The incidence of NEC varies from 0.3 to 2.4 per 1,000 live births, with geographic, ethnic, and neonatal care strategy variations (34–36). The incidence of the disease is strongly inversely correlated with the gestational age (GA) at birth, with approximately 90%–95% of cases occurring in infants born before 36 weeks' GA (1). A multicenter cohort analysis conducted in the United States revealed only marginal improvement in the overall incidence of NEC cases among Very Low Birth Weight (VLBW) over the past years, with a decrease from 9% in 2006 to 6% in 2017 (37). However, term infants can also be affected, mainly when associated with other conditions such as congenital heart disease, polycythemia, early-onset bacterial sepsis, hypotension, or maternal drug use (38, 39).

Since its description in the 1940s to 1950s, NEC is recognized as a multifactorial disease caused by various risk factors, including prematurity, formula feeding, microbial dysbiosis, immature intestinal barrier, underdeveloped motility, immune regulation, hypoxia, inadequate microcirculation, and intense inflammation (Table 1) (40–57). In this context, the upregulation of TLR4 is pivotal in NEC pathogenesis, directly contributing to the intestinal epithelial barrier impairment (5–7). Because of the central role of TLR-4 in the pathogenesis of the disease, many therapeutic options are moving toward drugs explicitly targeting the signaling pathway associated with this molecule. The inhibitor 2-acetamidopyranoside (C34), identified by Neal et al., mainly exhibits efficacy in inhibiting TLR-4 signaling both in vitro and in vivo. Mice treated with C34 showed preserved intestinal mucosa and a reduced incidence of the disease in an experimental model of NEC (58, 59). The pregnane X receptor (PXR), a xenobiotic sensor that acts as an intermediary for specific host-bacterial metabolites, can inhibit the TLR-4 pathway (60). In PXR knockout mice, symptoms following NEC induction were more severe. Activation of intestinal PXR by lithocholic acid (LCA), a PXR agonist, reduces NEC-induced intestinal inflammation (60, 61). Furthermore, Dai et al. recently demonstrated that high mobility group box 1 (HMGB1), a DNA-binding protein, inhibits enterocyte migration by activating the TLR-4 pathway. Hence, NEC is associated with elevated protein expression (62). In a rat model, glycyrrhizin, an HMGB1 inhibitor, was used to reduce the disease incidence through the TLR-4 and nuclear factor kB (NF-kB) pathways (63).

The disruption of the TLR4 pathway and the other risk factors are closely linked to alterations in the normal proliferation/apoptosis cycle within the intestinal tissue. Specifically, the Wingless and Int-1 (Wnt) signaling pathway, originating from Paneth cells, drives crypt division and plays a pivotal role (64–66). After apoptosis, which leads to the average turnover of epithelial cells, there is an autophagic clearance of apoptotic remnants and shedding of cell remnants into the lumen (66). Under normal conditions, there is a balance between cell proliferation and loss through processes such as apoptosis, autophagy, and shedding. In contrast, in experimental models and humans, NEC is defined by intestinal necrosis, epithelial cell loss, and increased apoptosis and autophagy (67–70). The loss of this delicate intestinal stem cell niche disrupts the balance between cell propagation and loss, while also impairing cell migration (71). Intestinal epithelial apoptosis triggers severe bowel necrosis in an experimental neonatal rodent model that resembles necrotizing enterocolitis in human newborns (72).

Clinical features, radiographic findings, and laboratory values based on Bell's staging criteria support the diagnosis of NEC. They were initially described in 1978 and subsequently refined by Walsh and Kliegman in 1986, extending the original 3-stage classification to the current 6-stage classification (73–75). Despite their well-recognized pitfalls, Modified Bell's criteria are widely employed to stage the severity of NEC, with higher stages associated with a greater risk of adverse outcomes (74).

The condition's severity is commonly classified into “medical NEC” vs. “surgical NEC”, a distinction that has critical implications, as surgically managed disease carries higher mortality and worse long-term outcomes (76).

In clinical practice, the early manifestation of NEC typically shows wide variation in infants, and its non-specific nature makes it challenging to make an early diagnosis. The disease exhibits various clinical symptoms and signs, including apnea, bradycardia, lethargy, irritability, and temperature instability. This holds for other conditions in preterm infants, such as sepsis (59, 77). Concurrent gastrointestinal symptoms, such as abdominal distension, blood in the stools (either gross or occult), gastric residual volumes, and bilious vomiting, may also support the diagnosis of NEC (78, 79). Radiographic findings and laboratory values help evaluate diseases and exclude other conditions from the differential diagnosis of NEC (80). Abdominal x-rays can indicate intestinal dilation, ileus, pneumatosis intestinalis, portal venous gas, or pneumoperitoneum, which is almost pathognomonic for NEC (81, 82). Additionally, abdominal ultrasound is increasingly recognized as a promising method for enhancing NEC diagnosis and guiding clinical decision-making (83, 84).

Acute complications include fulminant sepsis, peritonitis, abscess formation, thrombocytopenia, granulocytopenia, disseminated intravascular coagulation (DIC), and hypotensive shock (85, 86).

NEC medical management involves implementing measures such as bowel rest, nasogastric or orogastric drainage, fluid balance, parenteral nutrition, pain medication, and the empiric administration of broad-spectrum antibiotics. Critically affected patients often require ventilatory support, vasopressors for blood pressure stabilization, and blood transfusions (87, 88).

While numerous NEC cases are treated medically, around 20%–70% of affected infants require surgical procedures (89).

The only definitive indication for surgical intervention is the presence of gastrointestinal tract (GIT) perforation, as determined by radiographic evidence of pneumoperitoneum or paracentesis showing enteric content (90). However, surgery may be required when infants do not display improvement despite medical management or in patients with particular symptoms (91, 92).

Surgical management includes placing a peritoneal drain or performing a laparotomy, resecting non-viable segments of the gastrointestinal tract, and potentially creating ostomies or primary anastomoses (93–97).

Despite significant advances in neonatal intensive care, NEC-related mortality rates remain alarmingly high, peaking at up to 42% in infants weighing less than 750 g at birth (98, 99). Patients with “medical NEC” have a 21% mortality rate, while those with “surgical NEC” face a mortality rate of up to 35%–50% (100, 101). Additionally, infants with extensive gut resection often face complications such as wound dehiscence, nutrient imbalance, a high rate of abscesses, a high incidence of strictures, and short bowel syndrome (12, 76, 100, 102). Moreover, extended use of total parenteral nutrition (TPN) raises the risk of infections, cholestasis, and liver failure (98, 103). Additionally, NEC patients have been shown to experience worsened neurological outcomes in both the short and long term (13, 104).

NEC research should emphasize prevention over treatment, which can alter outcomes and reduce morbidity. Preventive strategies have centered on standardized protocols, breastfeeding and nutrition with human milk, antenatal maternal steroid administration, prophylactic probiotics, careful antibiotic use, and avoiding histamine type II receptor antagonists (22–29, 31–33, 105).

MSCs are a versatile type of adult stem cell (ASCs) localized in specialized niches within various tissues throughout the body, including the bone marrow, adipose tissue, and umbilical cord. MSCs play a crucial role in tissue homeostasis, repair, and regeneration. They are multipotent cells capable of differentiating into several cell types, including bone, cartilage, fat, and connective tissue, making them a valuable resource for regenerative medicine (14).

MSCs can be easily isolated in vitro by plastic adherence and expanded in culture as fibroblast-like cells expressing CD90, CD73, and CD105 to reach an appropriate number for clinical use (14, 118). Also, they need to lack the expression of hematopoietic cell surface markers CD34, CD45, CD11a, CD19, and HLA-DR and to adhere to plastic surfaces under culture conditions (119, 120). Clinical and preclinical data reported the therapeutic benefits of MSC transplantation in repairing injured tissues and modulating the inflammatory response to damage (121, 122). Importantly, due to the lack of HLA class II and co-stimulatory molecule expression, MSCs are immune-evasive, enabling MSC transplantation across histocompatibility barriers and the creation of off-the-shelf therapies, consisting of exogenous MSCs in culture (123).

One of the defining characteristics of MSCs is their immunomodulatory and anti-inflammatory properties (124). They can sense inflammatory signals via TLRs and secrete multiple anti-inflammatory molecules to support tissue regeneration, regulate immune responses to protect tissue from excessive damage, and promote the survival of tissue-derived precursor cells when injected in response to injury (125–137).

MSCs interact with and regulate the activity of innate and adaptive immune cells, inhibiting the proliferation of B, T, and natural killer (NK) cells through direct interactions and the release of soluble molecules, such as transforming growth factor (TGF)-β1, indoleamine 2,3-dioxygenase (IDO), and prostaglandin E2 (PGE2). Consequently, these molecules halt immune cells in the G0 stage to prevent their proliferation. Additionally, MSCs modulate T-cell activation by suppressing the production and secretion of inflammatory cytokines and preventing dendritic cells' activation and maturation (132, 138).

For this reason, MSCs are frequently used in patients to prevent heightened immune cell activation and to modulate the inflammatory response, promoting tissue regeneration in conditions like Crohn's disease and graft-versus-host disease (GvHD) following allogeneic hematopoietic stem cell transplantation (139, 140). MSCs have also been utilized clinically in regenerative medicine due to their ability to differentiate into bone and chondrocytes, thereby repairing cartilage and similar tissues (141). Recent findings increasingly suggest that the paracrine activity of MSCs is the primary mechanism underlying their therapeutic efficacy. Rather than relying solely on direct differentiation, MSCs promote tissue repair primarily through the secretion of paracrine factors that enhance the survival of injured cells and activate tissue-resident progenitor populations, thereby preventing the formation of non-functional scar tissue (142–145). Their regenerative potential is therefore attributed to modulation of the stem cell niche and secretion of bioactive molecules, including anti-inflammatory cytokines, immunoregulatory mediators, and growth factors such as insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and basic fibroblast growth factor (bFGF), all of which contribute to processes such as angiogenesis, cell survival, and proliferation (131, 134). A recent review by Che et al. emphasized the invaluable role of MSC-derived paracrine signaling in inflammatory bowel disease (IBD), highlighting how secreted bioactive molecules, such as HGF, VEGF, and epidermal growth factor (EGF), contribute to epithelial repair, angiogenesis, and mucosal regeneration (146). Furthermore, MSCs have been shown to enhance the expression of tight junction proteins (e.g., zonula occludens 1 (ZO-1), occludin), promote goblet cell function and mucosal healing, and stimulate the proliferation and differentiation of intestinal epithelial cells (146). Collectively, these findings suggest that MSCs also represent a promising therapeutic approach for NEC. In support of this hypothesis, several preclinical studies have demonstrated that MSC treatment significantly reduces the severity of NEC in rodent models (114, 115). The beneficial effects observed in these studies are thought to be mediated mainly by MSC-derived paracrine factors, including interleukin (IL)-6, IL-10, and TGF-β, which contribute to the resolution of inflammation and restoration of the intestinal barrier (147–149).

McCulloh et al. have revealed that various types of stem cells exert a therapeutic effect by enhancing the intestinal barrier and decreasing intestinal permeability. However, it remains unclear whether the improved gut barrier function is influenced by common or unique pathways among different stem cell types (114).

BM-MSCs are a subpopulation of MSCs found in the medullary stroma of bone marrow. The characteristics of BM-MSCs are closely linked to the age and condition of the donors, as the quantity and differentiation ability decline with age (150). BM-MSCs derived from young and old donors (under 18 years and over 55 years) exhibit significant differences. The old BM-MSCs show increased expression of β-Gal, a hydrolase found in the lysosomes of aging cells, making it a marker of cellular senescence. Additionally, they display changes in cytoskeleton composition, which correlate with impairments in migration and the ability to respond to biological and mechanical signals (151). Even BM-MSCs from aged mice show a greater presence of cellular senescence markers, including the DNA double-strand break marker phosphorylated H2A histone family member X (γH2AX) and the DNA checkpoint response mediator p53-binding protein 1 (53BP1), compared to those from younger mice, which can enhance osteogenic activities and migration in mice instead (152). BM-MSCs release various paracrine factors that regulate fibrosis, proliferation, apoptosis, and angiogenesis in damaged tissues, thereby promoting the recovery of the injured area, modulating the immune system, and safeguarding cells from apoptosis (153). Their potential in treating neonatal diseases has been the subject of considerable research (154, 155). The administration of MSCs has been shown to be beneficial for inflammatory bowel disease. In animal models of colitis, MSC therapy not only reduced the Th1 cytokine response and enhanced the regulatory T cell (Treg) response. Furthermore, MSC therapy has been demonstrated to improve survival and quickly correct weight loss (156).

Kagia et al. have demonstrated that BM-MSCs can treat colitis in mice and improve overall survival compared to the control group. Additionally, colon health in the treated group was significantly better (157).

In a recent study, Abbuehl and colleagues found that freshly isolated murine BM-MSCs, unlike ex vivo expanded ones, can repair stromal niche damage after irradiation and enhance the engraftment of hematopoietic stem and progenitor cells (HSPCs) when co-transplanted intra-bone (158).

The administration of BM-MSCs has effectively reduced the incidence and severity of NEC in a murine model. Specifically, mice with NEC treated with BM-MSCs exhibited decreased levels of Caspase 3, a marker associated with apoptosis, and increased expression of ZO-1. ZO-1 is a protein found at the tight junctions of the intestinal barrier, and its elevated levels compared to the control group suggest that MSCs can help restore the proper functionality of the barrier. These findings indicate that BM-MSCs may have significant therapeutic potential for treating NEC in humans (15).

A study by Seok Lee and colleagues explored oral BM-MSC administration for premature infants to reduce invasive procedures. Administering BM-MSCs orally and intraperitoneally in neonatal mice reduced NEC-related histological injury after NEC induction. The TdT-mediated dUTP Nick-End labeling (TUNEL) assay assessed apoptosis levels, revealing decreased apoptosis in BM-MSC groups. Consequently, stem cell administration lowered TLR-4 expression (159). Previous studies have shown that stem cells can effectively combat NEC in mouse models, regardless of whether BM-MSCs are given before or after NEC onset. These findings suggest that BM-MSCs have potential for preventive and therapeutic uses, achieving notable results whether administered early or later. Additionally, the effects of BM-MSCs are similar whether given orally or intraperitoneally for NEC (159). Wang et al. argued that intraperitoneal injection is the best method for delivering stem cells to treat colitis, despite being an invasive procedure (160). The oral treatment administration is advantageous for vulnerable patients and deserves consideration (159).

Combining BM-MSCs with other factors can enhance their therapeutic potential in NEC prevention by improving their protective efficacy and modulating transcription factors that either suppress or increase the activity of BM-MSCs.

Heparin-binding EGF-like growth factor (HB-EGF) was initially recognized as a possible treatment for NEC almost twenty years ago (161). It safeguards intestinal injury by preventing injury to intestinal stem cells from damage, facilitating enterocyte proliferation and migration, enhancing gut barrier function, and minimizing intestinal apoptosis (162–165). Yang et al. investigated the potential for attaching HB-EGF to BM-MSCs to influence the activity of the stem cells. HB-EGF was found to enhance the proliferation and migration of BM-MSCs while simultaneously reducing their apoptosis. Furthermore, in a rat model of NEC, administering HB-EGF and BM-MSCs decreased the incidence of pathology and lowered levels of 70 kDa FITC-Dextran (FD70), a marker of intestinal permeability, compared to the control and stem cell-only groups (166).

A study by Chen et al. demonstrated that inhibiting the gene responsible for prolyl hydroxylase 2, which plays a role in activating hypoxia-related transcription factors, prompted BM-MSCs to enhance their paracrine effects by releasing protective factors such as TGF-β2. Additionally, the inhibition of prolyl hydroxylase 2 increases survival rates in rats with NEC treated with BM-MSCs by promoting epithelial regeneration (167).

The transplantation of MSCs in animal models of experimental NEC has proven feasible, safe, and effective. Notably, it has been reported that various types of stem cells can restore the integrity of the intestinal barrier (114). MSCs have been shown to protect against intestinal damage and reduce the incidence of NEC (168). Whether stem cell transplantation serves as a preventive or protective model for NEC is yet to be determined.

Despite the importance of BM-MSCs as a potential therapy for treating NEC, several other stem cell types show characteristics that may be beneficial in managing intestinal diseases. Stem cells are typically classified based on their origin and differentiation potential.

Embryonic stem cells (ESCs), derived from the inner cell mass of blastocyst-stage embryos, represent the gold standard of pluripotency (169). They can differentiate into all three germ layers (ectoderm, mesoderm, and endoderm), thus giving rise to a broad range of specialized cell types, including neurons, muscle cells, and different tissue-specific cells (170–172). Furthermore, they hold potential for personalized cell-based therapies, where patient-specific pluripotent stem cells can generate replacement tissues or organs (173). However, their clinical application remains ethically controversial, due to their derivation from the human embryo (174). Amniotic fluid-derived stem cells (AF-SCs) represent a promising alternative. These cells exhibit both mesenchymal markers (CD29, CD44, CD90) and embryonic-like characteristics, such as the stage-specific embryonic antigen (SSEA-4) and the octamer-binding transcription factor 4 (Oct4) (175). Their pluripotency, combined with easier collection and culture methods compared to ESCs, makes them particularly attractive for clinical applications. In NEC models, AF-SCs have shown protective effects by enhancing intestinal barrier function through modulation of endoplasmic reticulum stress and upregulation of tight junction proteins like claudin-7 (112). Both enteral administration and intraperitoneal injection of AF-SCs have shown efficacy in experimental NEC models (68, 176).

The enteric nervous system (ENS), often referred to as the “second brain”, interacts with the diverse range of microbes that populate the gastrointestinal tract, forming critical linkages with intestinal microbiota, the immune system, and the endocrine system to maintain a stable intestinal environment (177–179). It undergoes significant damage during the NEC development process, thus increasing the interest in neural stem cells (NSCs) as a potential therapy for NEC.

Neural stem cells, also derived from amniotic fluid (AF-NSCs), express nestin, an intermediate filament protein that can be considered a marker of immature neural cells (180). Despite their slow growth rate when cultured in vitro, AF-NSCs have shown potential in treating NEC through Wnt-dependent mechanisms (112, 181, 182).

Neonatal enteric neural stem cells (N-ENSCs) are specialized stem cells found within the ENS that control the complex network of neurons in the gastrointestinal tract. N-ENSCs can differentiate into various neural cell types within the ENS, including neurons and glial cells (183). N-ENSCs have emerged as a promising avenue for research as they can potentially repair or regenerate the ENS's damaged neural and glial cells. This regenerative capacity offers hope for developing innovative treatments for NEC (184).

Beyond BM-MSCs, other MSC sources have also gained attention. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are multipotent stem cells that can be isolated in a non-invasive manner and exhibit regenerative properties comparable to BM-MSCs, making them a promising alternative for stem cell research and therapies (185). In NEC models, UC-MSCs can promote intestinal integrity by activating endothelial nitric oxide synthase and secreting hydrogen sulfide (108). Placental-derived MSCs (P-MSCs) have also been shown to be a good source of stem cells with potent anti-inflammatory effects (186), able to promote intestinal regeneration via the Wnt/β-catenin pathway (108).

These diverse stem cell populations, ranging from pluripotent ESCs to more specialized AF-SCs and neural stem cells, offer multiple avenues for regenerative medicine approaches, particularly for intestinal pathologies such as NEC. Their varying degrees of potency, combined with distinct mechanisms of action ranging from barrier reinforcement to neural regeneration, could expand the knowledge aimed at the development of novel cell-based therapies. Continuous research into stem cells could lead to a deeper understanding of their therapeutic potential and the resolution of associated ethical issues.