Enhancing mesenchymal stem cell function with epiregulin in tissue engineering: From tissue regeneration to gut microbiota modulation

Abstract

Epiregulin (EREG), a ligand of the epidermal growth factor receptor, has emerged as a potent modulator of mesenchymal stem cell (MSC) function, particularly in inflammatory conditions. Recent evidence indicates that EREG signaling significantly enhances the migration, chemotaxis, and lineage-specific differentiation of MSCs, overcoming critical barriers to cell survival and engraftment in hostile microenvironments. This review summarizes recent advances in the synergistic application of EREG and MSCs in various tissue engineering applications, such as bone, skin, liver, and intestinal regeneration. We discuss the context-dependent nature of EREG activity and emerging biomaterial-based delivery strategies. We also explore the underappreciated role of the gut microbiota as a systemic regulator of EREG expression and a dynamic biomarker of therapeutic efficacy. By integrating mechanistic insights with translational prospects, we outline how EREG-primed MSC therapies can be optimized for clinical implementation in complex regenerative settings.

Key Words: Mesenchymal stem cells; Epiregulin; Bone tissue regeneration; Epidermal growth factor receptor; Regeneration; Tissue engineering; Inflammation; Gut microbiota; Prebiotics

Core Tip: Epiregulin enhances the migration, chemotaxis, and osteogenesis of mesenchymal stem cells under inflammatory conditions and can be used to treat various diseases associated with inflammation. In this opinion review, we discuss the prospects of using epiregulin to enhance the function of mesenchymal stem cells in tissue engineering applications, including bone, skin, liver, and intestinal regeneration, as well as gut microbiota modulation.

INTRODUCTION

Epiregulin (EREG) belongs to the epidermal growth factor (EGF) family and functions as a ligand for the EGF receptor (EGFR). Binding of EREG to EGFR stimulates cell proliferation and suppresses the production of pro-inflammatory mediators, thereby enhancing the regenerative potential of mesenchymal stem cells (MSCs). Recent findings support the hypothesis that EREG is a critical mediator of tissue regeneration mediated by MSCs[1-4]. This assumption is further supported by evidence demonstrating a significant decrease in the functional activity of MSCs in inflammatory conditions, including impaired migration, chemotaxis, proliferation, and differentiation[5]. Collectively, these observations establish EREG as a pivotal factor in optimizing MSC-based strategies for clinical translation under physiologically relevant conditions.

Despite their transformative potential in regenerative medicine, the clinical efficacy of MSC-based therapies is often limited by significant biological barriers. A primary limitation is the low survival rate and poor engraftment of transplanted cells. This is because the hostile microenvironment at the injury site, characterized by oxidative stress and inflammation, triggers the rapid death of the administered cells. This substantial cell loss significantly reduces the therapeutic efficacy and remains a major obstacle to the widespread clinical adoption of MSC treatments[6,7].

To address this critical bottleneck, EREG supplementation has emerged as a promising strategy to enhance the resilience of MSCs in hostile microenvironments. Mechanistic insights, such as those provided by Zhao et al[1], suggest that EREG-mediated activation of the EGFR initiates signaling cascades that significantly alter the immune microenvironment. Specifically, this interaction drives the polarization of macrophages from a pro-inflammatory M1 phenotype towards a reparative M2 state. By attenuating local inflammatory stress through this immunomodulatory switch, EREG effectively preserves the viability of MSCs and sustains their therapeutic functionality.

Collectively, EREG represents a promising approach for advancing MSC-based therapeutic strategies across multiple tissue and organ systems. A bibliometric analysis reveals a significant disparity in research attention: While studies investigating MSC-mediated tissue regeneration have shown consistent growth over the past two decades (Figure 1A), research exploring the regenerative potential of EREG remains limited and has fluctuated considerably (Figure 1B). This review summarizes the current evidence supporting the hypothesis that the strategic integration of EREG with MSC therapies can significantly improve biomedical outcomes, particularly in tissue engineering applications, thereby accelerating their translation into clinical practice.

Figure 1 Number of publications indexed in the PubMed database from 2000 to 2025. A: Publications related to tissue and organ regeneration using mesenchymal stem cells; B: Publications related to tissue and organ regeneration using epiregulin.

ROLE OF EREG IN TISSUE REGENERATION

EREG is a pleiotropic mediator whose biological effects are strictly determined by tissue context and microenvironmental cues. Under physiological conditions, EREG plays a key role in maintaining epithelial barrier integrity and facilitating tissue renewal. Notably, EREG accelerates cutaneous wound healing by stimulating the migration and proliferation of keratinocytes and fibroblasts, and by promoting angiogenesis[8]. However, in pathological settings, this potent mitogenic activity can be detrimental. In colorectal cancer, for example, EREG promotes tumor progression by increasing proliferation, invasion, and resistance to chemotherapy and targeted therapies[9,10]. Similarly, in psoriasis, EREG contributes to epidermal hyperproliferation and the perpetuation of inflammatory processes[11]. Therefore, EREG is an example of a context-dependent signaling factor, as its biological impact is defined by the cell type, receptor expression profile, and the physiological or pathological status of the tissue.

Elucidating the specific mechanisms of EREG in regenerative contexts requires robust experimental models. In this regard, the study by Zhao et al[1] is notable for its well-thought-out methodology and clear focus on the key question: What role does EREG play in periodontal tissue regeneration? The authors adopt a comprehensive approach, combining in vitro experiments using mouse bone marrow MSCs (BMSCs) with an in vivo Wistar rat model of periodontitis. This dual approach enabled the evaluation of both the cellular mechanisms of EREG action and its effects in an inflammatory microenvironment. However, as the authors rightly acknowledged in the discussion section, the study has certain limitations. In particular, the long-term effects of EREG remain insufficiently studied, and the data are limited in their translatability to humans. These limitations highlight the need for further research.

Given that its functions are highly context-dependent, it is important to consider the role of EREG in a broader context. On the one hand, EREG acts as a marker of dysregulation in several pathologies. For example, in the mesenteric adipose tissue of patients with Crohn’s disease, its expression correlates with the progression of the inflammatory process[12]. On the other hand, numerous studies have confirmed the regenerative potential of EREG[13-16]. For instance, Lemmetyinen et al[14] demonstrated that EREG knockout mice exhibit heightened sensitivity to sodium dextran sulfate-induced colitis, highlighting its pivotal role in restoring the colonic epithelium following injury. Similar findings have been reported for the skin, lungs, and other tissues, where EREG is predominantly expressed by fibroblasts, keratinocytes, and epithelial cells in response to injury[17-19].

Despite its well-established role in soft tissue regeneration (e.g., the intestines, lungs, and skin), accumulating data confirms the osteoinductive potential of EREG. The key mechanism underlying EREG activity involves ligand-dependent activation of EGFR, which initiates the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/protein kinase B cascade signaling pathways[20,21]. Activation of these pathways leads to increased expression and nuclear translocation of the key osteogenic transcription factors Runt-related transcription factor 2 and Osterix, thereby promoting the differentiation of MSCs into osteoblasts[22] (Figure 2). Notably, unlike high-affinity ligands (e.g., EGF), EREG has a low binding affinity, promoting the formation of specific EGFR dimers and reducing the rate of receptor internalization. This ensures the prolonged activation of downstream signaling pathways, which is critical for maintaining differentiation signals in tissue engineering constructs[23]. Thus, the available data do not allow us to categorize EREG as either “beneficial” or “harmful”; rather, it functions as a molecular switch that is activated under stressful conditions and modulates the balance between inflammation and regeneration. Its effect is determined by the cellular context, the stage of the pathological process, and its interactions with other signaling pathways.

Figure 2 Schematic representation of the molecular mechanisms underlying epiregulin-induced osteogenic differentiation of mesenchymal stem cells. Epiregulin binds to the epidermal growth factor receptor (EGFR) on the cell membrane, inducing dimerization and activation of the receptor. Due to its lower binding affinity than that of high-affinity ligands (e.g., epidermal growth factor), epiregulin promotes the formation of specific EGFR dimers and reduces the rate of receptor internalization, thereby ensuring the prolonged activation of downstream signaling pathways. Activation of the EGFR triggers two major signaling cascades: (1) the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway, involving sequential activation of Ras, Raf, MEK, and extracellular signal-regulated kinase; and (2) the phosphatidylinositol 3-kinase/protein kinase B pathway involving phosphatidylinositol 3-kinase, phosphatidylinositol 3,4,5-trisphosphate, and protein kinase B. Both pathways converge in the nucleus, where they promote the expression and nuclear translocation of the key osteogenic transcription factors Runt-related transcription factor 2 and Osterix. These transcription factors then bind to DNA and initiate the transcriptional program that drives mesenchymal stem cell differentiation into osteoblasts. This sustained signaling mechanism is critical for maintaining osteogenic differentiation signals in tissue engineering constructs. Akt: Protein kinase B; EGFR: Epidermal growth factor receptor; EREG: Epiregulin; ERK: Extracellular signal-regulated kinase; MAPK: Mitogen-activated protein kinase; PI3K: Phosphatidylinositol 3-kinase; PIP3: Phosphatidylinositol 3,4,5-trisphosphate; Runx2: Runt-related transcription factor 2.

In this sense, the work of Zhao et al[1] is particularly valuable, as it is the first to demonstrate the involvement of EREG in regulating MSC function under conditions of periodontal inflammation—a complex microenvironment in which fibroblasts, osteoblasts, cementoblasts, and immune cells interact. Furthermore, the authors justify the importance of their study by citing evidence of the proangiogenic and immunomodulatory properties of EREG, as well as its ability to stimulate the proliferation, migration, and differentiation of oral soft tissue cells[24-26]. These functions make EREG a promising target for the development of regenerative strategies in biomedicine.

EREG AND MSCs: A NEW APPROACH TO TISSUE ENGINEERING

As mentioned previously, the biological activity of EREG is highly context-dependent. Depending on the specific characteristics of the microenvironment, it can either promote regenerative processes or inflammation. Consequently, the efficacy of combining EREG with MSCs depends on the histological characteristics of the target organ or tissue. Table 1 summarizes the most recent studies (2020–2026) on the application of this strategy in various tissue types (bone, skin, liver, and intestinal tissue).

Table 1 Research on epiregulin and mesenchymal stem cells in regenerative medicine (2020–2026).

Tissue/organ	Methodology	Results	Ref.
Bone	In vitro: Osteogenic differentiation of BMSCs in the presence of TNF-α. In vivo: Model rats with periodontitis	EREG enhances osteogenesis via the EGFR/ERK1/2 pathway, accelerating mineralization and bone formation in periodontal bone defects	[1,27,29]
Skin	Multi-omics analysis of UVB-induced photoaging in human keratinocytes treated with hUC-MSC-CM	hUC-MSC-CM reduced apoptosis by ~4.5%, decreased ROS production, enhanced keratinocyte migration, and modulated EREG expression through EGFR-dependent signaling pathways	[30]
Liver	In vitro: Human hepatocyte cell line (THLE-2) treated with EREG; CCK-8 proliferation assays. In vivo: Mouse ALPPS model; PHLF model; CCl4-induced acute/chronic liver injury; MSC administration [(1–5) × 106 cells/kg]; MSC-EV delivery (109–1012 particles/kg). Analysis: ScRNA-seq and CyTOF of the liver immune microenvironment; ELISA for growth factors	EREG was found to significantly promote hepatocyte proliferation (THLE-2) and improve survival in PHLF mice (median survival increased from 22 hours to 36 hours). THBS1+ monocytes were identified as the source of EREG. MSCs suppressed hepatocyte apoptosis and reduced inflammatory cytokines. They also enhanced liver regeneration via the secretion of HGF, IGF-1, IL-6, and VEGF; reduced fibrosis via the downregulation of TGF-β1; and improved liver function	[31,32]
Intestinal tissue	In vitro: HIOs cultured with EREG; scRNA-seq. In vivo: Transplantation of HIOs under the murine kidney capsule and injection of tomato lectin into the tail vein	EREG promotes the simultaneous differentiation of the epithelium, mesenchyme, enteric neuroglial cells, endothelial cells, and organized smooth muscle. EREG-HIOs form functional neuromuscular units that exhibit spontaneous, peristaltic-like contractions	[15,16]

ALPPS: Associating liver partition and portal vein ligation for staged hepatectomy; EGFR: Epidermal growth factor receptor; ELISA: Enzyme-linked immunosorbent assay; EREG: Epiregulin; ERK1/2: Extracellular signal-regulated kinase 1/2; HGF: Hepatocyte growth factor; HIOs: Human induced pluripotent stem cell-derived intestinal organoids; hUC-MSC-CM: Human umbilical cord mesenchymal stem cell-conditioned medium; IGF-1: Insulin-like growth factor-1; IL-6: Interleukin-6; MSC: Mesenchymal stem cell; MSC-EV: Mesenchymal stem cell derived extracellular vesicle; PHLF: Post-hepatectomy liver failure; ROS: Reactive oxygen species; scRNA-seq: Single-cell RNA sequencing; TGF-β1: Transforming growth factor β; TNF-α: Tumor necrosis factor-α; VEGF: Vascular endothelial growth factor.

Bone tissue

While accumulating evidence highlights the role of EREG in regulating MSC function across multiple regenerative contexts, a recent study by Zhao et al[1] specifically elucidates how EREG modulates the functional activity of mouse BMSCs under inflammatory conditions, offering a mechanistic framework with broad implications for tissue engineering. To recreate the pathophysiological microenvironment in vitro, the chronic inflammation characteristic of destructive periodontal diseases was simulated using recombinant tumor necrosis factor-α[27]. Under these conditions, EREG triggered EGFR activation, followed by the downstream phosphorylation of ERK1/2. This signaling cascade directly enhanced BMSC migration, chemotaxis, and osteogenic differentiation[1].

The validity of these findings was confirmed through a comprehensive methodological approach. Genetic engineering techniques, including partial and complete EGFR knockdown via transfection, demonstrated that the effects of EREG are strictly receptor-dependent. Functional assessments included a scratch wound healing assay to evaluate migratory and regenerative capacity, while alizarin red staining was used to quantify osteogenic differentiation. Molecular validation was achieved through quantitative real-time polymerase chain reaction and western blotting to assess the expression of key osteogenic markers. Collectively, these findings demonstrate that EREG significantly enhances BMSC proliferation, migration, and lineage-specific differentiation under inflammatory stress[1].

These findings were further substantiated in vivo using a rat model of induced periodontitis. Local administration of recombinant human EREG (rhEREG) promoted structural restoration of periodontal tissues, including regeneration of alveolar bone, cementum, and the periodontal ligament. Interestingly, a previous study by the same group on human dental pulp stem cells demonstrated that EREG suppression enhances osteogenic differentiation[28]. Although these findings appear contradictory, this discrepancy may be explained by differences in cell type and microenvironmental context. In the murine BMSC model, tumor necrosis factor-α-driven inflammation critically primes the EGFR-ERK1/2 axis, whereas human dental pulp stem cells were cultured under non-inflammatory conditions, likely activating alternative signaling pathways. Thus, the biological impact of EREG is determined not only by the target cell population but also by the pathological or homeostatic state of the microenvironment.

Broader evidence supporting the role of EREG in skeletal remodeling comes from Dong et al[29], who demonstrated upregulated EREG expression during BMSC osteogenesis, concurrent with its downregulation during adipogenic commitment. The systemic administration of recombinant human EREG (rhEREG) in ovariectomized mice increased osteoblast numbers, stimulated new bone formation, and attenuated osteoclast-mediated resorption, highlighting the capacity of EREG to rebalance bone remodeling under pathological conditions.

Collectively, these studies position EREG as a potent modulator of MSC-mediated bone regeneration, particularly under inflammatory conditions. However, its effects are highly context-dependent, necessitating further investigation into stem cell heterogeneity, signaling pathway crosstalk, and disease-specific microenvironments. The successful translation of EREG-based therapies into clinical tissue engineering and regenerative medicine will ultimately depend on the reproducibility of their therapeutic outcomes and rigorous validation of the underlying mechanisms in clinically relevant models.

Skin tissue

While the role of EREG in skin biology is increasingly recognized, its therapeutic potential in combination with MSCs has been demonstrated in models of photoaging. Multi-omics analysis of UVB-induced photoaging in human keratinocytes (HaCaT) revealed that EREG is among the key differentially expressed genes affected by UVB irradiation (20 mJ/cm²)[30]. Treatment with human umbilical cord MSC-conditioned medium (hUC-MSC-CM) significantly reduced photoaging phenotypes by modulating EREG expression alongside other important regulators, such as MYC, fibroblast growth factor 1, interleukin-8 (IL-8), c-FOS, c-JUN, and p53. hUC-MSC-CM treatment reduced apoptosis by around 4.5%, alleviated G1-phase cell cycle arrest, decreased reactive oxygen species production, and enhanced keratinocyte migratory capacity. Transcriptomic and proteomic profiling revealed that hUC-MSC-CM exerts its protective effects via EGFR-dependent signaling pathways, with EREG identified as a key mediator of keratinocyte proliferation and skin regeneration[30]. These findings suggest that paracrine factors derived from MSCs, including EREG, can effectively counteract UVB-induced skin damage and cellular senescence. This positions hUC-MSC-CM as a promising therapeutic approach for preventing photoaging and regenerating skin tissue. However, further in vivo studies and standardization of MSC secretome composition are necessary for clinical translation.

Liver

Liver diseases, including acute liver failure, cirrhosis, and chronic hepatitis, pose a significant global health challenge and have limited treatment options aside from transplantation. However, recent advances in our understanding of liver regeneration mechanisms have identified two promising new avenues of treatment: EREG-mediated hepatocyte proliferation[31] and MSC-based regenerative therapy (Table 1)[32].

EREG, a potent hepatocyte mitogen and EGFR ligand, has emerged as a critical regulator of liver regeneration. Chen et al[31] demonstrated that tissue monocytes secreting EREG (THBS1+) are recruited by C5a to promote rapid liver regeneration in both murine models and patients undergoing associating liver partition and portal vein ligation for staged hepatectomy. Mechanistically, EREG activates the EGFR on hepatocytes, thereby triggering the mitogen-activated protein kinase/ERK signaling cascade that drives hepatocyte proliferation. In vitro studies confirmed that EREG significantly stimulated the proliferation of human hepatocyte THLE-2 cells (P < 0.05), and in vivo administration improved the survival of mice with post-hepatectomy liver failure, increasing the median survival time from 22 h to 36 h (P = 0.048)[31]. These findings establish EREG as a potent endogenous mitogen capable of accelerating hepatocyte regeneration.

MSCs have demonstrated therapeutic potential in various liver disease models via paracrine mechanisms. Recent comprehensive reviews have highlighted that MSCs secrete a cocktail of trophic factors, including hepatocyte growth factor, insulin-like growth factor-1, IL-6, and vascular endothelial growth factor, which promote hepatocyte proliferation, suppress fibrosis, and modulate immune responses[32]. Although therapeutic outcomes remain variable, clinical trials have confirmed the safety and efficacy of MSC therapy in decompensated cirrhosis and acute liver failure[32].

The combination of EREG and MSCs is a promising therapeutic strategy. EREG directly stimulates hepatocyte proliferation via the EGFR, which is the key receptor regulating liver regeneration[33]. Meanwhile, MSCs create a favorable microenvironment through their immunomodulatory and antifibrotic effects, as well as through the secretion of additional trophic factors. This multi-targeted approach can overcome the limitations of monotherapy and improve the treatment of liver diseases.

Intestinal tissue and gut microbiota

The molecular mechanisms underlying the function of EREG as an EGFR ligand in the large intestine have been studied extensively[12,13,15,16]. As discussed in the section “Role of EREG in tissue regeneration”, this ligand has significant regenerative capabilities, facilitating the differentiation of intestinal enteroids from intestinal stem cells (ISCs) in vitro (Table 1)[15,16]. However, EREG may also exert pathogenic effects under conditions of excessive inflammation, such as those observed in Crohn’s disease[12]. This suggests that regeneration of the gastrointestinal tract (GIT) poses a particular challenge, with EREG emerging as a crucial factor in restoring the epithelium and maintaining the integrity of the mucosal barrier.

A fundamental characteristic of the intestinal epithelium is its ability to self-renew under homeostatic conditions and to regenerate rapidly following injury. Under physiological conditions, intestinal epithelial homeostasis is primarily maintained through the canonical Wnt signaling pathway in Lgr5+ ISCs[34-36]. Following injury, the regeneration process is regulated by the Hippo signaling pathway, in which the downstream effector Yes-associated protein (YAP) temporarily reprograms Lgr5+ ISCs (Figure 3). YAP suppresses excessive Paneth cell differentiation while simultaneously activating a regenerative program that stimulates stem cell proliferation, enhances cell survival, and activates the EGFR signaling pathway[37,38]. The key mediator of YAP-dependent regeneration is the EGFR ligand EREG. Studies have shown that, in the absence of YAP, exogenous administration of EREG can restore organoid formation and compensate for the loss of YAP function in ISCs[39]. Furthermore, increased expression of stromal EREG is observed around surviving YAP-depleted crypts following radiation-induced damage, indicating the existence of a non-cell-autonomous compensatory mechanism[40]. It is important to note that EREG can modulate signaling pathways in the intestinal epithelium, switching cells between homeostasis and regeneration. The flexibility of signal regulation makes EREG a promising therapeutic target for regenerative medicine and intestinal tissue engineering. This is particularly relevant for the treatment of radiation-induced enteropathy, inflammatory bowel disease, and recovery following surgical resection.

Figure 3 Schematic representation of the mechanisms underlying regeneration of the intestinal epithelium. Left panel (homeostasis): Intestinal epithelial homeostasis is maintained through canonical Wnt signaling in Lgr5+ intestinal stem cells (ISCs) located at the crypt base. R-spondin and Wnt3a, which are produced by the niche, activate the Wnt pathway, thereby supporting ISC self-renewal and differentiation into epithelial lineages, including Paneth cells. Right panel (regeneration): Following injury, the Hippo pathway effector Yes-associated protein (YAP) transiently reprograms Lgr5+ ISCs. This suppresses excessive Paneth cell differentiation and activates a regenerative program that stimulates proliferation, survival, and epidermal growth factor receptor (EGFR) signaling. Epiregulin (EREG) serves as a critical mediator of YAP-dependent regeneration by binding to EGFR on ISCs. Stromal cells surrounding crypts secrete EREG in response to injury, particularly radiation damage, providing non-cell-autonomous compensation that can rescue organoid formation even in YAP-deficient conditions. Central inset: EREG binds to the EGFR receptors on Lgr5+ ISCs, thereby activating downstream signaling cascades. The ability of EREG to regulate the transition between homeostatic and regenerative states makes it a promising therapeutic target for treating radiation enteropathy, inflammatory bowel disease, and post-surgical intestinal restoration. EGFR: Epidermal growth factor receptor; EREG: Epiregulin; ISC: Intestinal stem cell; YAP: Yes-associated protein.

The regenerative potential of the intestine is determined by both the epithelial cells that line the GIT and the resident bacterial communities that are primarily located on the surface of the colonic epithelium[41]. Often considered a virtual endocrine organ, the gut microbiota exerts a systemic effect on the body by synthesizing vitamins, hormones, mediators, and metabolites that cross the intestinal barrier and modulate the functions of distant organs[42]. A key characteristic of the gut is its close symbiotic interaction with a complex bacterial community; any impact on the GIT significantly alters the microbiota composition[43,44]. In this regard, the gut microbiota profile can serve as a biomarker of the efficacy of therapeutic interventions based on MSCs or growth factors such as EREG. Dynamic monitoring of the abundance of individual operational taxonomic units, particularly the symbiotic microorganisms Lactobacillus and Bifidobacterium, allows differentiation between inflammatory and regenerative processes occurring in the large intestine.

Recent studies have shown that the gut microbiota indirectly regulates EREG expression via Toll-like receptor signaling pathways[45]. Dysbiosis is a pathological condition of the gut microbiota characterized by decreased alpha diversity and the replacement of commensal microflora by opportunistic pathogens. The latter synthesize large amounts of lipopolysaccharides (LPS) which, in the presence of increased intestinal permeability, are translocated into the systemic circulation. The activation of Toll-like receptor 4 on hepatocytes by bacterial LPS initiates inflammatory signaling cascades that lead to the pathological overexpression of EREG[46,47].

Conversely, the effects of EREG on the gut microbiota are indirect, operating through the modulation of the intestinal epithelium, as suggested by Childs et al[15,16]. By stimulating the proliferation and differentiation of epithelial cells, EREG helps to restore the barrier function of the colon, thus reducing bacterial translocation and normalizing the composition of the microbiota[15]. Furthermore, the potential of the gut microbiota as a biomarker has been identified. Changes in EREG expression are accompanied by shifts in the Firmicutes/Bacteroidetes ratio, as well as changes in the abundance of probiotic bacteria belonging to the Lactobacillus and Bifidobacterium genera[46]. Therefore, the gut microbiota profile can serve as an indicator of the efficacy of EREG-mediated regenerative therapy in treating intestinal and other diseases.

In recent years, the effects of MSCs on the gut microbiota have received considerable attention. One study demonstrated the therapeutic efficacy of human umbilical cord MSC (hUMSC) therapy in a mouse model of dextran sodium sulphate-induced colitis[48]. Analysis of 16S rRNA sequencing and the metabolomic profile of short-chain fatty acids (SCFAs) revealed that hUMSC therapy normalized the gut microbiota composition to a level comparable to that of the control group (Figure 4). At the phylum level, there was an increase in Firmicutes and a decrease in Bacteroidota and Proteobacteria. At the genus level, the abundance of the potentially symbiotic bacterium Akkermansia increased[49], as did the abundance of the commensal genera Blautia, Anaerotruncus, Lachnospiraceae_NK4A136_group, Faecalibaculum[50], and Clostridia_UCG_014[51]. Conversely, the abundance of Alloprevotella, which was elevated in colitis, decreased. Furthermore, hUMSC therapy was accompanied by increased SCFA production, particularly butyrate, a positive marker of anti-inflammatory and regenerative processes in the colon (Figure 4)[48]. Notably, emerging evidence suggests that the therapeutic efficacy of MSCs can be further enhanced by combining them with prebiotics. For example, novel prebiotic compounds based on poly(3-hydroxybutyrate), a biocompatible and biodegradable microbial polyester, have demonstrated the capacity to favorably modulate the composition of the gut microbiota and stimulate the growth of beneficial bacterial taxa[52]. Combining poly(3-hydroxybutyrate)-based prebiotics with MSC therapy could create an optimized microenvironment for tissue regeneration, supporting beneficial microbial communities while simultaneously enhancing stem cell engraftment and function.

Figure 4 Schematic representation of the effects of human umbilical cord mesenchymal stem cell therapy on the gut microbiota-short-chain fatty acid-immune axis in dextran sodium sulphate-induced colitis. The administration of human umbilical cord mesenchymal stem cells promotes the enrichment of beneficial bacterial genera, such as Akkermansia, Blautia, Faecalibaculum, and Clostridia, while suppressing pathogenic taxa such as Alloprevotella and Proteobacteria. This restored microbial community enhances the production of short-chain fatty acids, particularly butyrate, thereby strengthening the integrity of the epithelial barrier and exerting anti-inflammatory effects. Adapted from Liu et al[48]. hUMSCs: Human umbilical cord mesenchymal stem cells; MSCs: Mesenchymal stem cell; SCFAs: Short-chain fatty acids.

A similar study demonstrated that mice with systemic lupus erythematosus, a condition characterized by autoimmune and inflammatory reactions, exhibit significant gut microbiota dysbiosis[53]. In this study, the transplantation of hUMSCs into systemic lupus erythematosus-affected animals led to an increase in the abundance of the symbiotic bacteria Lactobacillus johnsonii[54], Romboutsia (R. ilealis, R. timonensis, and R. hominis)[55], and Acetobacter[56]. These bacteria produce SCFAs and tryptophan metabolites, which contribute to the restoration of the intestinal barrier integrity[53]. These findings suggest that the gut microbiota plays an important role in mediating the therapeutic effects of MSCs. In combination with EREG, MSCs may exert synergistic effects by creating a favorable niche for restoring the physiological composition of the gut microbiota. Monitoring these microbial changes may therefore serve as a biomarker of treatment efficacy.

THERAPEUTIC PROSPECTS

One of the key limitations of tissue engineering is the disruption to the functional activity of stem cells in a pathologically altered microenvironment, particularly in cases of chronic inflammation. One promising approach to overcoming this barrier is the use of combined therapeutic strategies involving MSCs and growth regulatory factors. EREG, in particular, has been shown to restore the proliferative and migratory potential of stem cells in an inflammatory environment, as demonstrated in a study by Zhao et al[1].

A promising approach to combined therapy involves co-encapsulating MSCs and rhEREG in biocompatible, biodegradable hydrogels (Figure 5). Over the past 5 years, accumulating evidence has supported the effectiveness of such delivery systems for both the individual components and their combinations[57-59]. For instance, one study developed a composite hydrogel based on type B gelatin and polyglutamic acid with immobilized EREG [200 ng/mL; EREG-loaded hydrogel (HG-Epi)][57]. In vivo testing in a rat skin wound model revealed accelerated epithelialization compared with the control group, which was attributed to enhanced keratinocyte proliferation and extracellular matrix remodeling[57].

Figure 5 Schematic illustration of the epiregulin-mesenchymal stem cell-hydrogel therapeutic platform for bone/periodontal regeneration. Biodegradable hydrogel scaffolds serve as a delivery system for mesenchymal stem cells (MSCs) and epiregulin (EREG). EREG binds to epidermal growth factor receptors on MSCs, activating mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2, and phosphatidylinositol 3-kinase/protein kinase B signaling pathways that promote cell proliferation, migration, and osteogenic differentiation. The synergistic actions of the MSC secretome and exogenous EREG modulate the inflammatory microenvironment (M1→M2 macrophage polarization), stimulate angiogenesis, and enhance deposition of mineralized extracellular matrix. This integrated approach enables targeted regeneration of bone and periodontal tissues in inflammatory conditions. Akt: Protein kinase B; EGFR: Epidermal growth factor receptor; EREG: Epiregulin; ERK1/2: Extracellular signal-regulated kinase 1/2; MAPK: Mitogen-activated protein kinase; PI3K: Phosphatidylinositol 3-kinase.

This mechanism was further validated by in vitro experiments using the HaCaT cell line, in which EREG stimulated the proliferation of epithelial cells and increased the expression of keratins, as well as matrix metalloproteinase 2 and 9, which are critical for cell migration and dynamic stromal remodeling[60]. However, the dose-dependent effect of EREG is also a critical factor. While studies such as that by Migliario and Renò[57] have demonstrated the effectiveness of 200 ng/mL EREG in hydrogels for skin wound healing, the optimal concentration for bone and periodontal tissue regeneration may differ significantly. EGFR signaling often demonstrates a biphasic response: Insufficient EREG levels fail to trigger osteogenesis, while excessive concentrations can lead to aberrant signaling, fibrosis, or even tumor formation[61]. Long-term safety also remains a concern, as sustained excessive activation of the EGFR pathway has been associated with hyperplastic conditions and malignant neoplasms in epithelial tissues. Therefore, it is important to establish a therapeutic window that maximizes regeneration without inducing pathological proliferation for successful clinical application.

Rizzi et al[62] reported a promising strategy for EREG delivery using poly(lactide-co-glycolide) (PLGA) nanoparticles as a carrier instead of hydrogels. The local delivery of EREG from PLGA nanoparticles to HaCaT keratinocytes stimulated cell proliferation, as opposed to the control group. In vitro experiments on keratinocytes and ex vivo experiments on tooth enamel revealed that the degradation of 50:50 PLGA nanoparticles, combined with their adhesive properties, makes this system a potential tool for directly delivering EREG to the periodontal pocket to stimulate epithelialization in cases of gum recession[62]. Meanwhile, the work of Childs et al[15,16] demonstrated the formation of full-fledged cellular organoids, in which EREG plays a pivotal role in the differentiation of complex human intestinal organoids. In particular, EREG was shown to regulate the differentiation of five major cellular components: The epithelium with its crypts and villi; the mesenchyme; the enteric glial network; the endothelial cells; and the organized smooth muscle. This leads to the formation of structurally complete organoids[16]. Here, EREG acts as a paracrine/autocrine modulator, activating the EGFR in the epithelium and indirectly stimulating the differentiation of mesenchymal and neural precursors[16]. Thus, in the studies by Childs et al[15,16], EREG acts as an “organizing” factor during intestinal tissue development. Unlike other EGFR ligands, particularly EGF, it provides physiologically accurate regulation of intestinal development and homeostasis at the epithelial and organ complexity levels. These findings open up new avenues for tissue engineering therapy.

The therapeutic effect of MSCs in various pathologies, ranging from autoimmune and inflammatory diseases to orthopedic injuries, is primarily mediated by their secretome rather than by their differentiation into tissue-specific cells. MSC secretions include growth factors (such as EREG), anti-inflammatory cytokines (such as IL-10 and transforming growth factor β), and extracellular vesicles containing regulatory microRNAs. These molecules can modulate the local microenvironment by stimulating the proliferation and migration of resident progenitor cells, inducing angiogenesis, polarizing macrophages toward the anti-inflammatory M2 phenotype (Figure 5), and suppressing fibrosis and apoptosis[28].

However, the heterogeneity of MSCs affects their sensitivity to growth factors, necessitating source-specific adjustments. Integrating rhEREG with MSCs into a single therapeutic platform, such as a hydrogel, enables the synergistic enhancement of tissue regenerative potential. This is achieved by directly stimulating epithelial and stromal cell proliferation via EGFR-dependent pathways, optimizing the functional activity of the MSCs in an inflammatory microenvironment and prolonging the local delivery of both components. Carrier immunocompatibility and degradation kinetics should match tissue regeneration rates. For instance, the acidic degradation products of PLGA may counteract the benefits of MSCs, favoring smart, enzyme-responsive systems.

This approach shifts the focus from traditional cell replacement therapy to “smart” regenerative medicine, where the emphasis is on the targeted modulation of the microenvironment through secretions and exogenous bioactive molecules rather than on cell transplantation itself. Nevertheless, successful clinical translation requires the validation of long-term stability, potential off-target effects, and carrier resorption.

CONCLUSION

Thus, the molecular mechanisms of EREG involvement in inflammatory diseases under the influence of MSCs provide an experimental basis for the clinical development of combined regenerative biomaterials, opening up opportunities for the development of standardized therapeutic systems capable of accelerating the translation of tissue engineering achievements into clinical practice.

ACKNOWLEDGEMENTS

This work was supported in part by the Russian Science Foundation (Grant No. 25-26-00587) for the section “Intestinal tissue and gut microbiota”. All other sections was carried out within the framework of government assignment of the Ministry of Science and Higher Education of the Russian Federation through the Research Center of Biotechnology of the Russian Academy of Sciences.