Inflammation-driven ISC damage

The innate immune system of the preterm intestine is underdeveloped and characterized by unusually high expression of pro-inflammatory genes, such as Toll-like receptor 4 (TLR4) (Ref. Reference Nanthakumar, Meng, Goldstein, Zhu, Lu, Uauy, Llanos, Claud and Walker10) and low expression of negative regulatory factors like IκB (Ref. Reference Claud, Lu, Anton, Savidge, Walker and Cherayil11). After excessive activation of TLR4, the receptor recruits and activates TIR-domain-containing adaptor-inducing interferon-β (TRIF) through the intracellular adaptor protein TRIF-related adaptor molecule (TRAM). Activation of TRIF subsequently induces the expression of the pro-apoptotic molecule p53-upregulated modulator of apoptosis (PUMA). PUMA belongs to the BH3-only subgroup of the BCL-2 family and is primarily involved in the regulation of the mitochondria-dependent intrinsic apoptotic pathway (Ref. Reference Vogler, Braun, Smith, Westhoff, Pereira, Pieper, Anders, Callens, Vervliet, Abbas, Macip, Schmid, Bultynck and Dyer12). Following activation of the TLR4–TRIF signalling pathway, increased PUMA expression promotes the activation of the pro-apoptotic proteins BAX and BAK, leading to increased mitochondrial membrane permeability, cytochrome c release, and subsequent activation of caspase-9 and caspase-3, ultimately inducing ISC apoptosis. Meanwhile, elevated PUMA expression can also inhibit the proliferative capacity of crypt cells, resulting in reduced intestinal epithelial renewal (Ref. Reference Dirisina, Katzman, Goretsky, Managlia, Mittal, Williams, Qiu, Yu, Chandel, Zhang and Barrett13). Related studies have shown that PUMA knockdown or genetic deletion of PUMA can block lipopolysaccharide (LPS)-induced crypt cell apoptosis and restore ISC proliferative capacity, further demonstrating that PUMA is a critical downstream molecule mediating TLR4-induced ISC dysfunction. Therefore, during the development of NEC, excessive microbial stimulation may activate the TLR4–TRIF–PUMA signalling axis on the surface of ISCs, leading to ISC depletion and reduced regenerative capacity, ultimately promoting crypt repair failure and persistent intestinal barrier dysfunction (Ref. Reference Neal, Sodhi, Jia, Dyer, Egan, Yazji, Good, Afrazi, Marino, Slagle, Ma, Branca, Prindle, Grant, Ozolek and Hackam14).

The harmful effects of TLR4 hyperactivation on intestinal regeneration are complex. Besides indirectly suppressing the regeneration of ISCs through the TRIF-PUMA axis, the resulting inflammatory environment – especially high TNF-α levels – has direct toxic effects on Lgr5+ ISCs, which is a key mechanism of ISC failure in NEC. The TNF-α-driven inflammatory environment is highly toxic to Lgr5+ ISCs at the crypt base. These ISCs are essential for maintaining the epithelium and repairing tissue after injury, and their self-renewal and proliferation depend heavily on Wnt/β-catenin signalling (Ref. Reference Sato, Vries, Snippert, van de Wetering, Barker, Stange, van Es, Abo, Kujala, Peters and Clevers15). Under conditions of sustained TNF-α exposure, ISC survival and function are severely compromised. Studies show that epithelial injury caused by TNF-α decreases Lgr5+ ISC numbers, and restoring epithelial health requires reactivating Wnt/β-catenin signalling to conserve and expand the ISC pool (Ref. Reference Wu, Xie, Miao, Li, Wang, Wang and Yu16). These findings strongly suggest that ongoing inflammation in NEC, especially high TNF-α levels, directly impairs ISC regenerative capacity, making mucosal injury difficult to repair and more likely to worsen.

Ischemia–hypoxia-caused metabolic stress and mitochondrial damage

Intestinal ischemia is a major trigger for preterm NEC. A hypoxic environment influences disease progression through the dual roles of hypoxia-inducible factor-1α (HIF-1α), while ISC regenerative ability is central to mucosal repair. Regulation of ISC regeneration by HIF-1α is a key mechanism in intestinal repair. Ischemic preconditioning (IPC) activates HIF-1α and works together with Wnt/β-catenin signalling to increase ISC markers like Lgr5 and Msi1 by boosting crypt regenerative capacity and enhancing organoid-forming efficiency (Ref. Reference Chen, Lee, Tsai and Tseng17). Collectively, HIF-1α is involved in NEC development through specific dual actions and coordinates ISC regeneration by regulating multiple pathways.

Dysbiosis and the loss of microbial metabolites

Regulation of ISC fate by the intestinal microbiota is mainly indirect, operating through remodelling of the stem cell niche and influencing metabolic signalling pathways. ISC self-renewal and lineage specification rely on coordinated interactions among macrophages, mesenchymal niche cells (MNCs), and Paneth cells. Commensal microbes encourage monocytes to differentiate into CD206+ M2 macrophages; these macrophages support niche cell proliferation and provide essential cues for ISC differentiation towards the Paneth lineage (Ref. Reference Kim, Li, Fei, Horne, Lee, Loe, Miyake, Ayar, Kim, Surette, Philpott, Sherman, Guo, Pierro and Kim18). In addition, Paneth cell maturation and functional maintenance are also highly dependent on microbial regulation. Beneficial bacteria like Lactobacilli improve Paneth cell function via the CD206+ macrophage–Wnt signalling axis, while Paneth cells, in turn, secrete Wnt/Notch ligands and antimicrobial peptides to further stabilize the ISC niche, creating the microenvironment necessary for ISC fate regulation (Refs Reference Kim, Li, Fei, Horne, Lee, Loe, Miyake, Ayar, Kim, Surette, Philpott, Sherman, Guo, Pierro and Kim18, Reference Yang and Shi19).

Microbial metabolites are another major group of ISC regulatory mediators, with lactate playing a particularly important role. Lactate produced by bacteria such as lactobacilli binds specifically to G protein–coupled receptor 81 (Gpr81) on Paneth and stromal cells, activates Wnt3/β-catenin signalling, and significantly promotes Lgr5+ ISC proliferation and differentiation towards the Paneth lineage. In contrast, Lactobacillus plantarum strains lacking lactate dehydrogenase, and thus unable to produce lactate, fail to induce ISC expansion, highlighting the central regulatory role of lactate (Refs Reference Salzman, Underwood and Bevins20, Reference Lee, Kim, Kim, Lee, Kim, Kang, Yang, Baek, Sung, Park, Hwang, Kim, Liu, Kamada, Gao and Kweon21). Another key metabolite, butyrate, has developmentally dependent effects on ISCs: in the metabolically immature intestine of preterm infants, high concentrations of butyrate directly damage ISCs by activating p-MLKL-mediated necroptosis and disrupting the balance between proliferation and differentiation, whereas in the mature intestine, butyrate slightly modulates ISC proliferation to maintain epithelial homeostasis (Ref. Reference Wang, Tao, Salimi-Jazi, Lin, Sun, Liu, Sinclair, Mostaghimi, Dunn and Sylvester22).

Activation of reserve stem cells and cellular plasticity

Injury sensing and initiation of repair signalling

Role of Wnt signalling in ISC proliferation and regeneration in NEC

The Wnt/β-catenin pathway forms the central molecular axis responsible for maintaining crypt architecture and supporting ISC self-renewal, and it is especially important during postnatal intestinal development and injury repair (Ref. Reference Kriz and Korinek44). Growing evidence shows that this pathway is suppressed at multiple levels during NEC, leading to a significant decline in intestinal regenerative capacity.

In experimental NEC mouse models and intestinal samples from infants with acute NEC, the number of Lgr5+ ISCs is significantly decreased, nuclear translocation of β-catenin is hindered, and the expression of key Wnt pathway genes is downregulated, collectively indicating a broad reduction in Wnt signalling activity. Organoid studies further show that cultures derived from NEC-injured intestines have impaired long-term growth, fewer buds, and abnormal differentiation. In contrast, adding exogenous Wnt7b restores organoid growth and rebuilds crypt-like structures. These findings confirm that defective Wnt signalling is a core mechanism behind regenerative failure in NEC (Ref. Reference Li, Lee, Cadete, Zhu, Koike, Hock, Wu, Botts, Minich, Alganabi, Chi, Zani-Ruttenstock, Miyake, Chen, Mutanen, Ngan, Johnson-Henry, De Coppi, Eaton, Maattanen, Delgado-Olguin, Sherman, Zani and Pierro45).

Compared with adult intestine, the immature neonatal intestine has limited compensatory capacity and relies more heavily on appropriate Wnt signalling input. Consequently, even moderate suppression of Wnt signalling can quickly deplete the ISC pool and lead to irreversible regenerative failure.

The biological effects of Wnt signalling are not determined by individual ligands alone but result from coordinated regulation by Wnt ligands, R-spondins (RSPOs), and niche-derived signals. Wnt ligands mainly provide a permissive signal that allows ISCs to respond to RSPOs. In contrast, RSPO acts as the primary amplifier driving ISC expansion and determining stem cell pool size (Ref. Reference Yan, Janda, Chang, Zheng, Larkin, Luca, Chia, Mah, Han, Terry, Ootani, Roelf, Lee, Yuan, Li, Bolen, Wilhelmy, Davies, Ueno, von Furstenberg, Belgrader, Ziraldo, Ordonez, Henning, Wong, Snyder, Weissman, Hsueh, Mikkelsen, Garcia and Kuo46). In NEC, reduced Wnt activity may therefore not only directly suppress ISCs but also indirectly weaken the RSPO–LGR5 amplification axis, thus worsening regenerative defects.

Notch signalling balances ISC proliferation and differentiation

The Notch pathway is a key regulatory system that controls ISC homeostasis and maintains the integrity of the crypt proliferative zone. In a healthy intestine, Notch receptors (Notch1–4) are activated when they bind to Delta-like or Jagged ligands on neighbouring cells, causing the Notch intracellular domain (NICD) to be released. The NICD then moves to the nucleus and forms a transcription complex with RBPJ and members of the Mastermind-like (MAML) family to regulate target gene expression (Refs Reference Liang, Li and Wang47, Reference Wang, Zang, Liu and Aster48).

In crypt base columnar (CBC) stem cells, Notch signalling also has direct effects. Pharmacologic or genetic inhibition of Notch causes rapid depletion of CBC cells, increased apoptosis, and significantly lowers organoid-forming efficiency. In contrast, the transcription of the stem cell–specific marker Olfm4 directly depends on activation of the Notch–RBPJ axis (Ref. Reference VanDussen, Carulli, Keeley, Patel, Puthoff, Magness, Tran, Maillard, Siebel, Kolterud, Grosse, Gumucio, Ernst, Tsai, Dempsey and Samuelson49). Therefore, Notch signalling acts as a key factor in maintaining ISCs and preserving the integrity of the crypt proliferative zone.

Beyond promoting proliferation, Notch signalling controls epithelial cell fate decisions by preventing early activation of secretory lineage programs. Atoh1 (also called Math1) is the key transcription factor that drives secretory lineage commitment (Ref. Reference Santos, Lo, Mah and Kuo50)and its removal causes a complete loss of goblet, Paneth, and enteroendocrine cells. Meanwhile, absorptive enterocytes are mostly unaffected (Ref. Reference Yang, Bermingham, Finegold and Zoghbi51).

In NEC, the immaturity of the preterm intestine makes the Notch–Atoh1 axis especially vulnerable to inflammatory and microenvironmental disturbances. When Notch signalling is suppressed or disrupted, progenitor cells prematurely and excessively differentiate into secretory lineages, especially goblet cells. This results in a lower proportion of functional absorptive epithelial cells and hinders barrier repair (Refs Reference Pellegrinet, Rodilla, Liu, Chen, Koch, Espinosa, Kaestner, Kopan, Lewis and Radtke52, Reference van Es, van Gijn, Riccio, van den Born, Vooijs, Begthel, Cozijnsen, Robine, Winton, Radtke and Clevers53). Such lineage skewing is particularly harmful in NEC, where rapid replacement of mature epithelial cells with barrier and transport functions is critically needed during early disease stages.

EGFR signalling promotes epithelial migration and proliferation

Disruption of the intestinal epithelial barrier is a key feature of NEC, evidenced by increased permeability, damage to the mucosal structure, and unregulated inflammatory responses. EGF signalling helps repair the barrier and restore function through multiple coordinated mechanisms. Exogenous EGF administration reverses these pathological changes by binding to EGFR on intestinal epithelial cells. On one hand, EGF upregulates the transcription factor Atoh1, accelerates goblet cell maturation, restores MUC2 production, and strengthens the mucus barrier; on the other hand, it remodels tight junction architecture, normalizes occludin and claudin-3 expression and localization, and significantly reduces intestinal permeability (Refs Reference Clark, Doelle, Halpern, Saunders, Holubec, Dvorak, Boitano and Dvorak54–Reference Dvorak, Halpern, Holubec, Williams, McWilliam, Dominguez, Stepankova, Payne and McCuskey56). Additionally, EGF modulates epithelial homeostasis by increasing anti-apoptotic Bcl-2 and decreasing pro-apoptotic Bax, thereby limiting excessive epithelial apoptosis, while simultaneously suppressing pro-inflammatory cytokines such as IL-18 and TNF and upregulating IL-10. This collectively reinforces barrier repair and immune balance (Ref. Reference Nair, Warner and Warner55).

In the specific pathological context of NEC, hypoxia and inflammation directly damage ISCs and disrupt crypt architecture. Under these conditions, EGF signalling serves as a vital natural protective mechanism. Heparin-binding EGF-like growth factor (HB-EGF) effectively shields all intestinal epithelial lineages in neonatal rat NEC models, reduces crypt cell apoptosis, and supports ISC survival and activation after injury. These effects rely on EGFR activation and are mediated via downstream MEK1/2 and PI3K signalling pathways, which increase cellular resistance to apoptosis and maintain the integrity of the crypt proliferative zone. These findings have been further confirmed in isolated ISC and organoid models, where HB-EGF directly reduces hypoxia-induced injury and encourages TA cell expansion, confirming the direct protective and regenerative roles of EGF family members on the stem cell compartment (Ref. Reference Chen, Yu, James, Zhang, Yang, Radulescu, Zhou and Besner57).

Beyond its primary epithelial protective effects, HB-EGF also influences the enteric nervous system (ENS) and exogenous stem cells, thereby broadening the functional range of EGF signalling in NEC regeneration. NEC is linked to the loss of neuronal nitric oxide synthase (nNOS)–positive neurons within the ENS, whereas HB-EGF encourages the differentiation of enteric neural stem cells (NSCs) into neurons, preserves myenteric plexus architecture in a nitric oxide–dependent manner, decreases neuronal apoptosis, and enhances intestinal dysmotility (Ref. Reference Zhou, Wang, Olson, Yang and Besner58). These effects are closely associated with ErbB receptor signalling and Nardilysin-mediated pathways, which sustain ENS integrity and indirectly support intestinal homeostasis (Ref. Reference Wei, Zhou and Besner59).

Crosstalk among core regenerative pathways in NEC

The proliferation and regeneration of ISCs are not independently controlled by a single signalling pathway but are regulated by an integrated network of multiple signalling cascades. As a central regulator of ISC stemness and proliferative capacity, Wnt signalling not only directly promotes ISC expansion but also serves as an important regulatory hub connecting different signalling pathways. Activation of Wnt/β-catenin signalling induces the expression of stem cell-associated genes, including Lgr5 and Ascl2, thereby maintaining the undifferentiated state of ISCs (Ref. Reference Kriz and Korinek44). Meanwhile, Wnt signalling can enhance Notch activity by regulating intercellular communication within the crypt. For example, Wnt activation increases the expression of Notch ligands, such as Dll1 and Dll4, thereby enhancing the responsiveness of ISCs and neighbouring progenitor cells to Notch signalling and maintaining a highly proliferative state (Ref. Reference Nauman and Stanley60). Therefore, Wnt signalling not only directly regulates ISC proliferative capacity but also shapes an appropriate crypt microenvironment through modulation of Notch signalling input, thereby supporting continuous ISC renewal (Ref. Reference Beumer and Clevers61).

Notch signalling further regulates ISC fate determination based on the Wnt-driven proliferative program and establishes a reciprocal regulatory relationship with Wnt signalling. Upon activation, Notch signalling releases the Notch intracellular domain (NICD), which translocates into the nucleus and induces the expression of downstream target genes, such as Hes1. This process maintains the proliferative state of ISCs and early progenitor cells while suppressing Atoh1-mediated differentiation towards secretory lineages (Ref. Reference Liang, Li and Wang47). However, Notch signalling is not merely a molecular ‘switch’ determining cell fate; it also maintains the undifferentiated state of ISCs and enhances cellular responsiveness to Wnt signalling, thereby stabilizing the Wnt-driven stem cell maintenance program (Ref. Reference Wu, Yu, Yu, Wu, Yuan, Duan, Cai, Xiong, Lin and Guo62). Thus, Wnt and Notch form a bidirectional regulatory relationship: Wnt promotes Notch activation to maintain the proliferative crypt environment, whereas Notch preserves ISC sensitivity to Wnt stimulation, together ensuring the stability of the ISC pool.

Building on the Wnt–Notch regulatory network, EGF signalling further contributes to ISC expansion and epithelial renewal. EGF primarily promotes cellular proliferation, migration, and survival; however, its effects depend on the pre-existing stemness state and proliferative potential of ISCs (Ref. Reference Clark, Doelle, Halpern, Saunders, Holubec, Dvorak, Boitano and Dvorak54). In other words, Wnt and Notch signalling determine the capacity of ISCs to sustain proliferation and regeneration, whereas EGF signalling further amplifies this proliferative response by promoting progenitor expansion and epithelial structural recovery. Therefore, EGF does not independently drive ISC regeneration but instead exerts a synergistic effect based on Wnt-mediated maintenance of stemness and Notch-mediated regulation of ISC state.

During the development of NEC, inflammatory stimulation and disruption of the crypt microenvironment can disturb the balance of the Wnt–Notch–EGF signalling network, thereby impairing ISC maintenance and regenerative capacity. Excessive TLR4 activation not only directly induces ISC injury but also suppresses Wnt/β-catenin activity through inflammatory cytokine production, weakening ISC stemness maintenance and reducing cellular responsiveness to Notch signalling (Ref. Reference Zhao, Ravikumar, Leach, Kraushaar, Lauder, Li, Sun, Oravecz-Wilson, Keller, Chen, Maneix, Jenq, Britton, King, Santibanez, Creighton, Rao and Reddy63). Meanwhile, Paneth cell dysfunction and disruption of the intestinal stem cell niche further decrease the availability of supportive signals, including Wnt ligands, Notch ligands, and EGF, thereby impairing ISC proliferation, differentiation, and repair processes (Ref. Reference Quintero and Samuelson64). Because EGF-mediated regenerative effects rely on an intact Wnt–Notch regulatory network, disruption of this signalling balance limits the ability of EGF to effectively promote epithelial regeneration. Therefore, ISC dysfunction in NEC is not caused by abnormalities in a single signalling pathway but results from the combined effects of inflammatory injury signals and disruption of regenerative signalling networks, ultimately leading to ISC depletion, impaired crypt regeneration, and defective intestinal mucosal repair (Figure 1).

Figure 1.

Intestinal stem cell injury and regenerative repair in necrotizing enterocolitis. A. NEC-associated insults induce TLR4-centered inflammatory and metabolic injury, leading to ISC depletion and regenerative failure. B. Reserve stem cells, epithelial plasticity, and pro-regenerative signals initiate endogenous epithelial repair. C. Crosstalk among major regenerative pathways coordinates ISC stemness, proliferation, and injury-induced reprogramming. D. Recovery of the epithelial barrier and stem-cell niche restores crypt–villus architecture and mucosal homeostasis. The pattern diagram was drawn using the Figdraw (https://www.figdraw.com).

Figure 1. Long description

At the center of the diagram is a circular anchor labeled Intestinal stem cell, showing a U-shaped crypt structure. Four colored arrows point outward to four distinct panels.

Panel A (Top-Left, Red): N E C-associated I S C injury. Triggers include Prematurity, Formula feeding, Dysbiosis, and Ischemia/Hypoxia (O sub 2). These lead to T L R 4 overactivation, which branches into four pathways:

1. T R I F-P U M A axis leading to I S C-apoptosis.

2. N F-kappa B / T N F-alpha / I L-1 beta leading to inflammatory epithelial injury via macrophage cytokine release.

3. Decreased A K T / increased G S K-3 beta / beta-catenin degradation leading to reduced Wnt signaling.

4. Hypoxia / H I F-1 alpha / mitochondrial stress leading to dysbiosis and metabolite imbalance.

Below these, Niche disruption is shown through Paneth cell dysfunction, decreased Wnt ligands (Wnt3, R S P O), decreased Notch ligands (D l l 1), and decreased E G F support, culminating in Lgr5 plus I S C depletion and regenerative failure.

Panel B (Top-Right, Blue): Initiation of endogenous repair. Four boxes show repair signals: Bmi1 plus / Hopx plus reserve stem cells; Epithelial plasticity and dedifferentiation (D l l 1 plus progenitors to Y A P / T A Z activation); I L-22 / S T A T 3 signaling; and P G E sub 2 / E P 4 signaling. These converge on Reactivation of regeneration, showing a crypt where cells undergo dedifferentiation/reprogramming to become Lgr5 plus stem cells, followed by regeneration/differentiation.

Panel C (Bottom-Left, Green): Core regenerative pathway crosstalk. A network diagram shows T L R 4 / inflammatory disruption inhibiting Wnt and B M P. Wnt, Notch, and E G F / E G F R positively regulate I S C stemness and proliferative competence. Y A P / T A Z provides transient injury-induced reprogramming. B M P acts as a late-stage brake to restrain excessive regeneration.

Panel D (Bottom-Right, Purple): Late-stage tissue remodeling and niche reconstruction. Four pillars of recovery are listed:

1. Barrier restoration (Z O-1, Claudin, Occludin).

2. Mucus layer recovery (goblet cell, Muc2).

3. Paneth cell niche recovery (Wnt3, E G F, Notch ligands).

4. Multicellular niche reconstruction (Reparative macrophages releasing I L-10 and T G F-beta, and Mesenchymal stromal cells providing Wnt-associated signals).

These result in restored crypt-villus architecture and mucosal homeostasis.

BMP-mediated termination of regeneration and restoration of crypt–villus homeostasis

During the late phase of repair after NEC injury, the intestinal epithelium must not only undergo rapid regeneration but also promptly stop excessive stem cell proliferation to restore the normal crypt–villus structure and epithelial balance. Bone morphogenetic protein (BMP) signalling is recognized as a key inhibitory pathway that helps limit intestinal stem cell overactivation and directs tissue remodelling at this stage. Mechanistically, BMP signalling decreases the expression of stemness-related genes such as Lgr5 through Smad-dependent transcriptional regulation, thus reducing ISC identity. Importantly, this process occurs independently of the canonical Wnt/β-catenin pathway; instead, Smad1/Smad4 recruit histone deacetylase HDAC1 to target gene promoters, creating a repressive chromatin state that directly limits sustained activation of stem cell programs (Ref. Reference Qi, Li, Zhao, Xu, Liu, Li, Zhang, Wang, Yang, Xie, Li, Han and Chen65).

At the tissue architectural level, BMP signalling is also crucial for preventing abnormal crypt formation. Blocking BMP signalling, such as through transgenic expression of noggin, causes widespread ectopic crypt development, often misaligned with the normal crypt–villus axis, eventually leading to pathological changes similar to juvenile polyposis in humans (Ref. Reference Haramis, Begthel, van den Born, van Es, Jonkheer, Offerhaus and Clevers66). These results show that during the late repair phase of NEC, BMP signalling not only reduces stem cell numbers but also maintains spatial organization, helping to prevent abnormal crypt growth and harmful remodelling.

Although BMP-mediated suppression of stemness does not directly rely on Wnt/β-catenin signalling, the dynamic interaction between BMP and Wnt pathways is crucial for preventing unchecked proliferation during tissue recovery. This balance is maintained through the phosphatase and tensin homolog (PTEN)–phosphoinositide 3-kinase (PI3K)–Akt signalling cascade, which acts as a molecular link between these pathways. Mechanistically, BMP signalling modulates PTEN activity, thereby reducing PI3K–Akt signalling, suppressing Wnt/β-catenin activity, and limiting nuclear accumulation of β-catenin. Through this mechanism, BMP signalling functions as a molecular ‘brake’ on ISC self-renewal during late-stage regeneration (Ref. Reference He, Zhang, Tong, Tawfik, Ross, Scoville, Tian, Zeng, He, Wiedemann, Mishina and Li67). BMP signalling thus plays a dual role by directly repressing stemness-related transcriptional programs and indirectly restraining proliferative signalling via pathway crosstalk, ultimately protecting crypt architecture and tissue integrity.

Remodelling of the epithelial microenvironment: barrier restoration, mucus layer integrity, and Paneth cell niche recovery

Beyond intrinsic stem cell signalling, late-stage tissue remodelling after NEC critically depends on thorough reconstruction of the epithelial microenvironment. During the acute phase of NEC injury, excessive inflammatory responses, oxidative stress, and microbial invasion not only directly damage Lgr5+ ISCs, but also disrupt the complex cellular interactions within the crypt microenvironment and impair epithelial barrier integrity. Consequently, successful intestinal repair extends beyond epithelial regeneration and requires coordinated reconstruction of a functional regenerative microenvironment, including restoration of epithelial junctional integrity, recovery of the mucus barrier, and re-establishment of an ISC-supportive niche (Refs Reference Chen, Ye, Seidler, Tian and Xiao68, Reference Duckworth69).

Restoration of the intestinal epithelial barrier represents a critical component of late-stage tissue remodelling after NEC injury. During NEC development, inflammation-mediated epithelial apoptosis and disruption of junctional complexes increase intestinal permeability, facilitating the translocation of bacteria and microbial products across the epithelial barrier, thereby amplifying local inflammation and impairing ISC recovery. During the repair phase, reestablishing tight junction protein placement is essential for restoring barrier function (Ref. Reference Yang, Wei, Zhong, Guo, Liu, Wang and Tu70). Recovery of junctional proteins, including occludin, claudin family members, and zonula occludens-1 (ZO-1), reduces intestinal permeability and restricts microbial penetration into the crypt region, thereby providing a protective environment for ISCs and their surrounding niche. Studies show that internalization of tight junction proteins and increased intestinal permeability often occur before NEC begins. Conversely, stabilizing claudins and occludin at junctions during recovery helps prevent microbial translocation and lowers the risk of disease progression (Ref. Reference Bergmann, Liu, Tian, Kushnir, Turner, Li, Chou, Weber and De Plaen71).

At the same time, restoring the intestinal mucus layer is a crucial part of late-stage homeostatic recovery. The thick inner mucus layer created by MUC2 efficiently separates luminal bacteria from the epithelial surface, and damage to this layer allows bacteria to invade the crypts and triggers inflammatory responses (Ref. Reference Johansson, Phillipson, Petersson, Velcich, Holm and Hansson72). In NEC, dysfunction of goblet cells and impaired mucus production compromise this protective barrier, facilitating microbial proximity to the crypt region and potentially disrupting ISC function. Thus, restoration of goblet cell maturation and mucus-secreting capacity is essential not only for epithelial barrier recovery but also for limiting sustained microbial perturbation of the ISC niche. Restoring this barrier is therefore vital because it provides a physical protection that shields ISCs and their niche from microbial intrusion.

In addition, Paneth cells, as essential components of the niche for Lgr5+ ISCs, play a crucial role in restoring crypt homeostasis during repair. These cells not only regulate microbial composition through antimicrobial peptide secretion but also provide vital signals for stem cell maintenance, including EGF, Wnt3, and Notch ligands (Ref. Reference Sato, van Es, Snippert, Stange, Vries, van den Born, Barker, Shroyer, van de Wetering and Clevers73). Loss of Paneth cell function mimics NEC-related microbial imbalance and epithelial damage (Ref. Reference Lueschow, Stumphy, Gong, Kern, Elgin, Underwood, Kalanetra, Mills, Wong, Meyerholz, Good and McElroy74), emphasizing the importance of restoring the Paneth cell–ISC axis as a key yet often underestimated part of late-stage NEC tissue remodelling.

Beyond Paneth cells, restoration of the ISC niche requires coordinated interactions among mesenchymal stromal cells and immune populations (Ref. Reference Sun, Tan, Chen, Wu and Su75). Intestinal mesenchymal cells serve as critical regulators of ISC homeostasis by supplying niche-derived factors, including Wnt ligands, R-spondins, and BMP modulators, which support stem cell maintenance and crypt architecture. In parallel, immune cells, particularly macrophages, actively participate in tissue remodelling through the regulation of inflammatory resolution and secretion of regenerative mediators. During NEC recovery, the conversion of a pro-inflammatory environment into a reparative microenvironment is essential for rebuilding a functional ISC-supportive network. Reparative macrophages, for instance, release IL-10, TGF-β, and Wnt-associated signals that promote epithelial recovery and stabilize the stem cell niche. Collectively, late-stage repair after NEC represents a complex multicellular ecosystem reconstruction process involving epithelial cells, Paneth cells, mesenchymal stromal cells, and immune components, rather than a simple restoration of ISC abundance (Ref. Reference England, Lin, Sigal and Leedham76).

Enhancing ISC regenerative signalling

ISC self-renewal and injury-induced regeneration are governed by multiple highly coordinated signalling pathways, among which the Wnt, Notch, and EGFR signalling axes play critical roles in maintaining crypt architecture and promoting epithelial recovery. Therefore, restoring or enhancing ISC-associated regenerative pathways may represent a promising strategy for facilitating intestinal mucosal repair.

The Wnt/β-catenin signalling pathway is a central regenerative pathway that regulates ISC self-renewal, proliferation, and crypt formation. Previous studies have demonstrated that enhancing Wnt signalling or supplementing Wnt pathway agonistic factors such as R-spondin can promote intestinal organoid expansion and maintain ISC function, suggesting that modulation of the Wnt pathway may represent a promising strategy for promoting ISC recovery after NEC (Refs Reference Harnack, Berger, Antanaviciute, Vidal, Sauer, Simmons, Meyer and Sigal77, Reference Zhou, Zan, Zhu, Gao, Yan and Wang78). In recent years, human milk-derived exosomes have also been shown to possess potential regulatory effects on intestinal epithelial repair. Some studies suggest that these exosomes may promote intestinal epithelial cell survival and regeneration through activation of the Wnt/β-catenin signalling pathway and may contribute to ISC maintenance by improving the local regenerative microenvironment (Refs Reference Gao, Huang, Li, Zhang, Lee, Alganabi, Yamoto, Peng, He, Cao, Pierro, Shen and Zhu79, Reference Yan, Liu, Yao, Chen, Yu, Jiang, Chen, Chen and Han80). However, as Wnt signalling not only promotes tissue regeneration but also regulates cellular proliferation, sustained or excessive activation of this pathway may result in uncontrolled cellular expansion and potentially increase the risk of tumorigenesis. Therefore, ISC-targeted modulation of Wnt signalling remains largely at the experimental stage, and further studies are required to determine the optimal therapeutic timing, safety profile, and long-term biological effects of Wnt-based interventions in NEC.

Compared with the Wnt/β-catenin pathway, studies investigating the role of Notch signalling in NEC-associated ISC injury and repair remain limited. To date, only a small number of studies have explored Notch-related regulatory mechanisms in NEC. For example, inhibition of ANGPT2 was shown to improve NEC by blocking Notch signalling and attenuating intestinal epithelial inflammatory responses (Ref. Reference Dai, Jie, Bi, Qing, Chen and Le81). However, this study primarily focused on the role of Notch signalling in inflammatory regulation and epithelial injury, without directly investigating its effects on ISCs. Therefore, further studies are needed to elucidate whether Notch signalling contributes to intestinal repair after NEC through the regulation of ISC function.

Research on the role of EGFR signalling in ISC regulation has been relatively extensive, and some related strategies have entered the preclinical research stage. For example, heparin-binding EGF-like growth factor (HB-EGF) has been shown to reduce damage across multiple intestinal epithelial lineages, promote ISC survival and activation, and enhance the expansion of transit-amplifying cells (Refs Reference Chen, Yu, James, Zhang, Yang, Radulescu, Zhou and Besner57, Reference Watkins, Zhou, Matthews, Chen and Besner82). It also significantly increases the number of LGR5+/prominin-1+ ISCs, thereby preserving ISC homeostasis and promoting intestinal mucosal recovery after injury. In addition, hyaluronic acid can activate EGFR signalling on the surface of ISCs, promoting Lgr5+ stem cell proliferation and crypt fission, thereby facilitating intestinal growth and epithelial repair (Ref. Reference Riehl, Alvarado, Ee, Ciorba and Stenson83). Therefore, modulation of EGFR-related pathways may represent an important candidate strategy for promoting ISC recovery after NEC; however, further animal validation and preclinical studies are still required before clinical translation.

Restoring the ISC niche

The maintenance of intestinal stem cell (ISC) function is governed not only by their intrinsic regenerative capacity but also by dynamic interactions with the surrounding microenvironment and diverse extrinsic signals. Breast milk-derived bioactive components are important niche regulators that contribute to intestinal maturation in preterm infants. Recent studies have demonstrated that human milk oligosaccharides (HMOs) regulate transcriptional programs associated with the crypt–villus axis, promoting the differentiation of intestinal epithelial cells towards mature functional phenotypes, including the upregulation of absorptive enterocyte- and goblet cell-associated markers. These effects may restore an epithelial microenvironment that supports ISC maintenance and regeneration (Refs Reference Zito, Wu, Li, Botts, Feizi, Lee, Lee, Johnson-Henry, Surette, Sherman and Pierro84–Reference Slater, Park, Dennison, Mak, Bendik, Rehman, Mussler, Wiens, Nayak and Zilbauer86). In addition, lactoferrin has been shown to regulate ISC differentiation towards the absorptive lineage through activation of the Brg1/Notch1/Hes1 signalling pathway, suggesting that breast milk-derived proteins can restore the impaired ISC niche by modulating ISC fate decisions and promoting crypt–villus structural maturation (Ref. Reference Zhang, Li, Bi, Lu, Zhang, Shi, Qiao, Sun, Chen, Wang, Wang, Ren and Li87).

The intestinal microbiota represents an important extrinsic regulator of the ISC niche. Recent studies have demonstrated that the human milk-derived probiotic strain Ligilactobacillus salivarius YL20 improves ISC-associated functions, increases goblet cell numbers, promotes MUC2 expression, and enhances tight junction protein expression, thereby improving the ISC-supportive microenvironment (Ref. Reference Wang, Geng, Zhu, Huang, Zhang, Zhang, Zhao, Zhang, Du and Wang88). Similarly, Lactiplantibacillus plantarum JY039 enhances ISC regeneration by increasing the expression of ISC markers, including Olfm4 and Ascl2, promoting proliferation-related markers such as Ki67, c-Myc, and cyclins, while restoring tight junction integrity and modulating gut microbial composition (Ref. Reference Guo, Zhao, Gao, Sun, Zhao, Zhang and Jiang89). However, probiotic application in preterm infants remains challenging due to the immaturity of the intestinal barrier, which may increase the risk of probiotic translocation and subsequent bacteremia or sepsis. Moreover, probiotic strains exhibit considerable functional heterogeneity, and their efficacy is influenced by strain specificity, dosage, timing of administration, and host developmental status. Currently, several probiotic preparations have entered clinical studies for NEC prevention or intestinal maturation; however, further investigations are required to determine optimal strain selection and safe dosing strategies.

Microbial metabolites are another major group of ISC regulatory mediators, with lactate playing a particularly important role. Lactate produced by bacteria such as lactobacilli binds specifically to Gpr81 on Paneth and stromal cells, activates Wnt3/β-catenin signalling, and significantly promotes Lgr5+ ISC proliferation and differentiation towards the Paneth lineage. In contrast, L. plantarum strains lacking lactate dehydrogenase, and thus unable to produce lactate, fail to induce ISC expansion, highlighting the central regulatory role of lactate (Refs Reference Salzman, Underwood and Bevins20, Reference Lee, Kim, Kim, Lee, Kim, Kang, Yang, Baek, Sung, Park, Hwang, Kim, Liu, Kamada, Gao and Kweon21). Another key metabolite, butyrate, has developmentally dependent effects on ISCs: in the metabolically immature intestine of preterm infants, high concentrations of butyrate directly damage ISCs by activating p-MLKL–mediated necroptosis and disrupting the balance between proliferation and differentiation, whereas in the mature intestine, butyrate slightly modulates ISC proliferation to maintain epithelial homeostasis (Ref. Reference Wang, Tao, Salimi-Jazi, Lin, Sun, Liu, Sinclair, Mostaghimi, Dunn and Sylvester22). The bidirectional effects of butyrate on intestinal homeostasis highlight the importance of considering intestinal developmental stage and maturity when applying microbiota-based interventions. Future microbiome-targeted strategies should not simply aim to increase the abundance of specific metabolite-producing bacteria or promote the production of individual metabolites, but rather focus on restoring overall microbial homeostasis and optimizing microbial metabolic functions. Integrating microbiome profiling with metabolomic and other multi-omics approaches may facilitate the identification of protective microbial communities and their metabolic signatures, while preventing the excessive accumulation of potentially harmful metabolites in the immature intestine. Such precision-based approaches may enable the safe and effective remodelling of the ISC-supportive niche after NEC.

Stem cell therapies targeting ISCs in NEC

ISCs are the principal cellular drivers of intestinal mucosal repair and regeneration. Amniotic fluid stem cells (AFSCs) promote intestinal epithelial proliferation and activate the body’s own ISC programs, helping to reduce NEC-related injury, with these protective effects closely linked to modulation of the Wnt signalling pathway (Ref. Reference Balsamo, Tian, Pierro and Li90). Compared to mesenchymal stem cells (MSCs), which mainly provide immunomodulatory functions, AFSCs are more focused on developmental and growth-related signalling. They increase Lgr5 mRNA expression and raise the number of Ki-67+ proliferating epithelial cells. Preconditioning with AFSCs not only lowers the incidence of NEC but also enhances barrier repair by activating endogenous ISC function and stimulating epithelial growth, offering targeted preventive protection for high-risk newborns (Ref. Reference Li, Lee, Cadete, O’Connell, Alganabi, Lee, Ganji, Miyake, Botts, Johnson-Henry, Maattanen, Sherman and Pierro91).

Notably, AFSCs further support ISC recovery by activating the Rspo3/AMPKα axis, thereby reducing epithelial injury and inflammation and expanding the mechanistic framework for direct stem cell therapy (Ref. Reference Ning, Wang, Li, Liu, Chen, Hui and An92). Human placental-derived stem cell (hPSC) therapy, in contrast, mainly targets the restoration of the damaged ISC niche. In NEC models, hPSCs reestablish components of the crypt niche, including LGR5+ ISCs, Paneth cells, and SOX9+ cells, normalize the villus–crypt architecture, decrease epithelial apoptosis, and promote proliferation, collectively aiding mucosal regeneration (Ref. Reference Weis, Deal, Mekkey, Clouse, Gaffley, Whitaker, Peeler, Weis, Schwartz and Atala93).

Stem cell–derived extracellular vesicles (EVs) have also become promising cell-free therapeutic agents capable of modulating ISC function and repairing NEC. Thrombin-preconditioned MSC-derived EVs (thMSC-EVs) can significantly increase the expression of the ISC marker OLFM4 and the tight junction protein claudin-4, while reducing stress-related molecular signatures (Ref. Reference Hwang, Sung, Kim, Yang, Koh, Ahn and Chang94). AFSC-derived EVs (AFSC-EVs) strongly promote ISC proliferation and epithelial regeneration by selectively activating the Wnt/β-catenin pathway (Ref. Reference Li, Lee, O’Connell, Antounians, Ganji, Alganabi, Cadete, Nascimben, Koike, Hock, Botts, Wu, Miyake, Minich, Maalouf, Zani-Ruttenstock, Chen, Johnson-Henry, De Coppi, Eaton, Maattanen, Delgado Olguin, Zani, Sherman and Pierro95). EVs derived from ISCs (ISCs-EVs) treatment indicates direct support for ISC engraftment, self-renewal, and differentiation. Additionally, ISCs-EVs promote angiogenesis and reduce inflammation, thereby indirectly creating a supportive microenvironment for ISC repair (Ref. Reference Zhang, Li, Wan, Bu, Jin, Yuan and Zhou96).

Challenges and future perspectives for ISC-targeted therapies in NEC

Despite the encouraging regenerative effects observed in experimental NEC models, several challenges must be addressed before stem cell-based therapies can be translated into neonatal intensive care (Ref. Reference Di, Wu, Liu and Shi97). A major challenge is defining the optimal therapeutic window. Current preclinical studies have applied stem cell-based interventions at different stages of NEC, including prophylactic administration before disease induction and rescue treatment after intestinal injury, suggesting that the therapeutic effect may depend on disease stage rather than a fixed time point (Refs Reference Zani, Cananzi, Fascetti-Leon, Lauriti, Smith, Bollini, Ghionzoli, D’Arrigo, Pozzobon, Piccoli, Hicks, Wells, Siow, Sebire, Bishop, Leon, Atala, Lythgoe, Pierro, Eaton and De Coppi98, Reference McCulloh, Olson, Wang, Vu, Gartner and Besner99). However, translating these findings into clinical practice remains challenging because NEC is frequently diagnosed only after epithelial injury and inflammation have already developed. Therefore, future clinical application will likely require dynamic assessment of disease evolution rather than relying solely on clinical diagnosis. Integration of early predictive biomarkers, including inflammatory indicators, intestinal injury markers, and microbiome or metabolomic signatures, may enable identification of high-risk infants and guide personalized intervention timing (Ref. Reference McCulloh, Olson, Zhou, Wang and Besner100).

Moreover, safety concerns associated with stimulating intestinal regeneration should be carefully considered. Although enhancing ISC activity may promote epithelial repair, excessive or uncontrolled activation of regenerative programs may disrupt the physiological balance between ISC self-renewal and lineage differentiation, potentially resulting in abnormal epithelial maturation or long-term alterations in intestinal homeostasis (Refs Reference Zeng, Wang, Zhuo, Luo, Sha and Chen101–Reference Choi and Augenlicht103). In addition, direct stem cell transplantation carries potential risks, including ectopic engraftment, abnormal differentiation, immune responses, and unpredictable long-term effects in developing infants. Therefore, future strategies should focus on restoring regenerative capacity rather than simply increasing stem cell activity (Ref. Reference Beumer and Clevers61).

Compared with direct cell transplantation, stem cell-derived extracellular vesicles and other cell-free approaches may provide improved safety profiles by minimizing risks associated with cellular engraftment (Refs Reference Zhang and Cheng104, Reference Tan, Li, Wang, Li, Shahzad and Zheng105). However, standardized production, dose optimization, and long-term safety evaluation remain critical challenges. Ultimately, stem cell-based therapies may require precision application based on disease stage, intestinal maturity, and molecular biomarkers before they can be incorporated into clinical management of NEC.