Exploring the therapeutic potential of MSC-derived secretomes in neonatal care: focus on BPD and NEC

Abstract

Background

Every year, millions of infants are born prematurly, many of whom develop serious complications like bronchopulmonary dysplasia and necrotizing enterocolitis. Despite improvements in neonatal care, there are few therapies that actively promote healing or prevent long-term damage. In recent years, secretions from mesenchymal stem cells, rich in reparative proteins and tiny extracellular particles have shown promise as a safe and effective way to support tissue repair without the risks of live-cell therapy.

Methods

This review brings together findings from animal studies and early-stage clinical trials to explore how these stem cell-derived secretions work and how they might be used in the clinic. We examine how different sources of mesenchymal stem cells, such as bone marrow or umbilical cord affect the quality and function of their secretions. We also look at key biological pathways they influence, including inflammation control, blood vessel growth, and tissue regeneration. In parallel, we assess the designs and outcomes of current clinical trials involving preterm infants.

Results

In animal models, these secretions have repeatedly shown the ability to reduce lung and gut injury, calm inflammation, and boost repair mechanisms. Products from umbilical cord tissue appear especially potent, delivering high levels of protective molecules while being low in immunogenic risk. Several small clinical studies report that the approach is safe and well-tolerated in preterm infants, with some signs of benefit. However, clinical use is still limited by variability in production methods and the lack of standardized dosing.

Conclusions

Mesenchymal stem cell secretions could offer a powerful new way to treat fragile preterm infants, providing regenerative support without the risks of cell transplantation. But before they become part of routine care, we need clearer guidance on how to manufacture, measure, and safely deliver these therapies in newborns.

Introduction

Preterm birth, defined by the World Health Organization as birth before 37 weeks of gestation, remains a major global public health issue, affecting approximately 1 in 10 live births worldwide in 2019 [1–4]. Despite significant advances in neonatal intensive care, premature infants continue to face high risks of severe complications, including bronchopulmonary dysplasia (BPD), necrotizing enterocolitis (NEC), retinopathy of prematurity (ROP), and periventricular leukomalacia (PVL). These conditions not only compromise survival during the immediate postnatal period but also impose enduring impacts on neurodevelopment, pulmonary function, and overall quality of life.

Current interventions including respiratory support, corticosteroids, and surfactants, are largely symptomatic, with limited capacity to reverse or prevent the underlying cellular and molecular disruptions associated with prematurity [5–7]. This persistent therapeutic gap has spurred growing interest in regenerative strategies that can target the inflammatory, ischemic, and degenerative mechanisms driving neonatal diseases. Among the most promising candidates are secretomes derived from mesenchymal stem cells (MSCs), which are mixtures of cytokines, growth factors, and extracellular vesicles (EVs) that mediate paracrine effects while bypassing the risks associated with live-cell transplantation [8, 9]. These cell-free therapeutics have demonstrated robust ability to modulate inflammation, promote tissue repair, and enhance organ development in preclinical models of neonatal disease [10, 11]. This shift from cell-based therapies to cell-free regenerative strategies represents a critical inflection point in neonatal medicine. Unlike live-cell approaches, which are constrained by risks of engraftment, immunogenicity, and logistical barriers, MSC-derived secretomes offer a promising regenerative platform capable of engaging multiple therapeutic pathways via paracrine signaling alone. As neonatal medicine advances toward greater precision, MSC-derived secretomes promise a modular, scalable, and safer modality that aligns with the delicate and evolving physiology of preterm infants. Beyond neonatal applications, MSC-derived secretomes are gaining recognition across regenerative medicine as scalable, acellular therapeutics with potential applications in pulmonary, gastrointestinal, cardiovascular, and autoimmune diseases. Their safety profile, manufacturing flexibility, and ability to engage conserved repair pathways position them as a promising therapeutic class not only for premature infants but also for broader age groups and clinical conditions.

Unlike stem cell transplantation, cell-free therapies such as MSC-derived secretomes eliminate the risks of immune rejection, tumorigenicity, and logistical complexities associated with live-cell handling, offering a safer and more scalable alternative for neonatal regenerative interventions. This review critically explores the therapeutic potential of MSC-derived secretomes in the context of prematurity-related disorders, with a particular focus on BPD and NEC.

Characterization of MSCs and their secretomes

Overview of MSC sources and properties

MSCs are multipotent stromal cells capable of self-renewal and differentiation into various mesodermal lineages such as osteocytes, chondrocytes, and adipocytes and myocytes [12–15]. While embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) exhibit full pluripotency, MSCs possess more restricted multipotency, predominantly differentiating into mesodermal tissues [16]. MSCs were first isolated from bone marrow in 1999 and have since been derived from multiple tissues, including adipose tissue, amniotic fluid, umbilical cord blood, and Wharton’s jelly [17–19]. MSC can undergo multilineage differentiation both in vitro and in vivo [20]. Phenotypically, MSCs adhere to plastic under standard in vitro conditions and display a fibroblast-like morphology. They express surface markers including CD105, CD90, and CD73, but lack hematopoietic markers, such as CD34, CD45, CD14, CD19, and HLA-DR [21, 22]. The origin of MSCs significantly influences their secretory profile and therapeutic potential. Umbilical cord-derived MSCs (UC-MSCs), especially from Wharton’s jelly, are favored for neonatal applications due to their non-invasive harvest, immune-privileged phenotype, and high proliferative capacity. In contrast, bone marrow-derived MSCs (BM-MSCs) show donor-age-related functional decline and require invasive extraction [23, 24]. Table 1 compares MSC characteristics from different tissue sources.

Table 1.

Comparative characteristics of MSC sources and their secretome potency in neonatal and regenerative therapies

MSC Source	Harvest method	Secretome potency	Advantages	Disadvantages	Common Clinical Use	Ref
UC-MSCs (WJ)	Non-invasive extraction of Wharton’s Jelly post-delivery (umbilical cord matrix); GMP-grade expansion	High – Strong anti-inflammatory, angiogenic, and neuroprotective factor secretion; high IL-10 and VEGF levels	Highest MSC yield, rapid proliferation, immunoprivileged, strong regenerative paracrine signaling	Requires tissue dissection and GMP processing; potential donor variability	Used in BPD, SCI, MI, OA, liver failure; Phase 1/2 trials ongoing	[25] [26]
BM-MSCs	Invasive bone marrow aspiration	Good anti-inflammatory and regenerative secretome, but affected by donor age	Well-studied clinically Can be autologous Low immunogenicity	Invasive harvest Cell yield and quality decline with age Slower proliferation	Investigated in severe BPD, IVH, NEC Some compassionate use cases reported	[27]
UC-MSCs	Dissection of arteries, extraction of gelatinous Wharton’s Jelly tissue post-delivery	High – Strong angiogenic, anti-inflammatory, neuroprotective, and osteo/chondrogenic marker expression	Highest MSC yield in primary culture (4.9–6.6 million) High proliferation and viability Least contamination Expresses pluripotency & MSC markers Higher osteo/chondrogenic differentiation	Requires proper tissue processing Possible inter-donor variability Expanded MSCs may show heterogeneity	Widely used in trials for BPD, SCI, MI, liver failure, OA, and wound healing. Also effective in EV-based therapies	[26]
BM-MSCs	Invasive bone marrow aspiration (Allogeneic source used; expanded to passage 8 in vitro by certified provider)	High – Promotes IL-10 (anti-inflammatory), suppresses TNF-α (pro-inflammatory); reduces apoptosis, TLR4 expression, and gut barrier damage	Effective via oral or intraperitoneal (IP) route Low dose (1 × 10⁵ cells) as effective as high dose (1 × 10⁶) Anti-inflammatory & regenerative effects	Invasive to obtain from human donors Cell expansion and standardization are needed Clinical data in humans is still limited.	Investigational use in NEC Effective in reducing tissue damage and inflammation in preclinical neonatal mouse models.	[28]
UCB-MSCs	Non-invasive: collected from cord blood after birth, expanded under GMP	High – Paracrine profile rich in IL-10, VEGF, IL-6; promotes pulmonary and neuroprotection	Safe profile, no engraftment risk, easy storage, low immunogenicity	Single-dose use; scalability limited by donor volume; not yet standard in clinical practice	Trials for BPD prevention, especially in extremely preterm neonates	[29, 30]
MSC-EVs	Isolated from MSC culture supernatant; includes exosomes (40–160 nm) and microvesicles (50–1000 nm); purified via ultracentrifugation or filtration	Very High – Contains miRNAs, mRNA, VEGF, TSG-6, mitochondrial fragments; regulates angiogenesis, immune modulation, anti-apoptosis, and oxidative stress	Cell-free (no engraftment risks) Low immunogenicity Can cross tissue barriers Stable and can be cryopreserved Can be loaded with drugs or miRNAs	Still preclinical in humans Standardization of dosing, cargo, and isolation is lacking Heterogeneity of vesicle content and size	Animal studies only (BPD rat models) Multiple delivery routes (IT, IP, IV) Shows promise in improving alveolarization, angiogenesis, and reducing inflammation and PH	[31]
BM-MSCs	Invasive bone marrow aspiration from healthy adult donors	Higher expression of VEGF and CXCL12 (angiogenic genes)	Promotes angiogenesis and vascular remodeling Effective in BPD lung repair. Widely studied historically.	Lower proliferation Greater donor variability. Invasive harvest procedure.	Preclinical trials for BPD, IVH, NEC Often used intratracheally in rodent models.	[32]
UCT-MSCs	Non-invasive extraction from postnatal umbilical cords, digested and cultured	Higher expression of IL-10, TSG-6, HGF, Ang-1 – greater anti-inflammatory and epithelial healing capacity	Stronger anti-inflammatory action Greater suppression of lung macrophage infiltration Better epithelial wound healing	Requires collagenase digestion Newer in clinical use vs. BM	Growing use in BPD research; superior paracrine healing makes it an ideal candidate for clinical translation	[32]

MSC, mesenchymal stem cell; UC-MSC, umbilical cord-derived MSC; WJ, Wharton’s jelly; BM-MSC, bone marrow-derived MSC; UCB-MSC, umbilical cord blood MSC; UCT-MSC, umbilical cord tissue MSC; EV, extracellular vesicle; GMP, good manufacturing practice; SCI, spinal cord injury; MI, myocardial infarction; OA, osteoarthritis; BPD, bronchopulmonary dysplasia; NEC, necrotizing enterocolitis; IVH, intraventricular hemorrhage; IP, intraperitoneal; IT, intratracheal; IV, intravenous; TSG-6, TNF-stimulated gene 6; HGF, hepatocyte growth factor; Ang-1, angiopoietin-1; CXCL12, C-X-C motif chemokine ligand 12; VEGF, vascular endothelial growth factor; IL-10, interleukin-10; TNF-α, tumor necrosis factor-alpha; TLR4, toll-like receptor 4

Although most attention initially focused on MSCs for cell replacement therapies, growing evidence points to their therapeutic impact being mediated predominantly through paracrine mechanisms, rather than engraftment. Nonetheless, limited engraftment has been observed in specific settings such as ischemic myocardium, suggesting that tissue microenvironments can modulate MSC behavior [33].

As illustrated in Fig. 1, MSCs from diverse tissue sources including bone marrow, adipose tissue, amniotic fluid, and umbilical cord secrete a complex array of bioactive components, such as cytokines, growth factors, and microRNAs, via both CM and EVs, which collectively contribute to key therapeutic functions including anti-inflammation, antioxidation, angiogenesis, fibrosis attenuation, and barrier protection.

Fig. 1.

Sources, Components, and Biological Functions of the MSC Secretome

This foundational understanding of MSC origin and phenotype sets the stage for dissecting the molecular composition and clinical relevance of their secretomes, as detailed in the sections that follow.

Paradigm shift: from MSCs to cell-free therapeutics

Although MSCs were originally explored for their capacity to differentiate into tissue-specific cells. Recent insights reinforce that MSC-derived regenerative effects are predominantly mediated through secreted factors rather than direct tissue integration or lineage-specific differentiation [34–36]. This paradigm shift is supported by findings that MSC engraftment in injured tissues is minimal in most contexts [37]. Compared to whole MSCs, secretomes especially their EV component offer several advantages: reduced immunogenicity, simplified GMP manufacturing, and long-term storage capability without cryoprotectants [38, 39]. Beyond their differentiation capacity, MSCs secrete a complex array of bioactive molecules including cytokines, growth factors, and EVs that modulate inflammation, enhance angiogenesis, and support tissue repair. These functions are especially pertinent in neonatal diseases [40–42]. This secretome-centric view has become especially relevant in neonatal medicine, where acellular therapies offer a safer and more controlled alternative to whole-cell transplantation. Conditions such as BPD and NEC, which involve inflammation and impaired tissue development, are particularly responsive to the bioactive factors secreted by MSCs. Notably, MSC secretomes can be lyophilized and batch-standardized, overcoming variability constraints inherent to cell-based products. Technological advances have accelerated production scalability and precision. For example, Ulpiano et al. [43] demonstrated industrial-scale EV biomanufacturing using tangential flow filtration (TFF) and GMP-compatible automation. Simultaneously, CRISPR/Cas9-based MSC engineering has also enabled programmable EV payloads enriched in miR-21 and miR-146a, offering therapeutic targeting potential not achievable with native MSCs. Furthermore, efficacy and safety data from adult trials in myocardial infarction, osteoarthritis, parkinsons and stroke or other autoimmune/inflammatory diseases [21–45] can inform neonatal translation by validating dosing, immune tolerance, and storage platforms in non-neonatal settings.

However, the field is increasingly converging on the secretome as the principal effector, which raises new questions about standardization, potency, and quality control of these acellular therapeutics areas still underdeveloped in current literature [46]. Addressing these gaps is critical to realizing the full therapeutic potential of MSC-derived acellular products.Building on this conceptual shift, the following sections delve into the specific molecular constituents and clinical strategies underpinning the therapeutic use of MSC secretomes in neonatal disorders.

Composition of the MSC secretome

The MSC secretome encompasses a diverse array of molecules, including soluble proteins, cytokines, growth factors, and EVs, each of which contributs to immune modulation, angiogenesis, and tissue repair. Key functional categories include: (1) proangiogenic factors such as vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), and hepatocyte growth factor (HGF); (2) antiapoptotic molecules such as basic fibroblast growth factor (bFGF), transforming growth factor (TGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF); (3) anti-inflammatory mediators, including TNF-α-stimulated gene/protein 6 (TSG-6) interleukin-10 (IL-10), and heme oxygenase-1 (HO-1) [47, 48]. These anti-inflammatory factors play crucial roles in modulating the dysregulated immune responses seen in BPD and NEC. IL-10, TSG-6, and HO-1 each act on distinct inflammatory signaling cascades to suppress cytokine overproduction, limit oxidative stress, and restore epithelial homeostasis. A detailed mechanistic discussion of how these mediators influence macrophage polarization, NF-κB suppression, and microRNA regulation is provided in Sect. 4.8.

Many of these soluble factors are also encapsulated within EVs, which serve as carriers that extend MSCs’ biological influence through systemic circulation. These EVs act either by direct receptor-ligand interactions on recipient cells or by internalization, delivering regulatory proteins, lipids, and nucleic acids that modulate gene expression and cellular function [49–52,]. Numerous studies have systematically profiled the secretome components present in CM and EVs derived from in vitro MSC cultures [53], aiming to identify the principal factors underlying their regenerative potential. In this context, particular emphasis is placed on CM and EVs as the principal conduits through which MSCs exert their therapeutic effects, particularly in diseases such as BPD and NEC. The following sections explore each fraction in detail, examining their composition, mechanisms of action, and relevance to clinical translation.

MSC-derived CM

MSC-CM encompasses a complex mixture of soluble factors, including cytokines, growth factors, and EVs, which collectively exert potent regenerative effects. The production of MSC-CM begins with the in vitro expansion of MSCs under static or dynamic culture conditions to achieve adequate cell density [54]. Following phenotypic characterization, cells are transferred to serum-free basal media often termed a “starvation phase” to initiate the secretion of regenerative factors [55, 56]. Depending on the intended therapeutic profile, MSCs can be preconditioned with environmental modifications such as hypoxia, inflammatory cytokines (e.g., TNF-α), or growth factors, which have been shown to substantially alter the CM’s molecular composition and efficacy [57, 58]. For `ng increases VEGF and TGF-β1, while hyperoxic stress elevates STC-1 levels [59]. Similarly, TNF-α exposure enhances proangiogenic and immunomodulatory outputs, accelerating wound healing and immune cell infiltration in vivo [60, 61]. After a conditioning period typically 24–48 h the media is collected, filtered to remove cellular debris, and often concentrated via centrifugation or ultrafiltration [62]. Quality control is then performed using ELISA or proteomic profiling to confirm the presence of key molecules such as VEGF, HGF, and STC-1 [63, 64]. Functionally, MSC-CM derived from hUCMSCs is enriched in proteins linked to autophagy enhancement, mitochondrial stabilization, and anti-inflammatory signaling mechanisms highly relevant to neonatal lung injury [65]. In preclinical models BPD, MSC-CM has been shown to mitigate inflammation, inhibit fibrotic remodeling, and promote alveolar and vascular development [53, 66–71]. These effects are mediated in part through enhanced oxygen consumption and ATP production, which collectively reduce oxidative stress a key driver of BPD pathogenesis. By offering a cell-free, scalable therapeutic platform, MSC-CM circumvents the logistical and immunological challenges of live-cell transplantation. The tunability of its composition via preconditioning strategies further enhances its potential as a personalized, mechanism-targeted treatment modality in neonatal care.

MSC-derived EVs

EVs are membrane-bound nanostructures actively secreted MSCs and represent a potent fraction of the MSC secretome. Though CM inherently contains EVs, these vesicles can be selectively isolated using ultracentrifugation, TFF, or size exclusion chromatography (SEC) to enable targeted investigation of vesicle-specific bioactivity and therapeutic effects [72]. EVs, once considered cellular debris, are now recognized as essential mediators of intercellular communication [73]. They carry diverse molecular cargo including proteins, lipids, nucleic acids, and organelle fragments that influence gene expression, signaling cascades, and cellular function across recipient cells under both physiological and pathological conditions [74]. EVs function as carriers of diverse molecular cargo, including proteins, lipids, nucleic acids, and even organelles enabling complex intercellular communication and regulating a wide range of biological processes [75–77]. EVs are commonly classified into four subtypes on the basis of size: exosomes (30–150 nm), microvesicles (100–1000 nm), and apoptotic bodies (50–5000 nm), the latter arising during programmed cell death [78, 79, 80], and oncosomes (1–10 μm), a recently identified class derived from cancer cells [81]. MSC-EVs have emerged as critical effectors of paracrine signaling, facilitating tissue repair and immunomodulation without the risks associated with whole-cell transplantation [82]. In preclinical models of neonatal diseases, MSC-EVs have demonstrated potent therapeutic effects. In experimental BPD, MSC-EVs enhance alveolarization, attenuate lung inflammation, and reduce oxidative stress by delivering proangiogenic factors and anti-inflammatory microRNAs to injured tissues [83]. Similarly, in models of NEC, MSC-EVs preserve intestinal barrier integrity, suppress inflammatory cytokine production, and promote epithelial cell proliferation. MSC-EVs express classical EV surface markers such as CD63, CD9, and CD81, along with mesenchymal stem cell markers such as CD44, CD73, and CD90 [84]. Their cargo includes angiogenic proteins such as VEGF, immunomodulatory molecules such as TSG-6, and regulatory microRNAs that target inflammatory pathways [85–87]. Notably, MSC-EVs have been shown to reduce expression of TNF-α, IL-1β, IL-6, and IFN-γ, thereby contributing to a reparative anti-inflammatory environment [88–91]. Compared to direct MSC transplantation, EV-based therapies offer advantages such as reduced immunogenicity, improved stability, and simplified storage and delivery logistics [92]. MSC therapeutic potential primarily derives from their ability to migrate to injured tissues, secrete bioactive factors, and adaptively respond to inflammatory microenvironments [93, 94]. Accumulating evidence from preclinical models of BPD and NEC demonstrates that MSC-based therapies exert pleiotropic beneficial effects, including anti-inflammatory, anti-apoptotic, antioxidative, and anti-fibrotic actions [95–109].

Preparation method and quantitative impact

The therapeutic potency of EVs is strongly influenced by the method of preparation, which affects their purity, yield, and functional cargo. Ultracentrifugation, although widely used, can reduce vesicle integrity, while TFF offers higher recovery and scalability [110]. SEC enhances purity but often at the cost of yield [111–113]. These methodological differences translate into measurable variations in therapeutic content. For example, VEGF concentration and miRNA profiles (e.g., miR-146a, miR-21) can vary by up to 2-fold between isolation techniques [114–116]. Additionally, MSC passage number influences EV cargo composition, with late-passage cells exhibiting reduced angiogenic and immunomodulatory profiles [117–119]. These variations highlight the urgent need for standardization and quality control in EV production, especially for clinical-grade applications. Essential quality control strategies include quantifying protein content through assays like BCA or ELISA, assessing particle size and concentration via nanoparticle tracking analysis (NTA), and validating surface marker expression using flow cytometry or Western blotting. These parameters are fundamental to ensuring the reproducibility, safety, and therapeutic efficacy of EV-based interventions.

Together, MSC-EVs represent a powerful, cell-free therapeutic tool with broad regenerative applications. Their modular composition, biological potency, and manufacturing flexibility position them as a front-runner in next-generation neonatal therapeutics. In the following sections, we explore how EVs and other secretome components specifically modulate the cellular and molecular pathways implicated in BPD and NEC.

Comparative analysis of MSC sources

MSC-derived secretomes, which include bioactive molecules such as cytokines, growth factors, and EVs, have shown significant promise as cell-free therapeutic agents [120, 121]. Among the various stem cell therapies, MSC therapy is regarded as the most promising in terms of ethics, safety, and clinical practicability in regenerative medicine [95, 109]. EV production from UC-MSCs also results in increased particle yield (up to 10⁶ EVs per 10⁶ cells) and increased IL-10 and VEGF secretion profiles, supporting their superior anti-inflammatory efficacy in preclinical BPD models [122–124]. Comparative studies have shown that UC-MSC-derived secretomes exhibit distinct cytokine profiles compared to BM-MSCs, including higher levels of angiogenic and immunomodulatory factors such as TGF-β1 and HGF, which enhance their potential for epithelial repair and inflammation control [125, 126].

Meanwhile, BM-MSCs remain clinically relevant due to their well-established safety profile, immunomodulatory effects via IL-10 and TNF-α suppression, and historical use in neonatal trials [127, 128]. UCB-MSCs and UCT-MSCs have also emerged as viable candidates, showing strong neuroprotective and anti-inflammatory profiles, along with ease of non-invasive harvesting and reduced immunogenicity [129, 130]. A comparative overview is provided in Table 1, illustrating the source-specific secretome profiles, advantages, and clinical applicability.

Mechanisms of MSC-derived secretomes in treating BPD and NEC

Over the past decade, MSC-derived secretomes have shown considerable therapeutic potential in neonatal models of BPD and NEC. These therapeutic effects are largely attributed to secreted bioactive components, including cytokines, growth factors, EVs, and microRNAs that regulate inflammatory responses, facilitate tissue repair, and promote angiogenesis [53]. Nevertheless, the mechanistic evidence across various studies remains variable. Many preclinical investigations rely heavily on observational outcomes (such as histological assessments and cytokine level alterations) rather than mechanistic validation through knockout models, blocking antibodies, or pathway inhibitors. For example, while IL-10 is frequently cited for its anti-inflammatory effects, few studies isolate its role apart from other cytokines [131, 132]. Moreover, inconsistencies in secretome formulations, dosing protocols, and experimental models pose additional challenges for clearly attributing therapeutic outcomes to specific secretome components. To address these limitations, a deeper mechanistic exploration of key MSC-secreted mediators and their intracellular signaling pathways is provided below, with particular focus on inflammation, regeneration, angiogenesis, and epigenetic regulation relevant to BPD and NEC pathogenesis.

Anti-inflammatory signaling via IL-10, TSG-6, and MiRNAs

One of the hallmark properties of MSC secretomes is their robust anti-inflammatory activity. MSC-derived factors, notably IL-10 and TSG-6, facilitate an essential shift in macrophage polarization from the pro-inflammatory M1 phenotype toward the anti-inflammatory and reparative M2 phenotype [133, 134]. This phenotypic shift significantly reduces production of key inflammatory cytokines (e.g., IL-1β, IL-6, TNF-α), which are critical mediators in the pathogenesis of both BPD and NEC [135, 136]. IL-10 achieves its anti-inflammatory effects primarily by inhibiting NF-κB and JAK/STAT signaling, thus reducing transcriptional activation of inflammatory genes [137, 138]. In IL-10⁻/⁻ neonatal mice, MSC secretomes fail to protect against NEC-induced mucosal injury, directly validating IL-10’s role [139, 140]. Similarly, TSG-6 modulates macrophage activity and extracellular matrix remodeling by directly interacting with CD44 and hyaluronan, subsequently suppressing inflammatory cascades and tissue injury [141–143]. These anti-inflammatory actions not only suppress acute immune responses but also create a reparative microenvironment that facilitates downstream tissue regeneration and angiogenesis.

Angiogenesis and epithelial repair

MSC-derived secretomes contain abundant growth factors, including VEGF, HGF, IGF-1, and bFGF that drive endothelial cell proliferation, migration, and the formation of new vessels [73]. These factors act synergistically to stimulate endothelial cell proliferation, migration, and neovascularization, leading to improved tissue perfusion and enhanced epithelial regeneration [144]. HGF, in particular, has been shown to exert anti-apoptotic, mitogenic, and morphogenic effects on epithelial and endothelial cells, contributing to alveolar stabilization and vascular repair [145–147]. In preclinical lung injury models, HGF enhances epithelial survival and reduces fibrosis [148, 149], while in NEC models, it supports intestinal tight junction integrity and crypt regeneration [150–152]. Causal validation has been demonstrated in several models. For instance, in hyperoxia-exposed neonatal rats, administration of anti-VEGF antibodies abolished the alveolar rescue effects of MSC-derived EVs, confirming a critical mechanistic role for VEGF in lung remodeling [153]. Likewise, in NEC models, VEGF and IGF-1 secreted by MSCs were shown to mediate intestinal epithelial repair and barrier restoration, while VEGF neutralization abrogated these benefits, establishing direct causal linkage.

[154]. These findings go beyond correlative evidence, highlighting that secretome-mediated angiogenesis and epithelial repair are not only multifactorial but also mechanistically dependent on specific cargo components. Furthermore, beyond vascular repair, MSC-secretome bioactivity extends to immunomodulation and epigenetic reprogramming of epithelial and immune cell populations. These broader interactions likely act in tandem with angiogenic signaling to stabilize tissue architecture and resolve inflammation during neonatal injury responses.

Immunomodulation through tregs and epigenetic MiRNAs

MSC-derived secretomes exert potent immunomodulatory effects via both soluble factors and EV cargo, influencing multiple arms of the neonatal immune system. These secretomes have been shown to suppress T cell activation, inhibit dendritic cell maturation, and promote regulatory T cell (Treg) differentiation, thereby supporting immune tolerance and limiting tissue injury in inflammatory diseases [43, 155]. EVs specifically play a critical mechanistic role due to their cargo of epigenetically active microRNAs (miRNAs), including miR-21, miR-34a, and miR-146a. Among these, miR-146a stands out as a master regulator of inflammation. It targets TRAF6 and IRAK1, two essential intermediates in the NF-κB pathway, thereby reducing transcription of proinflammatory cytokines such as TNF-α and IL-6 [156, 157]. Functional relevance is supported by knockout and overexpression studies: miR-146a–deficient mice exhibit exaggerated NF-κB signaling and increased susceptibility to inflammatory injury [158, 159]. However, direct validation of these effects in neonatal disease contexts such as BPD and NEC remains limited. This illustrates a broader issue in the field: many secretome components are inferred to be active based on association, but their precise mechanistic roles particularly in human-relevant disease models remain incompletely defined.

Intracellular pathways activated by secretome components

Beyond immunoregulation, MSC-derived secretomes engage diverse intracellular signaling cascades in epithelial, endothelial, and progenitor cells that mediate tissue repair, regeneration, and fibrosis resolution. These pathways are activated by growth factors (e.g., VEGF, HGF, TGF-β), cytokines, and non-coding RNAs within the secretome cargo.

Table 2 summarizes the major pathways implicated in BPD and NEC, including their key effectors, cellular targets, and the level of mechanistic validation.

Table 2.

Key intracellular pathways mediating MSC secretome effects in BPD and NEC

Pathway	MSC Secretome Component(s)	Target Cells	Biological Effects	Evidence Type	Validation Status	Ref
PI3K/Akt	VEGF, HGF, IGF-1, miR-21	Alveolar cells, enterocytes	Enhances cell survival, proliferation, and mitochondrial function	Moderate (in vivo rodent models)	Well-supported by preclinical studies, but dose-response validation is needed	[160]
PTEN/Akt	VEGF, miR-21	Endothelial cells, epithelial cells	Reduces oxidative stress, promotes angiogenesis	Limited (preclinical only)	Few in vivo studies clarify direct causality in neonatal settings	[161]
WNT5a	WNT5a-loaded EVs, miR-146a	Intestinal stem cells, lung epithelium	Stimulates epithelial and alveolar morphogenesis	Moderate (in vitro + animal models)	Evidence supports mechanistic plausibility, but clinical trials are lacking	[162]
ERK1/2	IL-10, bFGF	Alveolar epithelial cells	Promotes epithelial repair, suppresses inflammation	Correlative (limited causal data)	Few knockout experiments confirm direct MSC-EV activation	[163]
JAK/STAT	IL-10, TSG-6, miR-146a	Macrophages, T cells	Drives anti-inflammatory reprogramming, induces Treg polarization	Limited knockout validation	Preclinical studies support mechanism, but KO validation is sparse	[164]
TGF-β/SMAD	TGF-β, bFGF	Fibroblasts, epithelial cells	Inhibits fibrosis, supports epithelial barrier integrity	Moderate (NEC + BPD preclinical studies)	Strong preclinical data, but clinical implementation is missing	[165]
NF-κB (indirect)	miR-146a, IL-10, TSG-6	Immune cells, epithelial cells	Suppresses cytokine production, downregulates inflammatory gene networks	Strong suppression shown; causal links lacking	Mechanistic studies confirm suppression, but direct causation remains uncertain	[156]

MSC, mesenchymal stem cell; CM, conditioned media; EVs, extracellular vesicles; VEGF, vascular endothelial growth factor; IGF-1, insulin-like growth factor-1; HGF, hepatocyte growth factor; bFGF, basic fibroblast growth factor; TGF-β, transforming growth factor-beta; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL-10, interleukin-10; HO-1, heme oxygenase-1; TSG-6, TNF-stimulated gene-6; miR-146a, microRNA-146a; NF-κB, nuclear factor kappa B; BPD, bronchopulmonary dysplasia; NEC, necrotizing enterocolitis

Among well-characterized repair pathways, PI3K/Akt and TGF-β/SMAD signaling have been shown to enhance cell survival, mitochondrial stability, and barrier integrity, with moderate-to-strong support from rodent NEC and BPD models [160, 165]. PTEN/Akt signaling, often modulated by miR-21 and VEGF, contributes to oxidative stress reduction and endothelial stabilization, although validation in neonatal settings remains limited [161].

Two emerging axes WNT5a and ERK1/2 have gained attention for their distinct roles in morphogenesis and epithelial repair. WNT5a-loaded EVs and miR-146a activate non-canonical WNT signaling in intestinal stem cells and alveolar progenitors, supporting epithelial branching and crypt-villus axis regeneration—key features disrupted in both NEC and BPD [162, 166, 167]. In NEC models, EV-mediated WNT5a signaling enhances epithelial restitution and tight junction integrity.

[162, 168–171], however, receptor-level validation (e.g., ROR2 blockade) remains lacking. Similarly, the ERK1/2 pathway, modulated by IL-10 and bFGF, supports alveolar epithelial proliferation while dampening injury-induced inflammation [172, 173]. Phosphorylation of ERK1/2 has been observed in neonatal lung following MSC-CM administration [174, 175]. Use of ERK inhibitors like U0126 reverses this benefit, implicating the pathway in epithelial rescue, though not yet confirmed in NEC-specific models [176]. Pathways such as JAK/STAT and NF-κB also play roles in immune reprogramming, but these are more fully addressed in Sect. 4.3 due to their centrality in Treg induction and miRNA-mediated inflammation suppression [156, 164].

Collectively, these cascades reflect how MSC secretomes reprogram cellular behavior via context-specific combinatorial signaling. Engineering secretome composition to amplify specific intracellular effects (e.g., WNT5a for epithelial morphogenesis or ERK1/2 for repair) may enable precision therapies for preterm infants.

Functional outcomes in BPD and NEC

To contextualize the therapeutic mechanisms of MSC-derived secretomes, Table 3 links secretome components to specific therapeutic effects in preterm neonatal disease models.

Table 3.

Mechanisms of action in preterm neonatal diseases by therapeutic complication

Therapeutic Effect	CM: Key Molecules	EVs: Key Mechanisms	Evidence Strength	Applies To	Current Limitations / Gaps	Ref
Anti-inflammatory	IL-10, STC-1	miR-146a-mediated NF-κB suppression; ↓ TNF-α, IL-1β	Correlative	BPD, NEC	Few studies have used knockout or blocking experiments.	[177]
Anti-oxidative	VEGF, HGF	Modulation of oxidative stress responses; PTEN/Akt pathway activation	Correlative	BPD	Limited in vivo validation in large animals/humans.	[34]
Anti-fibrotic	bFGF	TGF-β/SMAD pathway inhibition; ↓ fibrotic gene expression	Moderate	BPD	No human data; indirect signaling only.	[178]
Angiogenesis and Regeneration	VEGF-driven endothelial proliferation and migration, IGF-1	Cargo delivery (miR-21, WNT5a activation) to promote angiogenesis and epithelial repair	Moderate	BPD, NEC	Dose-response and causal proof are lacking.	[179]
Barrier Protection	—	ZO-1, occludin upregulation; epithelial proliferation	Moderate	NEC	No clinical validation: cargo content varies	[180]
Epigenetic Modulation	—	Delivery of regulatory miRNAs (miR-21, miR-34a, miR-146a) to modulate gene transcription	Limited; no in vivo KO models	BPD, NEC	Mostly based on in vitro studies.	[181]

CM, conditioned media; EVs, extracellular vesicles; VEGF, vascular endothelial growth factor; IGF-1, insulin-like growth factor-1;HGF, hepatocyte growth factor; bFGF, basic fibroblast growth factor; TGF-β, transforming growth factor-beta; STC-1, stanniocalcin-1; IL-10, interleukin-10; TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1 beta; ZO-1, zonula occludens-1;miR, microRNA; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; WNT5a, Wingless-Type MMTV Integration Site Family, Member 5 A; PTEN, phosphatase and tensin homolog; Akt, protein kinase B; SMAD, suppressor of mothers against decapentaplegic homolog; KO, knockout; BPD, bronchopulmonary dysplasia; NEC, necrotizing enterocolitis.↑, upregulation/increase; ↓, downregulation/decrease

In preclinical neonatal models, MSC-derived secretomes exert distinct therapeutic effects that cluster around six functional outcomes: anti-inflammatory activity, oxidative stress reduction, fibrosis attenuation, angiogenesis and regeneration, epithelial barrier protection, and epigenetic modulation [43, 53]. These effects are mediated through both soluble molecules in conditioned media and cargo within extracellular vesicles, though the strength of supporting evidence varies considerably across categories [182]. Anti-inflammatory and anti-oxidative functions have the strongest biological rationale, showing consistent improvements in lung and gut inflammation markers across BPD and NEC models [183]. However, most studies remain correlative, with relatively few leveraging genetic knockouts or pathway inhibition to isolate specific molecular contributions. Fibrosis reduction and vascular regeneration, particularly relevant in chronic lung injury, are moderately supported by functional data. These effects appear to involve both growth factor signaling and miRNA-mediated modulation of morphogenic pathways [184, 185]. Despite promising outcomes in small-animal models, reproducibility across species and disease contexts remains to be established. Barrier protection and epigenetic modulation, though conceptually compelling for NEC, are currently supported mainly by in vitro observations or indirect markers such as tight junction protein upregulation [186, 187]. The absence of long-term in vivo validation or clinical readouts highlights the need for standardized potency assays and improved mechanistic tracing. Taken together, while some secretome functions are backed by moderate mechanistic evidence, others require more rigorous experimental validation. Translating these findings into clinical-grade products will demand not only efficacy, but also reproducibility, scalability, and regulatory clarity particularly in fragile preterm populations.

Summary of registered clinical trials in neonatal care

Clinical Translation of MSC-Derived Secretomes in Neonatal Care Encouraged by promising preclinical outcomes, an increasing number of clinical trials have commenced to evaluate MSC-derived therapies in neonatal care, particularly targeting severe complications like BPD, NEC, and IVH. These trials aim to establish safety, feasibility, optimal dosing regimens, and preliminary efficacy of MSC-secretome therapies across various neonatal contexts. The diversity of MSC sources, routes of administration, and therapeutic approaches being explored highlights the exploratory and dynamic nature of the field. As of May 2025, a total of 21 clinical trials evaluating MSC derived secretome or EV based therapies for BPD and NEC have been registered on ClinicalTrials.gov and the WHO International Clinical Trials Registry Platform (ICTRP). These studies primarily utilize UC-MSCs or BM-MSCs, with administration routes including intratracheal, intravenous, and intraperitoneal delivery.

Of these, 5 trials have published preliminary safety outcomes. Notably, NCT03857841 reported no serious adverse events associated with intratracheal delivery of UNEX-42 (MSC-EVs) during a 12-month follow-up period. Similarly, NCT02381366 demonstrated a favorable safety profile and preliminary improvement in respiratory parameters in infants with BPD treated with UC-MSC–derived secretome products. In contrast, other trials such as NCT05490173 and NCT04255147 are ongoing or pending results, with endpoints focused on neurodevelopmental safety and inflammatory modulation.

These trials represent a growing global effort particularly in Asia, North America, and Europe to validate the feasibility of secretome-based cell-free regenerative therapies for high-risk neonatal populations. However, the limited long-term outcome data underscore the need for extended follow-up, standardized potency assays, and harmonized clinical endpoints.

A summary of ongoing and completed registered clinical trials related to MSC-derived secretome therapies in preterm infants is presented in Table 4 below.

Table 4.

Summary of registered clinical trials based on MSC secretomes potential for treatment of premature

NCT Number	Study Title	Conditions	Interventions	Phase	Status	Primary Endpoint	Reported Outcomes	Study URL
NCT03774537	Human Mesenchymal Stem Cells For Infants At High Risk For BPD	Bronchopulmonary Dysplasia	hUC-MSCs	Phase I	Completed	Safety, BPD incidence	Safe, early signal of efficacy	https://clinicaltrials.gov/study/NCT03774537
NCT03873506	Follow-Up Study of Mesenchymal Stem Cells for Bronchopulmonary Dysplasia	Bronchopulmonary Dysplasia	hUC-MSCs	Follow-up	Active, not recruiting	Long-term safety & pulmonary outcomes	Ongoing	https://clinicaltrials.gov/study/NCT03873506
NCT01207869	Intratracheal Umbilical Cord-derived Mesenchymal Stem Cells for Severe Bronchopulmonary Dysplasia	BPD, Extremely Premature Infants, IVH, PVL	ucMSCs	Phase I	Completed	Feasibility and safety	Dose-escalation tolerated	https://clinicaltrials.gov/study/NCT01207869
NCT06788470	Safety and Efficacy of Umbilical Cord-derived MSC Transplantation in BPD	Bronchopulmonary Dysplasia	MSC	Phase II	Recruiting	Safety; incidence of moderate-severe BPD	Not yet reported	https://clinicaltrials.gov/study/NCT06788470
NCT03558334	Human Mesenchymal Stem Cells For BPD	Bronchopulmonary Dysplasia	MSC	Not listed	Completed	Safety	Results not posted	https://clinicaltrials.gov/study/NCT03558334
NCT04255147	Cellular Therapy for Extreme Preterm Infants at Risk of BPD	Bronchopulmonary Dysplasia	Allogeneic UCT-MSCs	Phase I/II	Active, not recruiting	Safety and feasibility	No results posted yet	https://clinicaltrials.gov/study/NCT04255147
NCT02381366	Safety and Efficacy of PNEUMOSTEM® in Premature Infants with BPD (US Study)	Bronchopulmonary Dysplasia	UCB-MSCs	Phase I/II	Completed	BPD incidence, oxygen need	Safe, improved respiratory outcomes	https://clinicaltrials.gov/study/NCT02381366
NCT02673788	Follow-Up Study of Pneumostem® in Premature Infants with IVH	Intraventricular Hemorrhage	MSC	Follow-up	Completed	Neurodevelopmental status	Not published	https://clinicaltrials.gov/study/NCT02673788
NCT05490173	Neuroprotective Effects of Exosomes in ELBW Infants	Premature Birth, IVH, Hypoxia-Ischemia	MSC-derived Exosomes	Follow-up	Completed	Neurodevelopmental status	No published results	https://clinicaltrials.gov/study/NCT05490173
NCT02274428	Phase 1 Trial of PNEUMOSTEM® for IVH in Premature Infants	Intraventricular Hemorrhage	MSCs	Pilot	Recruiting	IVH incidence, neuroprotection	No reported outcomes	https://clinicaltrials.gov/study/NCT02274428
NCT02443961	MSC Therapy for BPD in Preterm Babies	Bronchopulmonary Dysplasia	MSC Therapy	Phase 1	Completed	Safety, hemorrhage grading	No significant adverse events reported	https://clinicaltrials.gov/study/NCT02443961
NCT06270199	MSC Use in Preterm BPD Patients	Bronchopulmonary Dysplasia	Allogenic fetal MSCs	Unknown	Unknown	BPD outcome	Unavailable	https://clinicaltrials.gov/study/NCT06270199
NCT03378063	Stem Cells for Bronchopulmonary Dysplasia	Bronchopulmonary Dysplasia	MSC Transplantation	Phase II	Not yet recruiting	Safety, lung development	Trial not yet started	https://clinicaltrials.gov/study/NCT03378063
NCT02023788	Long-term Safety of PNEUMOSTEM® in BPD Patients	BPD, Respiratory Infections	PNEUMOSTEM®	Unknown	Unknown	Unspecified	Unreported	https://clinicaltrials.gov/study/NCT02023788
NCT03645525	Intratracheal UC-MSCs for BPD	Bronchopulmonary Dysplasia	hUC-MSC	Follow-up	Active	Infection rate, growth, neurodevelopment	No published data	https://clinicaltrials.gov/study/NCT03645525
NCT03631420	MSCs for Prevention of BPD in Infants	Bronchopulmonary Dysplasia	hUC-MSC	Phase I/II	Completed	Safety	Well tolerated	https://clinicaltrials.gov/study/NCT03631420
NCT01297205	PNEUMOSTEM® Treatment for BPD	Bronchopulmonary Dysplasia	UCB-MSC	Phase I	Completed	Safety; early efficacy	Results not published	https://clinicaltrials.gov/study/NCT01297205
NCT03392467	PNEUMOSTEM® for Severe BPD in Premature Infants	Severe Bronchopulmonary Dysplasia	PNEUMOSTEM®, Placebo	Phase I	Completed	BPD incidence	Positive safety profile	https://clinicaltrials.gov/study/NCT03392467
NCT01632475	Pneumostem® in Premature Infants with BPD	Bronchopulmonary Dysplasia	Pneumostem®	Phase II	Completed	Severe BPD reduction	No data released	https://clinicaltrials.gov/study/NCT01632475
NCT02890953	Pneumostem® for IVH in Premature Infants	Cell Transplantation	Pneumostem®, Normal Saline	Phase I	Completed	Safety	Confirmed safety over 2 years	https://clinicaltrials.gov/study/NCT02890953
NCT03857841	Stem Cell-derived EVs (UNEX-42) in Preterm Neonates	Bronchopulmonary Dysplasia	UNEX-42 (MSC-EVs)	Phase I	Completed	Feasibility, safety	No serious adverse events	https://clinicaltrials.gov/study/NCT03857841

MSC - Mesenchymal Stem Cell, hUC-MSCs - Human Umbilical Cord-Derived; Mesenchymal Stem Cells, ucMSCs - Umbilical Cord Mesenchymal Stem Cell, UCB-M SCs Umbilical Cord Blood-Derived Mesenchymal Stem Cells, UCT-MSCs - Umbilical Cord Tissue-Derived Mesenchymal Stem Cells, ELBW - Extremely Low Birth Weight, BPD - Bronchopulm onary Dysplasia, IVH - Intraventricular Hem orrhage, PVL - Periventricular L eukom alacia, EVs - Extracellular Vesicles, UNEX-42 - A proprietary formulation of MSC-derived extracellular vesicles used in trial NCT03857841, PNEUMOSTEM@ - A branded allogeneie UCB-MSC product designed for neonatal respiratory diseases, Placebo - An inactive substance used as a control in clinical trials

Some trials have incomplete registry data; missing phase/status may reflect protocol updates or registry discrepancies.

Collectively, these trials underscore a growing confidence in the safety and feasibility of MSC-secretome therapies in neonatal populations, particularly for BPD. While early-phase studies consistently demonstrate tolerability, long-term efficacy data remain limited. The trials also reveal a trend toward intratracheal or intravenous delivery of umbilical cord-derived MSC products, with increasing emphasis on evaluating neurodevelopmental outcomes. Harmonizing study designs and implementing potency-based release criteria will be key to translating these promising interventions into standardized clinical protocols.

Mechanistic validation of key secretome components

While multiple components of the MSC secretome have been linked to immunomodulation, angiogenesis, and tissue repair, the strength of mechanistic evidence varies widely. To distinguish well-validated mediators from correlative candidates, Table 5 below summarizes key factors, their target pathways, and the level of experimental evidence supporting their causal role in preterm lung and intestinal repair. This includes studies using knockout (KO) animals, neutralizing antibodies, and RNA interference.

Table 5.

Causal evidence for key MSC secretome mediators

Molecule	Target Pathway(s)	Functional Evidence	Species / Model	Evidence Level	Ref
IL-10	JAK/STAT, NF-κB	IL-10⁻/⁻ mice → ↑ NEC severity	Mouse NEC model	Strong (KO-based)	[53]
TSG-6	CD44–HA axis	Anti-TSG-6 antibody reverses benefit	Rat BPD model	Moderate	[188]
VEGF	PI3K/Akt, angiogenesis	VEGF blockade reduces alveolarization	Neonatal hyperoxia model	Moderate	[189, 190]
miR-146a	NF-κB, IRAK1, TRAF6	miR-146a mimic lowers cytokines	NEC-like macrophage cultures	Correlative only	[191]
bFGF	ERK1/2, TGF-β/SMAD	Small-molecule inhibition used in BPD	Neonatal rodent lung model	Moderate	[192]
miR-21	PTEN/Akt, cell survival	Expression profile; no in vivo validation	NEC & lung injury models	Correlative only	[193, 194]

Evidence Level Definitions: Strong: Demonstrated causality using genetic knockout (KO) models, blocking antibodies, or RNA interference in relevant disease models

Moderate: Supported by pharmacological inhibition or consistent functional outcomes in vivo but lacks direct genetic or mechanistic validation. Correlative: Associations identified in vitro or in vivo without direct intervention studies; causal roles inferred but not mechanistically confirmed

Among the MSC-secretome components, IL-10 and TSG-6 currently exhibit the strongest causal evidence in neonatal disease models [195]. VEGF and bFGF show promising mechanistic roles with pharmacological support but lack genetic confirmation [196]. In contrast, miRNAs such as miR-21 and miR-146a, while widely reported, remain correlative due to limited in vivo mechanistic validation [181]. As discussed in Sect. 2.2, emerging strategies such as CRISPR-based EV engineering support disease-specific targeting and programmable therapeutic design.

To establish definitive causal roles of individual MSC-secretome components, future studies should employ gene editing and targeted inhibition strategies. CRISPR/Cas9 and RNA interference (RNAi) can facilitate selective knockdown of key mediators, such as IL-10, VEGF, and miR-146a, in MSCs prior to secretome harvesting, with subsequent validation in neonatal BPD and NEC models. Neutralizing antibodies in in vivo systems can further delineate target-specific effects and enhance translational relevance.

To dissect the underlying molecular mechanisms, pathway-reporter assays (e.g., NF-κB, JAK/STAT luciferase systems) should be used to quantify signaling activation in recipient cells following secretome exposure. In parallel, single-cell RNA sequencing of fluorescently labeled EVs can uncover cell-specific uptake patterns and regulatory transcriptional responses.

Finally, standardized biomarker panels including IL-10, TNF-α, ZO-1, CD31, and Arg-1 should be validated as functional readouts for potency, efficacy, and batch consistency. These strategies are essential to bridge preclinical findings with clinical-grade therapeutic development.

Figure 2 maps the key pathways through which MSC-derived secretome components particularly IL-10, TSG-6, VEGF, and miR-146a modulate cellular targets involved in neonatal lung and intestinal injury. Target cells include alveolar epithelial cells, intestinal stem cells, and M1/M2 macrophages, illustrating both shared and distinct mechanisms underpinning therapeutic effects in BPD and NEC.

Fig. 2.

Mechanistic pathways through which MSC-derived secretomes exert therapeutic effects in neonatal diseases such as BPD and NEC

MSC-derived secretomes, encompassing EVs and CM, deliver regenerative bioactive cargo such as VEGF, IL-10, TSG-6, and reparative microRNAs (e.g., miR-21, miR-146a). These components orchestrate key molecular pathways including anti-inflammatory signaling, angiogenesis, epithelial barrier stabilization, and immunomodulation ultimately contributing to improved pulmonary outcomes in BPD and intestinal repair in NEC. Collectively, these effects support the therapeutic positioning of MSC-derived secretomes as a safe, cell-free alternative for treating inflammatory and degenerative conditions in preterm neonates.

Applications in preterm neonatal diseases

Among the diverse MSC sources, UC-MSCs exhibit superior therapeutic potency due to their higher proliferation, immune-privileged status, and strong anti-inflammatory secretome profile [130, 197]. In contrast, BM-MSCs, while extensively studied and clinically established, suffer from donor age-related decline in quality and invasiveness in harvesting [198–200]. Notably, UCB-MSCs provide neuroprotective benefits with minimal immunogenicity and have been validated for safety over a 2-year follow-up. These comparative strengths position UC-MSCs and UC-MSCs as leading candidates for clinical translation, particularly in BPD and NEC contexts where systemic inflammation and tissue injury dominate pathophysiology.

Role of MSCs in BPD treatment

Clinical challenge of BPD

BPD is a chronic inflammatory lung disease predominantly affecting extremely preterm infants who require prolonged respiratory support. It is characterized by arrested alveolarization, pulmonary vascular dysregulation, and persistent inflammation factors that collectively impair lung function and increase susceptibility to respiratory complications later in life [201].

Despite advances in neonatal intensive care, existing treatments such as corticosteroids, diuretics, and supplemental oxygen primarily manage symptoms without preventing or reversing lung injury. The incidence of moderate-to-severe BPD declines with advancing gestational age, from 68% at 24 weeks to 44% at 27 weeks, a trend consistently reported in clinical cohorts [202–205]. Given this vulnerability in extremely preterm neonates, there is an urgent need for regenerative approaches that target the root pathophysiological mechanisms of BPD. MSC-derived secretomes have emerged as promising cell-free therapeutic candidates in this context [206, 207]. By delivering anti-inflammatory cytokines, growth factors, and reparative microRNAs, secretomes promote vascular and alveolar repair while mitigating immune-mediated damage. Principal components such as VEGF, IL-10, TSG-6, and miR-21 coordinate these regenerative effects without the risks associated with live-cell therapies.

Preclinical evidence robustly supports the efficacy of MSC-secretomes in BPD models

Kuamar et al. [208] demonstrated that human UC-MSC-derived EVs significantly reduced alveolar damage, inflammation (TNF-α, IL-6), and oxidative stress in neonatal mice, improving lung compliance and alveolar-capillary integrity. Similarly, Sutsko et al. [209] showed that MSC-conditioned media enhanced epithelial proliferation and attenuated fibrosis in hyperoxia-exposed neonatal rats, with treated animals exhibiting a 40–50% reduction in lung inflammation and a two-fold increase in surfactant protein expression. Despite encouraging findings, key challenges remain, including optimizing dosing, timing of delivery, and identifying critical therapeutic components within the secretome. Several early-phase clinical trials (e.g., NCT03774537, NCT05490173, NCT03857841) are investigating MSC-secretome therapies for BPD. MSC-secretomes thus represent a transformative approach for BPD, offering multi-mechanistic, low-immunogenicity, and cell-free regenerative therapy option [30, 210].

Role of mesenchymal stem cells in NEC treatment

Clinical challenge of NEC

NEC remains one of the most devastating gastrointestinal disorders in preterm infants, with mortality rates between 15 and 30% [211]. Despite its high incidence, the pathogenesis remains incompletely understood, and current treatments are reactive, often resulting in significant morbidities such as short bowel syndrome and neurodevelopmental impairment [212]. NEC presents a multifactorial challenge involving uncontrolled inflammation, compromised barrier function, and dysregulated perfusion pathways that MSC-derived secretomes are uniquely equipped to target. Studies show that MSC-EVs can reduce proinflammatory cytokines (e.g., TNF-α, IL-1β), restore tight junction proteins like ZO-1, and enhance mucosal healing. However, these studies are often short-term and conducted in controlled settings that do not fully replicate the clinical complexity of NEC, including microbial exposure and feeding dynamics. Additionally, variability in MSC sources, isolation protocols, and EV characterization further complicates reproducibility. A more nuanced understanding of the specific EV cargo particularly miRNAs and cytokines responsible for these protective effects is urgently needed to develop targeted, consistent interventions for NEC [28, 213, 214].

Preclinical evidence supporting secretome therapy for NEC

CM and EVs restore intestinal barrier function, reduce inflammatory cytokines (TNF-α, IL-1β), and promote mucosal healing [36, 215]. Zhao et al. [216] found that EV-treated NEC piglets exhibited improved intestinal perfusion, reduced histological injury, and better survival outcomes. Secretome-derived microRNAs (e.g., miR-21, miR-146a) reprogram inflammatory gene networks and bolster tight junction proteins (e.g., ZO-1, occludin), critical for maintaining gut integrity [217]. MSC-secretomes thus offer a dual-action strategy: immune modulation and epithelial protection, presenting a compelling, cell-free therapeutic approach for NEC. Table 6 bellow describe translational breadth across relevant neonatal disease models.

Table 6.

Integrated mechanisms of MSC-Derived secretomes in the treatment of bronchopulmonary dysplasia (BPD) and necrotizing Enterocolitis (NEC)

Source of secretomes	Target cells	Molecular mechanism	Action effect	Disease Context	Ref
MSC-EVs (hUC, BM, adipose, amniotic)	Lung epithelial cells, alveolar macrophages, pericytes, thymic cells	Anti-inflammatory (IL-10, Arg-1, CCR2 axis); immunomodulation (TSG-6, VEGF); miRNA delivery (miR-21-5p); epigenetic reprogramming (WNT5a, PTEN/Akt)	Restores alveolarization, vascularization; reduces inflammation, fibrosis, lung injury	BPD	[218] [195]
BM-MSC EVs (rat)	Lung epithelial cells, endothelial cells, right ventricle	VEGF cargo → angiogenesis ↑, apoptosis ↓; enhanced capillary formation in HUVECs	Restores alveolar and vascular structure; reduces RV hypertrophy; promotes lung growth	BPD	[219]
hUC Wharton’s Jelly MSCs	Lung alveolar and vascular cells	VEGF, NPPB, Anxa5 ↑; TNF-α, CX3CL1, TIM-1 ↓; upregulation of wound healing (MMP-2, LIF) and survival proteins (WISP-1, osteoprotegerin)	Improved alveolarization, vascularization, reduced medial wall thickness; feasible intranasal delivery	BPD	[220]
hBM-MSCs	Intestinal epithelial, immune, and stromal cells	Anti-apoptotic (↑Bcl-2, ↓Caspase-3); cytokine suppression (↓IL-1β); tight junction support (↑ZO-1); secretion of VEGF, FGF, IGF-1, HGF	Reduced NEC severity enhanced intestinal regeneration, improved weight gain, and survival.	NEC	[214, 221]
BM-MSC-derived Exosomes	IEC-6 cells, neonatal rat intestine	miRNA-mediated anti-apoptosis; VEGF mRNA-mediated angiogenesis; wound healing ↑, permeability ↓	Reduced NEC incidence (to 13%); enhanced wound healing.	NEC	[222]
AF-MSC, BM-MSC, AF-NSC, E-NSC (rats)	Intestinal epithelial, stromal, and neuronal cells	Paracrine anti-inflammatory and regenerative signaling; COX-2 modulation	Reduced NEC severity (~ 19–22%); preserved gut integrity.	NEC	[223]

MSC: Mesenchymal Stem Cells; EVs: Extracellular Vesicles; BPD: Bronchopulmonary Dysplasia; VEGF: Vascular Endothelial Growth Factor; miRNA: MicroRNA; TSG-6: Tumor Necrosis Factor-Stimulated Gene 6;CCR2: C-C Chemokine Receptor Type 2;PTEN: Phosphatase and Tensin Homolog; Akt: Protein Kinase B; Arg-1: Arginase-1;IL-10: Interleukin-10;WNT5a: Wnt Family Member 5 A; HUVECs: Human Umbilical Vein Endothelial Cells; MLI: Mean Linear Intercept; MMP-2: Matrix Metalloproteinase-2;LIF: Leukemia Inhibitory Factor; WISP-1: WNT1-Inducible Signaling Pathway Protein 1;TNF-α: Tumor Necrosis Factor Alpha; CX3CL1: C-X3-C Motif Chemokine Ligand 1;TIM-1: T-cell Immunoglobulin and Mucin Domain 1; hBM-MSCs: Human Bone Marrow-Derived Mesenchymal Stem Cells; NEC: Necrotizing Enterocolitis; Bcl-2: B-cell Lymphoma 2; ZO-1: Zonula Occludens-1;VEGF: Vascular Endothelial Growth Factor; FGF: Fibroblast Growth Factor; IGF-1: Insulin-Like Growth Factor 1;HGF: Hepatocyte Growth Factor; IEC-6: Intestinal Epithelial Cell Line 6;miRNA: MicroRNA; AF-MSC: Amniotic Fluid Mesenchymal Stem Cells; BM-MSC: Bone Marrow-Derived Mesenchymal Stem Cells; AF-NSC: Amniotic Fluid Neural Stem Cells; E-NSC: Enteric Neural Stem Cells; COX-2: Cyclooxygenase-2,

The therapeutic relevance of MSC-derived secretomes extends beyond NEC and BPD. While the current review focuses on neonatal applications, similar cell-free strategies are being explored in adult contexts such as inflammatory bowel disease, ischemia-reperfusion injury, and systemic autoimmune diseases. These emerging studies support the concept that MSC-secretome therapy is not limited to early life but may represent a modular, scalable treatment platform across age groups and disease settings—especially where conventional immunosuppressive or epithelial-targeted therapies fall short.

Challenges and future directions for clinical translation

Despite compelling preclinical evidence supporting the therapeutic efficacy of MSC-derived secretomes in neonatal models of BPD and NEC, significant barriers remain in translating these findings into clinical practice. The biological complexity of the neonatal population, coupled with technical and regulatory challenges, has made direct clinical application both scientifically and logistically difficult.

Physiological differences between species, such as immune system immaturity, lung architecture, and intestinal development, limit the generalizability of rodent and porcine model data to human neonates [224]. Additionally, preclinical studies often employ early interventions and supra-physiological doses of secretome products that are not feasible or ethically appropriate in clinical settings. These factors create uncertainty regarding dose selection, treatment timing, and therapeutic consistency in human applications. Furthermore, there remains a critical lack of data on the pharmacokinetics and biodistribution of secretome components, particularly EVs, in preterm infants. Unlike adult systems, neonatal biodistribution is dynamic and organ-specific uptake may differ significantly depending on gestational age and disease state. This variability increases the risk of off-target effects, such as immunosuppression or aberrant angiogenesis, which could have lasting consequences in a developmentally vulnerable population. Compounding these issues are inconsistencies in manufacturing protocols, product characterization, and potency assays. Without standardized preparation and quality control frameworks, batch-to-batch variability and undefined bioactive cargo undermine clinical reproducibility and regulatory confidence. Ethical oversight is especially critical in this population, necessitating trial designs that prioritize long-term neurodevelopmental outcomes and safety monitoring. The following subsections address these core translational barriers ranging from biological heterogeneity and manufacturing standardization to pharmacokinetics, scalability, and clinical trial design and outline the key requirements for advancing MSC-derived secretome therapies toward responsible and effective use in neonatal medicine.

Key knowledge gaps

Several unresolved issues impede clinical application

Species-Specific differences

Key physiological distinctions remain a major obstacle, as animal models particularly neonatal rodents differ from human preterm infants in immune system maturity, lung and gut development, and disease pathogenesis [224]. As a result, extrapolation of efficacy and safety data is fraught with uncertainty.

Non-Standardized delivery conditions

In preclinical models often involving supraphysiological dosing or early interventions limit the relevance of these studies to real-world neonatal care.

Secretome heterogeneity

The composition of secretomes, particularly EVs. Their composition is influenced by donor characteristics, MSC tissue origin, passage number, and culture parameters [225–227]. This variability not only complicates mechanistic studies but also presents challenges for reproducibility and regulatory compliance [226].

Uncharacterized bioactive constituents

Although growth factors (e.g., VEGF, HGF) and immunomodulatory proteins (e.g., TSG-6, IL-10) have been implicated, their precise roles remain unclear, hampering potency assay development and rational dosing strategies [228, 229].

Preclinical model inconsistencies

inconsistencies in preclinical disease models and immunological differences between neonatal rodents and human infants introduce further challenges [230]. Additionally, variability in gestational age and clinical status among neonates makes stratification essential in future trials. Without accounting for these clinical heterogeneities, therapeutic responses may be diluted or misinterpreted.

Critical requirements for translation

While Sect. 2.6 outlined the strategic rationale for transitioning from MSCs to cell-free therapies, this section critically examines the practical challenges that must be addressed to realize clinical translation, particularly for neonatal applications. Although enthusiasm for secretome-based therapies is growing, translational reproducibility remains a key limitation. One fundamental challenge is the intrinsic heterogeneity of the MSC secretome. Batch-to-batch variability driven by MSC tissue source, donor age, passage number, and culture conditions complicates therapeutic consistency. Moreover, few studies provide molecular profiling of secretome products or establish validated potency assays, making it difficult to correlate specific bioactive cargo (e.g., miR-146a, VEGF, TGF-β) with therapeutic efficacy. This lack of standardization limits dose optimization and delays regulatory progress. Although early clinical trials such as NCT03774537 and NCT04255147 demonstrate encouraging safety data, most remain underpowered, short-term, and unable to establish long-term efficacy.

Standardization of secretome preparation and characterization

Bridging the translational gap requires GMP-compliant, scalable manufacturing protocols for EVs and CM [231–233]. In parallel, validated potency assays —such as macrophage polarization, IL-10 induction, or NF-κB inhibition are needed to support quality control and regulatory review. There is a vast variety of different methods of isolating EVs in the literature, based on multiple EV characteristics [234]. Currently, the most widely employed methods include:

UC: Considered the gold standard for EV isolation, UC offers moderate purity but has relatively low throughput and risks vesicle aggregation or damage due to high g-forces [235]. Density Gradient Ultracentrifugation (DGUC): DGUC refines the UC method by incorporating a density gradient (e.g., sucrose or iodixanol), resulting in significantly higher purity and lower protein contamination. It was shown to outperform all other tested methods in preserving EV integrity and minimizing co-isolated proteins in a recent comparative study [236]. TFF: A scalable, closed-loop system that enables EV concentration and buffer exchange with higher yield than UC, though it may retain soluble protein contaminants unless combined with orthogonal techniques [237]. SEC: Offers high purity and preserves EV bioactivity by minimizing shear forces. However, SEC is limited by sample volume and requires post-processing concentration steps [238]. Precipitation Methods (e.g., polyethylene glycol): Provide high yield and simplicity but at the cost of co-isolating protein and RNA contaminants, potentially interfering with downstream functional assays. It’s also cost-effective [239].

Dialysis: Often used as a post-processing step to remove low-molecular-weight solutes or excess culture medium; however, it lacks selectivity for vesicular components and is not sufficient as a standalone method. Bellow Table 7 highlighting the relative trade-offs between each preparation method in terms of key performance metrics.

Table 7.

Comparative evaluation of extracellular vesicle (EV) isolation methods for MSC-secretome preparation

Method	Yield	Purity	Throughput	Scalability	Bioactivity Preservation	Clinical Applicability	Ref
Ultracentrifugation (UC)	Moderate	Moderate	Low	Limited	Moderate	Limited (research-grade)	[235]
Density Gradient UC (DGUC)	Low-Moderate	High	Very Low	Poor	High	Low (complex setup)	[240]
Tangential Flow Filtration (TFF)	High	Moderate	High	Excellent	High	High (GMP-compatible)	[241]
Size Exclusion Chromatography (SEC)	Low-Moderate	High	Low	Moderate	High	High (needs concentration)	[111]
Precipitation (e.g., PEG)	High	Low	High	Good	Low	Low (contaminants)	[242]
Dialysis	Low	Very Low	Very Low	Poor	Variable	Not standalone	[243]

UC, ultracentrifugation; DGUC, density gradient ultracentrifugation; TFF, tangential flow filtration; SEC, size exclusion chromatography; PEG, polyethylene glycol; GMP, good manufacturing practice. Bioactivity Preservation refers to the retention of functional vesicle cargo (e.g., proteins, miRNAs) post-isolation. Yield indicates total vesicle recovery; Purity reflects enrichment free of protein/lipid contaminants; Throughput refers to time and volume efficiency; Scalability assesses potential for large-batch clinical translation

Table 7 highlighting the relative trade-offs between each preparation method in terms of key performance metrics.

Each technique differentially affects yield, particle size distribution, and cargo retention [244]. For instance, exosomes isolated from later-passage MSCs tend to exhibit reduced anti-inflammatory miRNA content (e.g., miR-146a, miR-21), while batch comparisons have revealed variability in TGF-β and VEGF levels even under identical culture conditions [245]. These cargo shifts can profoundly influence potency, particularly in dose-sensitive neonatal applications. Multi-modal cargo profiling including RNA mass spectrometry, and NTA is essential to define batch consistency and optimize dosing strategies. Yet, few comparative studies directly link preparation method, molecular content, and functional outcomes. Cross-platform benchmarking studies are urgently needed.

Potency Assays and Regulatory Alignment To facilitate clinical use, it is imperative to develop validated potency assays aligned with the biological functions of interest—such as macrophage polarization, cytokine suppression, or angiogenesis promotion. Functional immunoassays (e.g., IL-10 induction, NF-κB inhibition) and surrogate markers (e.g., CD9+/TSG-6 + vesicle counts) can be integrated into release criteria to support regulatory review.

In this context, the choice of EV isolation method plays a pivotal role in determining the therapeutic consistency of secretome products. Comparative studies show that critical cargo molecules such as VEGF and miR-146a vary by 1.8–2.3-fold depending on whether size exclusion chromatography (SEC) or tangential flow filtration (TFF) is used, which substantially alters angiogenic and immunomodulatory efficacy [Ref]. Similarly, late-passage MSCs consistently yield EVs with diminished miR-21 and TSG-6 levels, which are key mediators in inflammation suppression [117–119].

These observations highlight that batch-to-batch variability is not solely biological, but also methodological driven by protocol parameters, vesicle isolation techniques, and post-processing conditions. Although Table 7 provides a comparative overview of common isolation methods, a universally superior technique has yet to be identified. SEC, for instance, preserves bioactivity but suffers from volume constraints, while TFF offers higher yield and GMP scalability at the cost of potential protein contaminants.

To address these challenges, standardized EV isolation protocols and cargo profiling workflows should be implemented in line with the MISEV2018 guidelines suppression [246]. These include detailed reporting of isolation conditions, validation of bioactive contents (e.g., miRNAs, cytokines), and incorporation of functional potency assays such as IL-10 induction or NF-κB inhibition into product release criteria. Furthermore, inter-laboratory reproducibility studies that correlate isolation technique with therapeutic output are urgently needed to establish validated best practices for clinical translation.

Pharmacokinetics and biodistribution uncertainty

Pharmacokinetic (PK) profiling remains another major gap: the biodistribution, half-life, and organ-specific uptake of EVs in neonates are poorly characterized [247]. Biodistribution variability has been observed not only between EVs from different MSC sources but also depending on the delivery route and the disease state of the recipient [124, 248, 249]. This variability complicates the prediction of therapeutic efficacy and safety in neonatal populations. The small size and membrane composition of EVs while offering excellent tissue penetration pose challenges for precise in vivo tracking. Although genetic engineering approaches, such as NanoLuc-fused CD63, have been developed to improve EV imaging, these modifications can alter natural biodistribution patterns, raising concerns about data validity [250, 251].

Urgency of harmonized manufacturing standards

To achieve regulatory and clinical consistency, harmonization of isolation and characterization protocols is essential. Without it, optimizing therapeutic windows, dosing, and route of delivery in neonatal populations will remain speculative. Addressing these technical challenges is critical to designing future trials with realistic dosing strategies, reliable safety monitoring, and mechanistically justified efficacy endpoints.

Importantly, many of these translational barriers are not unique to neonatal diseases. Similar challenges in potency profiling, regulatory ambiguity, and donor variability have been reported in adult applications of MSC-secretomes for myocardial infarction, stroke, and autoimmune diseases. Thus, resolving these issues in the neonatal context may also set the foundation for broader implementation of MSC-secretome platforms across disciplines. The neonatal population due to its biological sensitivity and ethical constraints may in fact drive the highest standards for reproducibility, safety, and bioengineering rigor applicable to all age groups.

Figure 3 illustrates the multifaceted role of mesenchymal stem cell (MSC)-derived secretomes including extracellular vesicles (EVs) and conditioned medium (CM) in the context of neonatal diseases such as bronchopulmonary dysplasia (BPD) and necrotizing enterocolitis (NEC). Pathogenesis: Secretome targets inflammatory and fibrotic responses in the immature lung and gut epithelium. Engineering/Optimization: Cargo composition varies based on tissue source (bone marrow, adipose, placenta), donor sex, gestational age, and preconditioning strategies. Therapy: EVs and CM promote epithelial repair, angiogenesis, and immune modulation (e.g., M2 macrophage polarization). Translational Barriers: Challenges include heterogeneity in donor material, lack of potency assays, difficulties in pharmacokinetics, neonatal-specific GMP production, and regulatory ambiguity.

Fig. 3.

Multifunctional Role of MSC-Derived Secretomes in Neonatal Disease: From Pathogenesis to Clinical Translation

Economic viability and scalability

The clinical translation of MSC-derived secretomes offers significant promise, but economic viability and scalability remain critical for widespread adoption. Manufacturing these biologics must comply with GMP standards, increasing production costs. However, secretomes, particularly EVs and CM, provide cost advantages over live-cell therapies by eliminating the complexities of cell engraftment, immunogenicity, and preparation [36, 252]. EV therapies can be cryopreserved, reducing storage requirements and allowing for centralized manufacturing, thereby lowering distribution costs [253]. Standardizing secretome isolation and maximizing EV yield could further reduce production costs. However, challenges remain, such as high initial costs for manufacturing infrastructure, raw materials (e.g., MSC culture media), and the development of potency assays for regulatory approval. Cost-sharing mechanisms, like public-private partnerships and streamlined regulatory pathways, may make these therapies more affordable, especially for treating preterm neonates in both high- and low-resource settings.

A perspective on clinical trial design

A future Phase 3 multicenter, randomized, placebo-controlled trial will evaluate MSC-derived secretome therapy for BPD and NEC in extremely preterm neonates (< 28 weeks gestation). The trial will aim to validate the efficacy and safety of this regenerative therapy in high-risk populations. For BPD, MSC-derived EVs (intratracheal or intravenous) will be compared to standard care, with the primary endpoint being survival without moderate-to-severe BPD at 36 weeks postmenstrual age. For NEC, secretome therapy (oral or intraperitoneal) will assess its impact on reducing NEC incidence (Bell Stage ≥ II), surgical intervention rates, and mortality.

While these endpoints are clinically meaningful, several trial design challenges warrant attention. Variability in secretome composition across batches even under GMP conditions raises concerns about reproducibility and standardization [137]. Diagnostic thresholds for BPD and NEC can vary between institutions, potentially affecting consistency in primary outcomes. Moreover, the optimal timing, dose, and route of secretome delivery remain uncertain, particularly in NEC where early detection and intervention are critical. The absence of validated pharmacodynamic markers or potency assays further complicates dosing decisions [254, 255]. Secondary outcomes such as duration of mechanical ventilation, oxygen dependency, growth trajectory, and neurodevelopmental status at 12–24 months are important but will require harmonized follow-up protocols and blinded evaluations to ensure reliability [141]. To maintain trial integrity and patient safety, rigorous oversight through real-time adverse event tracking and an independent data safety monitoring board is essential [11, 122].

Conclusion and future perspectives

MSC-derived secretomes hold transformative potential for treating neonatal diseases such as BPD and NEC. Preclinical models have consistently demonstrated their ability to suppress inflammation, enhance epithelial repair, and stimulate angiogenesis.

However, despite these encouraging findings, the transition from bench to bedside remains constrained by several interdependent challenges ranging from biological heterogeneity and manufacturing inconsistencies to regulatory uncertainties and knowledge gaps in neonatal pharmacokinetics. A major barrier is the intrinsic variability of secretome composition, influenced by MSC source, donor characteristics, passage number, and culture conditions [256, 257]. This variability complicates both mechanistic interpretations and clinical reproducibility. Moreover, the therapeutic cargo particularly miRNAs, cytokines, and growth factors remains incompletely defined, hindering the establishment of reliable potency assays and standard release criteria. Although early-phase trials have demonstrated favorable safety profiles in preterm populations, most remain underpowered and lack long-term efficacy data. To address these limitations, several critical strategies are needed: Standardized GMP-compliant protocols for secretome preparation and quality control to reduce batch-to-batch variability. Validated bioactivity and potency assays reflecting core mechanisms of action, such as immunomodulation (e.g., IL-10, TSG-6) or angiogenic signaling (e.g., VEGF, HGF). Pharmacokinetic and biodistribution studies specific to the neonatal context, accounting for gestational maturity, disease states, and delivery routes. Clear regulatory classification of secretome-based products as biologics or ATMPs, to facilitate streamlined clinical approval and oversight.

Looking ahead, next-generation technologies offer promising solutions. Synthetic nanovesicles can replicate EV structure while improving stability and reproducibility. Engineered exosomes customized with tissue-specific ligands or therapeutic miRNAs offer enhanced targeting and potency. Secretome amplification platforms, including high-yield bioreactors and co-culture systems, may ensure scalability without compromising function. Emerging platforms such as organoid-tissue extracellular vesicles (OTEVs) and spatially resolved EV profiling further enable microenvironmental specificity and predictive potency mapping, enhancing precision in disease-targeted secretome therapies [258, 259]. Emerging AI-based molecular profiling could enable real-time quality control and personalized secretome matching for high-risk infants.

Future clinical trials must evolve toward adaptive, biomarker-guided designs, with long-term follow-up encompassing neurodevelopment, immune maturity, and organ function. The neonatal setting due to its biological sensitivity and regulatory stringency may serve as the proving ground for scalable, precision-engineered secretome therapies.

MSC-derived secretomes are poised to redefine regenerative medicine, delivering multimodal therapeutic benefits without the safety risks of cell transplantation. As foundational barriers are addressed, their clinical utility may extend far beyond neonatology into autoimmune, cardiovascular, and neurodegenerative diseases positioning secretomes as a cornerstone of next-generation, cell-free regenerative therapeutics.

Abbreviations

ATMP

Advanced Therapy Medicinal Product

BPD

Bronchopulmonary Dysplasia

bFGF

Basic Fibroblast Growth Factor

CM

Conditioned Media

EVs

Extracellular Vesicles

ELBW

Extremely Low Birth Weight

HA

Hyaluronan

hUC-MSCs

Human Umbilical Cord-derived Mesenchymal Stem Cells

HGF

Hepatocyte Growth Factor

IGF-1

Insulin-like Growth Factor-1

IL-10

Interleukin-10

IVH

Intraventricular Hemorrhage

JAK/STAT

Janus Kinase / Signal Transducer and Activator of Transcription

KO

Knockout (Genetically Modified)

miRNA

MicroRNA

MSC

Mesenchymal Stem Cell

NEC

Necrotizing Enterocolitis

NF-κB

Nuclear Factor kappa-light-chain-enhancer of activated B cells

PBMC

Peripheral Blood Mononuclear Cells

PI3K/Akt

Phosphatidylinositol 3-Kinase / Protein Kinase B

PTEN

Phosphatase and Tensin Homolog

PVL

Periventricular Leukomalacia

SEC

Size Exclusion Chromatography

STC-1

Stanniocalcin-1

T1DM

Type 1 Diabetes Mellitus

TFF

Tangential Flow Filtration

TGF-β/SMAD

Transforming Growth Factor-beta / SMAD signaling pathway

TRAF6

TNF Receptor-Associated Factor 6

TSG-6

TNF-Stimulated Gene-6

UC-MSCs

Umbilical Cord-derived Mesenchymal Stem Cells

VEGF

Vascular Endothelial Growth Factor

WNT5a

Wingless-Type Family Member 5 A

Author contributions

TA, KH, FW and JM, wrote and conceptualized the manuscript. LB and ZJ drafted and revised the draft manuscript. YW, XZ, XH, and XQ reviewed and edited the final manuscript. CW, LZ, and JT supervised the manuscript. LB and KH conceived the concept and design of the paper and contributed to the preparation of the Figure. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 82470757), National Key Clinical Specialty Scientific Research Project (No. Z2023032), National Natural Science Foundation of China (No. 82070758) and Research Project of Health Commission of Hunan Province in China (No. 202206012608).

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.