[67], 2013	Female landrace pigs (LAD occlusion MI)	Porcine EPCs	Centrifuged at 600 × g for 5 minutes + 0.2 μm filtration	IGF-1	ICI	4 mL of X-vivo (control). CM, CM + anti-IGF-1 antibody. CM + Ig. X-vivo + anti-IGF-1 antibody	The data showed IGF-1 as a key mediator of CM’s anti-apoptotic and pro-angiogenic benefits when delivered via ICI
Pavo et al[65], 2014	Porcine (LAD occlusion MI)	Porcine PBMCs (APOSEC)	Dialysis and lyophilization	Not explicitly mentioned	IMI	APOSEC (resuspended in 4 mL physiologic saline, 300 μL aliquots) after 30 days from MI	APOSEC treatment showed long-term improvement in LV pump function, suggesting therapeutic benefits independent of cell differentiation
Vilahur et al[66], 2017	Pigs (LAD ligation MI)	Porcine ASCs	Centrifuged and filtered	GFP	CM: IV. ASCs: ICI (77)	(1) ASCs (1 × 107 cells); (2) CM (30 mL); (3) ASCs + CM; and (4) Control (PBS 30 mL). All of them, 7 days after MI	Explored ASCs and their CM. The ASC-CM group exhibited enhanced micro-vascularization, overcoming ischemia-induced vessel rarefaction
Ellis et al[68], 2021	Mouse exposed to cold UW cardioplegic solution	ASC-S	ASC-S was centrifuged and then filtered using 3 kDa cutoff filters	Antioxidant SOD3, catalase, HGF, VEGF, and SDF-1	I/C infusion of UW ± ASC-S	10% ASC-S in UW solution. Control groups: UW alone or UW + basal media (ASC-BM)	ASC-S significantly improved cardiomyocyte survival, demonstrating its protective role in I/R injury
Könemann et al[64], 2020	Mice (cyclin T1 induced LV-hypertrophy	Murine Sca-1+ & Sca-1- CPCs	0.22 μm filtration + centrifuged for 10 minutes at 300 × g	Not mentioned	IV	Sca-1+ control; Sca-1- control; Sca-1+ Aldo; Sca-1- Aldo, untreated	Sca-1+ CM showed superior cardioprotection, reducing hypertrophy and fibrosis, though both cell types contributed to functional recovery
Huang et al[69], 2020	SD rat (LAD ligation MI) & Yorkshire pigs (LAD ligation MI)	hCSCs	Centrifuged at 1000 × g for 10 minutes	VEGF, HGF, and IGF	TS	artCP (diameter = 3.5 cm patch)	The patch improved myocardial repair by paracrine effects and structural support in rat model. The hCSC-seeded artCP (3.5 cm patch) was implanted via TS. The patch, which releases VEGF, HGF, and IGF, significantly improved cardiac function, demonstrating its scalability for clinical applications

ROA: Route of administration; LCX: Left circumflex; MI: Myocardial infarction; hESC-derived MSCs: Human embryonic stem cell-derived mesenchymal stem cells; ICI: Intra-cardiac injection; CM: Conditioned medium; LAD: Left anterior descending; EPCs: Endothelial progenitor cells; IGF-1: Insulin-like growth factor 1; PBMCs: Peripheral blood mononuclear cells; APOSEC: Apoptotic cell secretome; LV: Left ventricular; ASCs: Adult stem cells; GFP: Green fluorescent protein; IV: Intravenous injection; PBS: Phosphate-buffered saline; UW: University of Wisconsin Solution; ASC-S: Adult stem cell-derived secretome; SOD3: Superoxide dismutase 3; HGF: Hepatocyte growth factor; VEGF: Vascular endothelial growth factor; SDF-1: Stromal cell-derived factor 1; I/C: Intra-corporeal; hCSCs: Human cardiac stem cells; TS: Tissue section; artCP: Articular cardiac patch.

MECHANISTIC INSIGHT INTO THE PARACRINE AND THE PROLIFERATIVE ACTIONS OF MSC-DERIVED SECRETOME

The regenerative capacity of MSC-derived secretome and EVs in cardiac therapy has been a matter of extensive debate, with ongoing discussion over whether the observed recovery reflects true cardiomyocyte proliferation or functional improvement driven by paracrine mechanisms[70]. Early reports describing increased expression of proliferation markers such as Ki-67, phospho-histone H3, and Aurora B kinase suggested a regenerative effect[71,72]. However, subsequent lineage-tracing and fate-mapping studies have provided strong evidence that new cardiomyocyte formation in adult hearts is exceedingly rare, redirecting the mechanistic understanding toward functional repair rather than cellular replacement. Recent studies using stable isotope labeling and multi-isotope imaging demonstrated that cardiomyocyte renewal occurs at a rate of less than 1% per year, even following myocardial injury[73]. These findings were corroborated by later Cre-lox lineage-tracing studies, which confirmed that improved cardiac function after exosome therapy largely stems from functional preservation, reduced apoptosis, and enhanced perfusion, rather than de novo cardiomyocyte formation[73,74].

Compelling evidence supporting this functional paradigm comes from translational in vivo studies. In a porcine MI model, Gallet et al[75] demonstrated that exosomes secreted by cardiosphere-derived cells significantly reduced fibrotic scarring, attenuated adverse ventricular remodeling, and improved systolic performance in both acute and chronic settings. Similarly, Chen et al[76] reported that exosomes derived from cardiac progenitor cells conferred potent cytoprotective effects against ischemia/reperfusion injury through anti-apoptotic and pro-angiogenic mechanisms. Furthermore, Khan et al[77] provided mechanistic insights showing that embryonic stem cell-derived exosomes activate endogenous cardiac repair pathways, stimulating neovascularization and enhancing functional recovery post-infarction. Collectively, these studies confirm that the beneficial outcomes of exosome therapy primarily result from paracrine bioactivity, including modulation of inflammation, fibrosis, and angiogenesis, rather than cellular proliferation.

The molecular constituents of the MSC-secretome - particularly miRNAs, growth factors, and cytokines - orchestrate these paracrine responses. Exosomal miRNAs such as miR-199a, miR-17-92, and miR-590 can transiently induce cardiomyocyte cell-cycle re-entry by upregulating cyclin D1 and cyclin-dependent kinase 6, though without completing cytokinesis[78]. Proteins such as neuregulin-1 and follistatin-like 1 have also been implicated in partial proliferative signaling, particularly in neonatal cardiomyocytes, but show inconsistent effects in the adult myocardium[78,79]. This emerging evidence supports a dual-mechanism hypothesis: The predominant effects are paracrine-mediated functional recovery, complemented by a limited and transient molecular stimulation of cardiomyocyte reactivation.

Recent clinical imaging data using magnetic resonance imaging and positron emission tomography further confirm significant improvements in perfusion, contractility, and ventricular wall thickness following exosome administration, with minimal evidence of new cardiomyocyte formation[75,77,79]. These functional outcomes strongly reinforce the concept that MSC-secretome and extracellular vesicle (EV) therapies act through cytoprotection, vascular regeneration, and anti-fibrotic remodeling rather than bona fide cardiomyocyte renewal. Together, the available literature delineates a more refined mechanistic model of MSC-derived secretome therapy in cardiac regeneration - one in which functional restoration via paracrine signaling constitutes the dominant process. At the same time, true cardiomyocyte proliferation remains minimal and transient. Future research should focus on identifying molecular switches capable of safely sustaining cardiomyocyte cell-cycle progression and developing standardized lineage-tracing and potency assays to distinguish between these intertwined mechanisms.

CLINICAL EVIDENCE OF SECRETOME THERAPY BENEFITS

Encouraged by the experimental data, the SECRET-HF phase 1 trial (NCT05774509) was the first-in-human application of a cardiovascular cell-derived secretome for HF. To date, only a single patient has been treated and reported, and this case remains the sole published clinical evidence for MSCsecretome therapy in HF. A 59-year-old patient suffering from a non-ischemic dilated cardiomyopathy, NYHA class III, with a 25% LVEF, received three intravenous doses of CPC-derived secretome (20 × 10⁹ EVs/kg/dose). The patient exhibited no immediate adverse effects related to the procedure. However, the LVEF improved to 32%, the LV end-diastolic volume decreased from 110 mL/m² to 88 mL/m², and the NT-proBNP level dropped from 2261 ng/L to 1136 ng/L during the 6-month follow-up. The patient’s functional class also improved to NYHA II. These findings demonstrate the feasibility of intravenous secretome delivery and suggest that dose escalation may improve therapeutic outcomes. Table 2 provides a list of clinical trials for various cardiovascular conditions that utilized MSCs and MSC-derived secretome as therapeutic approaches.

Table 2 Clinical trials evaluating cell-free and cell-based therapies for different cardiovascular diseases.

NCT & title	Condition	Intervention	Phase & sample size (n)	Country	Primary outcome parameters	ROA	Dose
Transplantation of MSCs for the prevention of AMI-induced HF. NCT05043610	Prevention of AMI-induced HF	UC-derived WJ-MSCs	Phase 3, n = 390 (completed)	Iran	The primary endpoint for assessing the intervention’s efficacy will be the incidence of HF	I/C	All 130 patients will receive a single intracoronary infusion of 107 WJ-MSCs, alongside the conventional treatment provided to the same number of patients in the control group
Exosome-based Nanoplatform for Ldlr mRNA Delivery in FH (ENDFH). NCT05043181	FH	LDLR mRNA exosomes	Phase 1, n = 30 (not yet recruiting)	China	Safety and preliminary effectiveness of exosome-based LDLR mRNA nanoplatform for gene therapy in HoFH	Abdominal puncture under ultrasound guidance	Escalation phase: Six dose groups are planned (with single doses of 0.044 mg/kg, 0.088 mg/kg, 0.145 mg/kg, 0.220 mg/kg, 0.295 mg/kg, and 0.394 mg/kg, respectively), with three treatments per group. Extension phase: 12 subjects are enrolled and receive three weekly infusions of LDLR mRNA exosomes, with the single dose determined in the dose-escalation phase
Cardiovascular Clinical Project to Evaluate the Regenerative Capacity of CardioCell in Patients with AMI. NCT03404063	AMI	CardioCell	Phase 2/3; n = 105 (completed)	Poland	Improve the clinical outcomes in patients with AMI	Coronary-non-occlusive method (CIRCULATE catheter)	Active IMP consists of 30000000 WJMSCs suspended in 20 mL 0.9% NaCl and 5% albumin administered
Safety and Efficacy of Intracoronary Adult Human Mesenchymal Stem Cells After Acute Myocardial Infarction (SEED-MSC). NCT01392105	AMI	Autologous BM-derived MSCs	Phase 2/3; n = 80 (completed)	South Korea	Safety and efficacy of intracoronary autologous BM-derived hMSCs in patients with AMI	I/C injection	Single dose of autologous BM-derived MSCs. Dosage: 1 × 1000000 cells/kg

NCT: National clinical trial; ROA: Route of administration; MSCs: Mesenchymal stem cells; AMI: Acute myocardial infarction; FH: Familial hypercholesterolemia; UC: Umbilical cord: WJ-MSCs: Wharton’s jelly-derived mesenchymal stem cells; I/C: Intracoronary; LDLR: Low-density lipoprotein receptor; HoFH: Homozygous familial hypercholesterolemia; IMP: Investigational medicinal product; BM: Bone marrow; hMSCs: Human mesenchymal stem cells.

STANDARDIZATION AND QUALITY CONTROL OF MSC-DERIVED SECRETOME AND EXOSOME THERAPIES

Ensuring standardization and quality control (QC) in the production of MSC-derived secretomes and EVs is a central prerequisite for achieving reproducible, clinically translatable outcomes. The recently released MISEV 2023 guidelines[80] provide the foundation for such harmonization, outlining the minimal information required to characterize EVs at the physical, molecular, and functional levels. These recommendations highlight that EV products should be validated for size distribution and morphology, marker composition, and biological potency before they are considered for therapeutic application.

Building on these principles, studies emphasized that achieving standardization requires controlling every step of the MSC-secretome manufacturing process - from donor cell selection and culture conditions to medium composition, preconditioning strategies, and storage[81]. Variations in these parameters significantly alter the protein and miRNA composition of the secretome and, consequently, its biological effects. Therefore, reproducibility relies on defining critical process parameters and critical quality attributes throughout production. Similarly, studies underscored the need for harmonized Good Manufacturing Practice (GMP)-compliant procedures to ensure consistency, scalability, and product comparability across laboratories and manufacturing facilities[82].

To align with these findings, standardized evaluation should include: (1) Physical and molecular characterization, confirming EV morphology, size, and surface markers such as CD9, CD63, and CD81 while excluding endoplasmic reticulum contaminants like calnexin; (2) Potency assays directly reflecting the intended therapeutic effect (e.g., pro-angiogenic or cardioprotective bioassays); and (3) Quality testing for sterility, endotoxin levels, and particle-to-protein ratios. Process validation should be documented in accordance with GMP requirements, supported by well-defined standard operating procedures and batch-release criteria.

The choice of isolation technique remains another critical determinant of product quality. Table 3 summarizes commonly employed methods and their respective trade-offs in terms of yield, purity, and scalability. Combining complementary methods - such as ultrafiltration followed by size-exclusion chromatography - has proven effective in balancing recovery efficiency with purity[81,82].

Table 3 Summary of the most commonly employed isolation methods for exosomes with their respective advantages and limitations.

Isolation method	Advantages	Limitations	Purity/yield
Differential ultracentrifugation	Well-established and widely used	Labor-intensive; potential co-isolation of proteins	Moderate purity, variable yield
Size-exclusion chromatography	High purity; minimal protein contamination	Requires specialized equipment	High purity, moderate yield
Ultrafiltration	Scalable, GMP-compatible, rapid	Risk of membrane clogging; limited selectivity	Good yield, moderate purity
Polymer precipitation kits	Simple and cost-effective	High protein contamination; unsuitable for clinical-grade use	Low purity, high yield

GMP: Good Manufacturing Practice.

Incorporating in-process QC metrics - including nanoparticle tracking analysis, protein quantification assays, and endotoxin detection - enhances GMP compliance and facilitates inter-laboratory comparability. Collectively, integrating the MISEV 2023 standards, manufacturing optimization, GMP alignment, and clinical dose validation establishes a comprehensive framework for producing standardized, reproducible, and clinically robust MSC-secretome-derived EV products.

LIMITATIONS AND CLINICAL TRANSLATION CHALLENGES

While the SECRET-HF case report demonstrates promising preliminary safety and potential efficacy signals, it is critical to acknowledge that n = 1 case reports provide only preliminary evidence and cannot establish definitive safety profiles or therapeutic efficacy[83]. The reported improvements in LVEF, ventricular remodeling parameters, and functional class, though encouraging, require validation in adequately powered clinical trials with appropriate control groups to account for natural disease progression, placebo effects, and variability in standard-of-care treatments[83]. Complete phase I trial results from SECRET-HF, including dose-escalation data, comprehensive safety assessments across multiple patients, and standardized efficacy endpoints, are essential to determine whether the observed benefits in this single patient represent reproducible therapeutic effects[83].

CLINICAL FEASIBILITY OF EXOSOMES IN OTHER DISEASES

To contextualize the cardiovascular findings within the broader landscape of exosome-based therapeutics, it is instructive to examine clinical trial data from other medical fields, where MSC-derived exosomes have advanced further in clinical development[84]. These studies provide valuable insights into the safety, feasibility, and potential efficacy of exosome-based therapies across diverse pathological contexts, including spinal cord injury (SCI), Crohn’s disease, and coronavirus disease 2019 pneumonia, as discussed below[84].

SCI

A first-in-human, single-arm, open-label phase I clinical trial investigated the safety and potential effects of intrathecal administration of allogeneic human umbilical cord MSC-derived exosomes in nine patients with complete subacute SCI[85]. The study demonstrated that intrathecal administration of allogeneic human umbilical cord MSC-derived exosomes was safe and well-tolerated, with no early or late adverse events attributable to the study intervention. Furthermore, significant improvements were observed in American Spinal Injury Association pinprick and light touch scores, Spinal Cord Independence Measure total scores, and neurogenic bowel dysfunction scores at 12 months compared with baseline. This trial provided preliminary evidence of potential clinical benefits, though the absence of a control group limits definitive conclusions regarding efficacy[85].

INFLAMMATORY BOWEL DISEASE

In a phase II clinical trial, twenty-three patients with fistulas resistant to at least one course of anti-TNF-α therapy were enrolled, of whom 20 completed the study[86]. Exosome-based treatment, repeated 3 times at 2-month intervals, resulted in complete fistula tract closure in 12 patients (60%), and an additional four patients showed clinical improvement. Histopathological analysis revealed substantial reductions in local inflammation and signs of enhanced tissue regeneration, and immunohistochemical analysis confirmed these findings. This phase II trial represents one of the most advanced demonstrations of MSC-derived exosome efficacy in a controlled clinical setting[86].

CORONAVIRUS DISEASE 2019 PNEUMONIA

A phase I/II randomized, double-blind, placebo-controlled trial evaluated the safety and efficiency of exosome inhalation therapy in 30 patients with severe acute respiratory syndrome coronavirus 2-associated pneumonia[87]. Exosome inhalation was safe and well-tolerated, with no serious or non-serious adverse events observed in any group during the trial or the 30-day follow-up period. Significant improvements in oxygen saturation, C-reactive protein, and lactate dehydrogenase levels were observed in the treatment group as compared to the placebo group. This trial demonstrated the safety of repeated exosome administrations via inhalation and provided preliminary evidence of anti-inflammatory effects in acute respiratory disease[87].

SAFETY CONSIDERATIONS

The safety of MSC secretomes and exosomes therapies has gained growing empirical support from recent non-clinical and early-phase human studies. In a good laboratory practice-compliant toxicology study using cynomolgus monkeys, an intravenous administration of human umbilical-cord MSC-exosomes (3.85 × 10¹² per dose) caused no observable systemic or organ-specific toxicity, hematologic abnormality, or immunogenic reaction over the span of 14 days[88]. This study provided the first large-animal validation of exosome tolerability under regulatory standards.

The first-in-human phase I clinical trial by Akhlaghpasand et al[85] confirmed the clinical safety: Nine patients with complete subacute SCI received intrathecal injections of 300 μg of exosomal protein [approximately (0.5-2) × 10¹⁰] and were followed for 12 months. No early or late adverse events were observed, and neurological assessments showed functional improvement without immune or infectious complications[85]. Similarly, in a phase II study of refractory Crohn’s disease fistulas[86] localized MSC-exosome injections were well tolerated, with no treatment-related adverse events, and 60% of treated patients achieved complete fistula closure. Those results indicate a good short-term tolerability of exosome preparations across systemic and local routes of administration.

Despite this, long-term risks, such as immune sensitization or ectopic biodistribution, require further study. Studies of the biodistribution of systemically administered EVs show predominant localization in the liver and spleen, while the implications for long-term fate, functional impact, and off-target effects remain poorly defined[89]. Future trials should integrate a longitudinal immune distribution analysis.

REGULATORY CONSIDERATIONS

European Medicines Agency regulatory framework

Secretome and exosome therapies in Europe are regulated as advanced therapy medicinal products (ATMPs) under the regulation (EC) No 1394/2007[90]. The European Medicines Agency (EMA)’s Committee for Advanced Therapies assesses the quality, safety, and efficacy through the centralized procedure. These therapeutics are treated like biological medicines, requiring compliance with GMP and complete Chemistry, Manufacturing, and Controls data to ensure batch consistency[90]. The sponsors must characterize the starting cell material and secreted components and ensure donor traceability. The classification is case-dependent: A cell-free secretome was not considered an ATMP, whereas an mRNA-loaded exosome was classified as a gene therapy ATMP[91]. Marketing authorization requires EMA approval following Committee for Advanced Therapies/Committee for Medicinal Products for Human Use review after successful clinical trials.

Food and Drug Administration regulatory framework

In the United States, MSC secretome and exosome products are regulated as biologics under the Public Health Service Act section 351 and the Food, Drug, and Cosmetic Act[90]. An Investigational New Drug application is required for trials, and a Biologics License Application is required for marketing, both of which are reviewed by the Food and Drug Administration (FDA)’s Center for Biologics Evaluation and Research (CBER). These products exceed the minimal manipulation limits of 21 CFR 1271 and thus fall under the FDA biologics regulation. CBER’s oversight demands compliance with cGMP, detailed Chemistry, Manufacturing, and Controls, and validated potency and safety assays before clinical use[90]. Exosome therapies for severe conditions may qualify for the Regenerative Medicine Advanced Therapy designation, thereby expediting development[92]. Clinical evaluation proceeds through phases I-III with strict quality, reproducibility, and donor screening standards[90]. The FDA has issued warnings that no exosome therapies are currently approved. In the United States, therapies such as exosomes and secretomes are considered biologics, requiring full Investigational New Drug/Biologics License Application pathways and adherence to CBER and GMP standards. The progression of clinical trials (phases I-III) demands solid analytical methods, lot-to-lot consistency, and scalable manufacturing. Potency assays for complex secretomes remain challenging, and donor variability requires stringent screening. FDA enforcement underscores that no exosome therapies have yet been approved, highlighting the need for robust safety and efficacy data[90].

THE GROWING PIPELINE OF EXOSOME CLINICAL TRIALS

The clinical application of exosomes has expanded substantially, with a comprehensive systematic review identifying a growing pipeline of exosome-based clinical trials across multiple therapeutic areas from 2000 to 2023[84]. This expanding clinical experience encompasses diverse administration routes (intravenous, intrathecal, local injection, inhalation), various disease contexts (cardiovascular, neurological, inflammatory, oncological), and different exosome sources (BM-MSCs, umbilical cord MSCs, adipose-derived MSCs, cardiac progenitor cells)[84]. The accumulating safety data across these trials provides a foundation for confidence in the general tolerability of exosome-based therapeutics, though each specific application requires independent safety validation.

CHALLENGES IN TRANSLATION FROM PRE-CLINICAL TO CLINICAL STUDIES

Despite these encouraging early clinical findings, several fundamental challenges must be addressed to advance exosome-based therapies from preliminary trials to established clinical practice. Dosing optimization remains a critical knowledge gap, with current trials employing widely variable dosing strategies ranging from microgram quantities of total exosomal protein to billions of exosome particles, without standardized metrics for dose calculation or established dose-response relationships[84,87]. The optimal dosing regimen for cardiovascular applications specifically requires systematic investigation through well-designed dose-escalation studies[83].

Delivery route selection significantly impacts therapeutic outcomes, with local delivery (intramyocardial, intrathecal) potentially achieving higher regional concentrations but at the cost of greater procedural complexity. In contrast, systemic delivery (intravenous) provides broader distribution but may result in off-target effects and reduced cardiac bioavailability[83,86]. The SECRET-HF trial’s selection of intravenous delivery represents a practical approach for clinical scalability. Still, comparative studies evaluating different delivery routes in cardiovascular disease are needed to optimize therapeutic delivery strategies.

Efficacy endpoints require careful consideration in cardiovascular applications, where surrogate markers (LVEF, biomarkers, functional class) must be validated against hard clinical outcomes (mortality, hospitalization, major adverse cardiovascular events). The 6-month follow-up in the SECRET-HF case report, while providing valuable preliminary data, is insufficient to assess the long-term durability of the effects or their impact on disease progression[83]. Future trials must incorporate more extended follow-up periods and clinically relevant endpoints to establish meaningful therapeutic benefit[83,84].

Manufacturing and QC pose additional challenges, as exosome production must be scaled from research-grade preparations to GMP-compliant manufacturing processes capable of producing consistent, well-characterized products for multi-center clinical trials[83]. Standardization of isolation methods, potency assays, and release criteria remains an ongoing challenge in the field that must be addressed to ensure reproducibility and regulatory acceptance[83,84].

These challenges underscore the need for systematic, rigorously controlled clinical trials to establish the therapeutic role of MSC-derived exosomes in HF. While the preliminary evidence is encouraging, the field remains in early-stage clinical development, and a realistic assessment of the path forward requires acknowledgment of both the promise and the substantial work needed to achieve regulatory approval and clinical implementation[84].

CURRENT HURDLES TO MSC-DERIVED SECRETOME THERAPY

Despite the secretome-based therapy having progressed to the clinical phase of assessment, the use of MSC-derived secretome has limitations that need to be addressed to exploit its therapeutic potential fully[93]. Firstly, individual variations among MSC donors, including genetic background, age, sex, and health status, remain a significant challenge for standardizing secretome therapy[93]. For example, MSCs derived from aged donors typically exhibit lower expression of cardioprotective factors (VEGF, IGF-1) and increased expression of pro-inflammatory cytokines (IL-6, TNF-α)[18,93]. Similarly, the paracrine profile of the secretome from patients with chronic pathologies, such as diabetes, differs from that of young, healthy donor cells. This broad diversity necessitates detailed donor screening and characterization[93]. Similarly, the anatomical origin of MSCs represents another essential variable influencing the efficacy of their secretome. For example, BM-MSCs differ from umbilical cord MSCs or adipose-derived MSCs, and this diversity extends to their proteomic, lipidomic, and EV profiles. BM-MSCs usually secrete more hematopoietic support factors, such as stem cell factor and granulocyte-macrophage colony-stimulating factor, than adipose-derived MSCs, which exert a more powerful angiogenic effect due to higher VEGF and FGF-2 levels. On the other hand, umbilical cord MSCs typically exhibit a stronger immunoreaction to PGE2 and indoleamine 2,3-dioxygenase secretions. Such tissue-specific disparities remain a hurdle to optimizing secretome composition[93].

Another limitation in the use of secretome is the lack of an optimized protocol for uniform isolation and yield optimization. The current protocols have their advantages and practical limitations, which can introduce bias into the final product[94,95]. The absence of consensus on optimal isolation schemes leads to extensive inter-laboratory variation in EV purity, recovery, and the estimated bioactivity of the purified product[71]. Lastly, secretome-based therapy is not without its potential ethical issues and regulatory hurdles. The use of allogeneic MSC lines raises questions about donor consent for secondary therapeutic applications[96]. Also, the optimized secretome formulations are subject to intellectual property claims, and the fetal-derived cell secretome faces challenges in public perception[97]. It is essential to carefully assess the risk-benefit analysis of potential off-target effects or unintended systemic actions by secretome components, especially in high-risk groups such as older adults with HF[96,97].

COMPARATIVE OVERVIEW OF MSCS, AND THEIR DERIVATIVE SECRETOME AND EXOSOMES

MSC-derived secretomes contain a wide variety of soluble molecules in their biochemical mixture (cytokines, growth factors, lipids, nucleic acids, and EVs, etc.); such biomolecules aid in mediating cell signaling and tissue repair. However, exosomes are a more purified and refined form of vesicles (sized 30-200 nm) that contain a cargo of regulatory proteins, miRNAs, and mRNAs, which influence multiple cell functions (angiogenesis, apoptosis, and immunomodulation).

From a manufacturing perspective, secretome isolation involves collecting CM from cultured cells and removing living cells and debris by filtration or centrifugation. In contrast, exosome purification requires the same steps but also includes additional steps such as ultracentrifugation, ultrafiltration, or size-exclusion chromatography, which increase production complexity and cost but can yield better quality and uniformity. Secretome products, being acellular, allow simpler large-scale production using bioreactor-based culture systems while maintaining consistency across batches[98,99]. Storage and handling are markedly different between these modalities. The living MSCs require cryopreservation and viability assurance before administration[100]. In contrast, both secretome and exosome cell-free therapies can be stored frozen or lyophilized with minimal loss of potency[101,102].

The dosing strategies also vary between the three modalities. Living MSC infusions are typically quantified by cell count (10⁶-10⁷ cells/kg), while secretome and exosomes are standardized by total protein content or nanoparticle concentration. The regulatory and translational rules and considerations further distinguish these modalities from each other. For example, MSC-based therapies are classified as ATMPs under the EMA and FDA guidelines, which require rigorous donor screening, cell tracking, and production batch testing[100]. In contrast, exosome and secretome cell-free therapies fall under the category of biological or biologic-derived products and are regulated under newer regulatory pathways. The FDA and EMA currently mandate full preclinical toxicology and efficacy evaluations for these acellular modalities, given their heterogeneous composition[103]. However, MSC-secretome preparations are recognized as neither traditional biologics nor ATMPs, placing them in a unique intermediate status between biologics and cell-based therapies[104].

The tissue origin of MSCs can result in variation in the secretome and exosomes’ therapeutic and functional profiles, such as umbilical cord-derived MSCs, BM-MSCs, and adipose-derived MSCs. For example, umbilical cord MSCs produce higher levels of immunomodulatory molecules, conferring superior anti-inflammatory and antifibrotic properties. Moreover, BM-MSCs typically secrete higher levels of hematopoietic-supportive cytokines, making them more favorable for myocardial repair in ischemic conditions. In addition, adipose-derived MSCs exhibit a stronger angiogenic and anti-apoptotic profile and are more effective in promoting vascular regeneration and protecting cardiomyocytes from hypoxic injury.

Finally, from a clinical perspective, the optimal modality depends on the therapeutic goal and the disease context. Living biodrug MSC-based therapy remains preferred when long-term paracrine support or direct differentiation potential is needed, such as in severe myocardial injury. Secretome preparations are advantageous for cost-effective, scalable applications requiring broad trophic and angiogenic effects, including acute ischemia. In contrast, exosome-based products are favored for chronic or immune-mediated cardiomyopathies due to their reproducibility, stability, and capacity for targeted delivery and bioengineering modifications. In summary, the comparison favors exosome-based therapies in regenerative cardiology because of their scalability, reproducibility, and lower safety risks, while also addressing that the MSC-derived secretome offers a more cost-efficient and biologically diverse therapeutic mixture (Table 4).

Table 4 Head-to-head comparison of mesenchymal stem cell-based therapy, mesenchymal stem cell-derived exosome-based therapy, and mesenchymal stem cell-derived secretome-based therapy approaches.

Parameter	MSC therapy	MSC-derived secretome	MSC-derived exosomes
Composition	Live cells can secrete various bioactive factors	A cell-free mixture containing soluble proteins, cytokines, chemokines, nucleic acids, and small extracellular vesicles released by MSCs	Nanosized vesicles (30-150 nm) enriched with proteins, lipids, and regulatory RNAs (miRNAs) that reflect the molecular profile of their parent MSCs
Isolation and production	Obtained from donor tissue (bone marrow, adipose tissue, or umbilical cord) and expanded under GMP conditions	Produced by culturing MSCs and collecting the conditioned medium, in addition to centrifugation and filtration to remove cells	Generated through further purification of the secretome using ultracentrifugation, filtration, or chromatography
Storage and stability	Requires cryopreservation (-196 °C).	Can be stored frozen (-20 °C to -80 °C) or lyophilized	Similar storage profile to the secretome; stable under freezing or lyophilized conditions
Dosing units	Defined by viable cell number [e.g., (10-100) × 106 cells per dose]	Quantified by total protein concentration or biological activity (e.g., mg of secreted protein)	Expressed by vesicle count (e.g., 1010 particles) or total exosomal protein content (μg)
Mechanism of action	Primarily functions through paracrine signaling, releasing bioactive factors over time and supporting differentiation into cardiac or vascular cells	Acts exclusively through soluble paracrine mediators that promote tissue repair via anti-inflammatory, angiogenic, and regenerative signaling pathways	Delivers specific molecular cargo (miRNAs, proteins, lipids) into target cells, modulating gene expression and promoting tissue regeneration; can be bioengineered for targeted therapeutic effects
Manufacturing complexity	High: Requires GMP-level cell culture, cell banking, karyotype and sterility testing, and cryostorage logistics	Moderate: Requires MSC culture, conditioned medium collection, and downstream processing (e.g., filtration and concentration)	High: Includes all steps of secretome production, specialized purification, and molecular profiling
Scalability	Limited by donor cell proliferation and donor variability. Large-scale expansion increases cost and heterogeneity	Highly scalable; one MSC source can produce large volumes of secretome using culture systems with minimal donor dependence	Partially scalable - while MSC culture is scalable, large-scale exosome isolation and purification remain technically demanding
Cost implications	High production cost due to cell culture, quality control, and cryogenic storage	Lower cost; major expenses arise from culture media and concentration processes, but no live-cell storage is required	Moderate to high cost due to intensive purification techniques

MSC: Mesenchymal stem cell; GMP: Good Manufacturing Practice; miRNAs: MicroRNAs.

MANUFACTURING, PROCESS VALIDATION, AND GMP TRANSLATION OF MSC-DERIVED SECRETOME PRODUCTS

Large-scale translation of MSC-derived secretome and EV therapeutics demands rigorously standardized GMP frameworks to ensure reproducibility, safety, and regulatory acceptance. The successful transition of MSC and exosome research into ATMPs depends on defining end-to-end control over raw-material qualification, cell expansion, EV isolation, characterization, and product release[105]. A comprehensive GMP strategy must integrate both process consistency and analytical comparability, supported by validated documentation and traceability throughout the manufacturing chain[83,105].

Upstream, achieving reproducible secretome production begins with qualified donor MSCs, expanded under xeno-free, chemically defined conditions to minimize batch variation[81,105]. Controlling donor selection, passage number, and preconditioning parameters markedly affects secretome potency and composition[81]. The implementation of closed, automated bioreactor systems, such as hollow-fiber or stirred-tank configurations, facilitates dynamic control of oxygen, pH, and nutrient supply, supporting scale-up from 10⁶ cells to 10¹⁰ cells while maintaining product integrity[105,106].

At the downstream stage, GMP-compliant workflows for cardiovascular progenitor- or MSC-derived secretomes incorporate tangential-flow filtration for concentration, followed by size-exclusion chromatography for purification, both of which are compatible with clinical-grade production[83]. Critical quality attributes - including particle size, identity, potency, and sterility - are systematically assessed before batch release, establishing reproducible standards for early-phase trials[81,105]. Consistent quality manufacturing is achieved by integrating automated monitoring, digital documentation, and statistical process control to reduce operator variability[106].

Process validation is the cornerstone of GMP translation[83,105]. Validated analytical assays - nanoparticle-tracking analysis for concentration, protein quantification (BCA/Bradford), flow-cytometric profiling for surface markers, and sterility/endotoxin testing - form the basis of reproducible manufacturing[105,106]. These tests must be coupled with well-defined standard operating procedures and real-time documentation of key process parameters to meet regulatory expectations under both the EMA-ATMP and FDA-Regenerative Medicine Advanced Therapy frameworks[83,105].

Long-term stability and distribution are equally critical for scalability. Optimized lyophilized MSC-secretome formulations have been shown to preserve biological activity for up to 12 months at ambient temperature[107]. Stabilizing excipients such as trehalose and mannitol prevent vesicle aggregation and protein denaturation during freeze-drying, enabling the production of shelf-stable, transportable clinical products. Such lyophilized formulations overcome logistical barriers associated with cryogenic storage, thereby substantially reducing costs and complexity[105,107]. Studies demonstrate that the convergence of closed-system automation, process validation, and formulation stabilization is transforming MSC-secretome production into a fully scalable biomanufacturing platform. Establishing harmonized GMP standards and digital quality management systems will further streamline clinical translation and facilitate multinational regulatory approval. The frameworks proposed in these recent reports now provide a blueprint for producing consistent, safe, and efficacious secretome-derived advanced therapy products suitable for large-scale clinical deployment.

FUTURE PERSPECTIVES

A significant feature of secretome- and exosome-based treatment approaches is that, unlike a one-size-fits-all approach, personalized secretome therapy will be more flexible, using MSC-derived factors tailored to the specific molecular signatures of distinct cardiac pathologies, such as ischemic cardiomyopathy, dilated cardiomyopathy, or hypertrophic cardiomyopathy. Proteomic analyses have identified unique secretome profiles enriched in reparative cytokines, growth factors, and miRNAs that correspond to specific pathological conditions[108]. For example, ischemic HF may benefit more from VEGF- or HGF-enriched secretome that promotes angiogenesis, whereas non-ischemic fibrotic conditions may require TGF-β-modulating factors. Advanced omics platforms, including transcriptomics and single-cell proteomics, can aid in patient stratification and the development of MSC-derived paracrine products tailored to their specific needs, offering a better safety profile in a disease-specific manner[109].

Secondly, their therapeutic efficacy is expected to significantly support cardiac repair using a combinatorial approach with biomaterials, gene editing, or pharmaceuticals. For example, combining MSCs with anti-fibrotic agents, such as pirfenidone or losartan, can reduce myocardial fibrosis and improve cardiac function after myocardial injury[110]. MSCs may also be reprogrammed for enhanced healing using gene-editing tools, such as CRISPR, to upregulate pro-regenerative genes or suppress inhibitory pathways. The problem of poor cell retention and limited activity in the diseased heart is further addressed by biomaterials, such as injectable hydrogels or engineered cardiac patches[111]. Commercially available scaffolds such as CorMatrix ECM patches (derived from porcine small intestinal submucosa) and PeriCord, which combines Wharton’s jelly-derived MSCs with decellularized pericardium, have been used in regenerative cardiac strategies[112]. These platforms not only provide mechanical support but also enhance the localized delivery and activity of paracrine signals, making them ideal partners for MSC-based therapies.

EMERGING APPROACHES IN EXOSOME ENGINEERING AND DELIVERY

The development and use of artificial secretome, engineered nanoparticles, or vesicles represent a leap forward in cell-free therapy approaches, offering better control and scalability. Engineered exosomes with cardiac-targeting ligands have emerged as a promising strategy to enhance myocardial specificity and therapeutic efficacy. Surface modification techniques, including biological, chemical, and physical methods, enable exosomes to be decorated with cardiac-homing peptides that specifically recognize ischemic myocardium[113]. For instance, exosomes engineered to overexpress cardiac-targeting peptide-Lamp2B fusion proteins have demonstrated enhanced distribution to the heart both in vitro and in vivo settings, resulting in reduced cardiomyocyte apoptosis, decreased inflammation, enhanced angiogenesis, and reduced infarct size in mouse models of MI[113]. Chemical modification approaches, such as conjugation with cardiac homing peptides via dioleoylphosphatidylethanolamine N-hydroxysuccinimide linkers, have shown improved homing efficacy and enhanced therapeutic outcomes in injured hearts. Physical modification through membrane fusion techniques, such as combining MSC-derived exosomes with monocyte or platelet membranes, has enabled exosomes to acquire inflammatory targeting characteristics, improve uptake by cardiomyocytes and endothelial cells, and reduce clearance by the mononuclear phagocyte system. These exosome mimetics can be engineered to carry the desired cargo profile and surface characteristics, ensuring the targeted delivery of necessary bioactive molecules[114,115]. These novel platforms may address many issues related to the use of natural MSC secretome, such as batch-to-batch variation and instability during storage, while enabling a tailored design for clinical needs.

Inhalation delivery systems represent a groundbreaking non-invasive approach for cardiac repair that addresses many limitations of conventional delivery methods. The recently developed stem cell-derived exosome nebulization therapy has demonstrated remarkable promise by delivering lung spheroid cell-derived exosomes via inhalation[116]. This innovative approach overcomes the barriers associated with intravascular and intraperitoneal administration - such as rapid clearance, off-target accumulation, and low cardiac retention - while avoiding the invasiveness of intramyocardial, pericardial, or epicardial injections. In preclinical studies, inhaled exosomes successfully crossed the air-blood barrier and accumulated in ischemic cardiac tissue within 60 minutes, with repeated dosing significantly increasing exosome retention in the heart after 48 hours[116]. In acute MI models, mice treated daily with leukemia stem cell-derived exosomes for 7 days showed significantly improved left ventricular ejection fraction, reduced infarct size, and modulation of inflammation and tissue remodeling compared with control groups. The therapy induced cardiomyocyte proliferation in the infarct zone, reduced cardiomyocyte apoptosis, and resulted in a thicker left ventricular wall with enhanced contractile capacity. Notably, stem cell-derived exosome nebulization therapy demonstrated efficacy in large animal models, with pig studies showing substantial improvements in heart function and an excellent safety profile, as all animals survived to the 28-day endpoint with no significant abnormalities in hematology, liver, or kidney function[116]. Mechanistically, RNA sequencing revealed that these exosomes are enriched in miRNAs, particularly miR-100, which promotes neovascularization and protects cardiac tissues under stress by suppressing fatty acid uptake through CD36 downregulation in endothelial cells. This approach offers several key advantages: Non-invasiveness, cell-free therapy, potential for repetitive dosing with lower overall doses, and adaptability for exosomes from various cell types or engineered exosomes with specific therapeutic cargo[116].

Artificial intelligence and machine learning applications are emerging as powerful tools for secretome optimization. Artificial intelligence-driven approaches can analyze complex multi-omic datasets to identify optimal MSC culture conditions, predict secretome composition, and determine the most effective cargo combinations for specific cardiac pathologies. Machine learning algorithms can also facilitate QC by predicting batch-to-batch consistency and identifying critical quality attributes that correlate with therapeutic efficacy. These computational approaches may accelerate the development of next-generation secretome-based therapeutics by enabling the rational design of exosome cargo, optimizing production protocols, and developing personalized treatment strategies based on patient-specific disease signatures.

The development of next-generation nanodelivery systems to achieve sustained release of secretome in the myocardium continues to advance. Researchers are exploiting the acidic environment of the ischemic myocardium to develop pH-sensitive nanoparticles for the controlled release of their payload[117]. Magnetically guided systems are being developed for targeting secretome-loaded particles to the infarct site. Responsive hydrogels, such as RGD-modified or thermosensitive gels, can also trap MSC-derived exosomes and cytokines, slowly releasing them over time, thereby enabling prolonged efficacy[118]. Novel formulations, such as secretome sprays and injectable secretome-loaded biomaterials, are being explored for less-invasive delivery to the heart[119]. Hybrid systems combining exosomes with biomaterials, such as hydrogel formulations containing MSC-derived exosomes, have shown enhanced cardiac retention and prolonged therapeutic effects following MI[113]. These biomaterial-based exosome formulations address the challenge of rapid clearance, improve localized delivery, promote endogenous angiogenesis, and reduce cardiac fibrosis[113].

STUDY LIMITATIONS

This narrative review, while comprehensive, has several inherent limitations that should be acknowledged. As it is not a systematic review, it may be subject to selection bias in the inclusion of studies and in the interpretation of findings. The heterogeneity in study designs, MSC sources, secretome preparation methods, and outcome measures across reviewed studies limits direct comparisons and meta-analysis. Additionally, the rapidly evolving nature of this field means that some recent developments may not be fully captured, and emerging data from ongoing clinical trials could modify current conclusions.

CONCLUSION

These latest progresses in targeting strategies, non-invasive delivery systems, artificial intelligence-guided optimization, and advanced biomaterial platforms make MSC secretome therapy for HF more accurate, safe, scalable, and long-lasting. The convergence of these innovative approaches - from engineered exosomes with cardiac-specific ligands to breakthrough inhalation delivery methods and artificial intelligence-powered personalization positions secretome-based therapeutics at the forefront of next-generation regenerative medicine for cardiovascular disease. As these technologies mature and clinical translation advances, they hold tremendous promise for transforming the treatment landscape for patients with HF.