Insights into Modeling Inflammatory Bowel Disease from Stem Cell Derived Intestinal Organoids

Abstract

Inflammatory bowel disease (IBD), encompassing Crohn’s disease (CD) and ulcerative colitis (UC), is a multifactorial, immune-mediated condition marked by chronic gastrointestinal inflammation. This condition significantly impairs patients’ quality of life and represents a major public health challenge globally. Pathogenesis of IBD arises from complex interplay among genetic predisposition, environmental factors, immune dysregulation, and microbial dysbiosis. Although significant strides have been made in unraveling these mechanisms, existing therapeutic options remain inadequate in addressing the full spectrum of clinical needs, underscoring the urgent demand for innovative strategies. Regenerative medicine has emerged as a promising frontier, offering novel tools for therapeutic development. We briefly consolidated current knowledge on IBD pathogenesis and treatments, emphasized the pivotal potential of human intestinal organoids (including adult stem cell-derived organoids and pluripotent stem cell- derived organoids) as a robust platform for mechanistic studies and treatment exploration. Leveraging this technology, we aim to advance personalized and next-generation therapies for IBD.

Graphical Abstract

Introduction

Inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), constitutes a complex category of long-term inflammatory conditions influenced by a multifaceted interaction of genetic, environmental, and immune components. In recent years, there has been considerable interest in the epidemiological features of IBD. Emerging global studies indicate a marked increase in the incidence of IBD during the 21 st century, particularly in industrialized nations. While some regions, such as North America and Europe, have reported stabilization or even a decline in IBD incidence since the 1990 s, a contrasting trend is observed in newly industrialized countries. Specifically, regions across Africa, Asia, and South America, though characterized by lower baseline incidence rates, are experiencing a gradual but consistent rise in IBD cases. These findings underscore the evolving global burden of IBD and highlight the need for tailored public health strategies to address this growing challenge [1, 2].

CD and UC, two primary forms of IBD, have demonstrated alarming annual growth rates of 11.1% and 14.9%, respectively, in regions such as Brazil and Taiwan. This surge in IBD incidence is closely associated with rapid economic development and the adoption of Westernized lifestyles, which are believed to play a pivotal role in driving the rising prevalence of the disease [3]. The economic and personal toll of IBD is substantial. The high cost of treatment places a significant financial burden on healthcare systems and patients alike. Moreover, IBD profoundly impacts patients’ quality of life, often manifesting through debilitating symptoms such as abdominal pain, chronic diarrhea, unintended weight loss, and persistent fatigue. These physical challenges frequently extend to impair daily functioning and mental health, underscoring the multifaceted nature of the disease and the urgent need for comprehensive management strategies [4–6].

Given the significant public health challenges posed by IBD, understanding its pathogenesis is a critical prerequisite for developing effective therapies. The pathological mechanisms of IBD are primarily associated with impaired intestinal barrier function, dysregulated immune responses, and alterations in the gut microbiota. Research has demonstrated that during active IBD, the permeability of Intestinal epithelial cells (IECs) is markedly increased. Moreover, the damage to IECs and their diminished regenerative capacity further contribute to disease progression [7, 8]. The interplay of these factors disrupts intestinal homeostasis, creating a vicious cycle that exacerbates the disease course.

Current treatment strategies for IBD primarily involve pharmacological and surgical interventions. Conventional pharmacological therapies include anti-tumor necrosis factor (TNF) antibodies, immunomodulators, and steroids. While these approaches have improved patients’ quality of life to some extent, they are not without limitations. For instance, monoclonal antibodies have revolutionized the treatment of CD; however, their efficacy is constrained by primary and secondary non-response rates. Despite advancements, the rate of surgery for drug-refractory CD has not significantly declined in recent years [9, 10]. Patients who are unresponsive to these treatments often face severe intestinal damage, necessitating the resection of affected bowel segments. Therefore, the development of personalized experimental models tailored to the heterogeneity of patients is essential for advancing therapeutic strategies.

Regenerative medicine has emerged as a promising field with immense potential in IBD research. Rapid advancements in stem cell-based technologies, particularly in vitro differentiation techniques, have led to the development of organoids—a groundbreaking area rooted in tissue developmental pathways and stem cell biology [11–13]. Organoids provide a robust platform for studying human organ development, drug screening, and disease treatment development. They offer an excellent in vitro model for investigating IBD pathogenesis and systemic interactions, making them a powerful tool for both mechanistic studies and therapeutic innovation in IBD.

In this context, this article reviews the progress in regenerative medicine as it pertains to IBD research and treatment, offering new perspectives on exploring disease mechanisms and developing personalized therapeutic approaches. By leveraging the potential of stem cell-based technologies, we aim to bridge the gap between fundamental research and clinical translation, ultimately advancing the management of IBD.

Histology Foundation of Intestine

In mammals, the intestine serves as the primary organ for nutrient absorption while simultaneously acting as a critical barrier against mucosal toxins, bacteria, and other harmful substances ingested with food and water [14]. These dual functions are meticulously balanced, a testament to the intricate cooperation among various intestinal cell types. The intestinal tissue, derived from all three embryonic germ layers, comprises ectoderm-derived intrinsic and extrinsic neural cells, mesoderm-derived stromal and smooth muscle cells, and endoderm-derived epithelial cells, collectively ensuring its complex functionality.

The physiological integrity and functionality of the intestine largely depend on the equilibrium between IECs and the gut microbiota. IECs form the critical interface between the intestinal lumen and underlying tissues, consisting of multiple specialized cell types, including absorptive enterocytes, goblet cells, enteroendocrine cells, undifferentiated stem cells, Paneth cells, and microfold (M) cells. The “intestinal barrier”, composed of IECs and local immune cells such as T cells and plasma cells, forms a natural defense against intestinal pathogens [15]. Notably, IECs are interconnected by tight junctions, creating an impermeable barrier that prevents direct contact between luminal bacteria and their metabolites and immune cells in the lamina propria. Tight junction proteins, such as Occludin and Claudins, play a pivotal role in maintaining this barrier’s integrity [16]. IECs function not only as a physical barrier, akin to a “fortress wall,” but also as active participants in immune regulation. Their basolateral surfaces interface with immune cells in the lamina propria, where they process and present antigens, thereby shaping the intestinal immune response. This dual role is crucial for maintaining intestinal homeostasis and oral tolerance [17]. In addition to the physical barrier, a biochemical barrier is equally vital. Goblet cells secrete a mucus layer that coats the intestinal epithelium, preventing bacterial adhesion. The primary component of mucus, MUC2 [16], forms a mesh-like structure that confines bacteria to areas distant from the epithelial surface. Paneth cells, located at the base of intestinal crypts, secrete antimicrobial peptides (AMPs), such as defensins and lysozyme, which directly kill or inhibit bacterial growth, further protecting the epithelium from pathogenic invasion. The homeostasis of intestinal stem cells (ISCs) is essential for the continuous renewal and differentiation of the intestinal epithelium, ensuring the maintenance and repair of the epithelial barrier. Lgr5 + stem cells, residing at the base of crypts, undergo division approximately every 24 h to generate new epithelial cells [18]. These Lgr5 + cells give rise to a diverse array of epithelial cell types, including enterocytes, goblet cells, enteroendocrine cells, and tuft cells, highlighting their central role in intestinal regeneration and barrier integrity.

Beyond the physical and biochemical barriers, the precise regulation of immune cells is indispensable for intestinal homeostasis. The lamina propria harbors a vast array of immune cells, including macrophages, dendritic cells (DCs), T cells, and B cells. These cells remain in a state of “vigilance,” poised to rapidly identify and eliminate invading pathogens. However, to avoid excessive immune responses against commensal microbiota and dietary antigens, these immune cells are tightly regulated to maintain a dynamic balance [19]. For instance, DCs extend their processes across the epithelial barrier to sample antigens from the intestinal lumen. Under steady-state conditions, these DCs tend to express co-inhibitory molecules such as PD-L1 and secrete immunosuppressive cytokines like IL-10, promoting the differentiation of regulatory T cells (Treg cells) [20]. Treg cells, in turn, suppress the activation of other immune cells, preventing excessive inflammatory responses. Additionally, B cells play a critical role in producing IgA, a secretory antibody that neutralizes pathogenic microbes in the lumen and prevents their adhesion to epithelial cells without activating the complement system, thereby averting inflammatory reactions [21]. Recent studies have also identified stromal-like cells in the intestinal submucosa and muscular layers that collaborate to modulate immune responses [22].

The gut microbiota itself actively contributes to immune homeostasis. Commensal bacteria regulate the host through various mechanisms, including the production of metabolites, hormones, and interactions with the immune and nervous systems. Certain commensals generate short-chain fatty acids (SCFAs), such as butyrate, acetate, and propionate, which have been shown to enhance the barrier function of IECs, suppress inflammation, and promote Treg cell differentiation [23]. Alterations in the composition of the gut microbiota are associated with other intestinal disorders, including irritable bowel syndrome (IBS) and celiac disease [24]. Furthermore, commensal bacteria competitively inhibit the growth of pathogenic microbes, maintaining the balance of the intestinal microbiota.

The functional homeostasis of the intestine also relies on the regulation of the enteric nervous system (ENS), often referred to as the “second brain” due to its autonomy from the central nervous system. The ENS comprises neurons and glial cells that form intricate networks within the submucosal and muscular layers of the gut. It is divided into two main plexuses: the submucosal plexus (SMP), located between the submucosa and circular muscle layer, and the myenteric plexus (MP), situated between the circular and longitudinal muscle layers [25, 26]. The ENS plays a pivotal role in regulating essential intestinal functions such as nutrient absorption, gastric acid secretion, electrolyte transport, mucosal immunity, and epithelial cell proliferation [25–27]. In fact, all functional cells in the intestine, including epithelial cells, smooth muscle cells, interstitial cells of Cajal (ICCs), vascular cells, and immune cells, are innervated by the ENS. This innervation allows the intestine to dynamically monitor and respond to external stimuli. Notably, dysfunction or loss of enteric neurons is observed in various intestinal diseases [28].

Intestinal motility is governed by the coordinated contraction of smooth muscle, whose rhythmic activity is regulated by ICCs in the myenteric plexus (ICC-MY or ICC-MP). ICCs exhibit spontaneous slow-wave electrical activity [29], characterized by periodic membrane potential oscillations of depolarization and repolarization. While these slow waves are not action potentials and do not directly induce muscle contraction, they modulate the excitability of smooth muscle cells. Intestinal motility is further fine-tuned by neural and hormonal inputs, ensuring precise control of gut activity.

With advancements in regenerative medicine, researchers have leveraged a deep understanding of intestinal development and tissue engineering techniques to recreate intestinal tissues in vitro. These organoid and tissue models provide powerful tools for studying intestinal diseases and developing novel therapeutic strategies, opening new avenues for research and clinical translation.

Derivation of Intestinal Organoids

Intestinal Development and Genetic Modification

The development of gut undergoes a precisely orchestrated developmental program, transforming from a simple, undifferentiated tube in the early embryo into a highly specialized, compartmentalized structure in the adult. During early gastrulation, the definitive endoderm is formed, giving rise to the primitive gut tube. Subsequent anterior-posterior and dorsal-ventral patterning establishes distinct regional identities along the developing gut, prefiguring the future stomach, small intestine, and large intestine [30]. Concurrently, enteric neural crest-derived progenitors progressively colonize the developing gut, establishing the enteric nervous system (ENS). The fetal period is characterized by villus formation in the small intestine, crypt formation in the colon, and cytodifferentiation of the epithelium into specialized cell lineages, each with distinct functions in digestion, absorption, hormone secretion, and host defense. Although subtypes of gut all formed, postnatal maturation proceeds involving further expansion of the crypt-villus axis, establishment of the gut microbiota, and refinement of intestinal function, culminating in the fully functional adult gastrointestinal tract.

Advancements in Intestinal Organoids

In recent years, 3D culture systems have enabled the generation of organoids from various tissues, such as the brain [31, 32], retina [33–35], kidney [36–38], liver [11, 39], and intestine. These organoids have been widely utilized in disease modeling, therapeutic development, drug screening, toxicity testing, and biomolecule delivery. Michele Manganelli et al. employed iPSC-derived organoids to investigate hormone receptor expression, underscoring their utility in drug screening and therapeutic development [40]. Intestinal organoids, based on their cellular origin, can be classified into two major types: adult stem cell-derived organoids (ASC organoids) and human induced pluripotent stem cell-derived intestinal organoids (HiOs). ASC organoids, which are three-dimensional tissue structures, have garnered significant attention. These organoids are typically derived from stem cells isolated from the small intestine or colon [41, 42]. The crypt region serves as the functional unit for organoid formation, as it harbors the intestinal stem cell niche, comprising Lgr5 + stem cells and Paneth cells [43]. Lgr5 + stem cells, located at the base of intestinal crypts, continuously proliferate to generate various epithelial cell types, including enterocytes, goblet cells, and Paneth cells, which are essential for epithelial renewal and repair [38, 44]. Remarkably, studies have shown that both murine and human intestinal crypts can maintain their stem cell properties in vitro without the addition of exogenous growth factors [45]. These stem cells self-organize into polarized epithelial layers on a basement membrane extract, folding to form three-dimensional crypt-villus structures with a lumen that mimics the intestinal lumen in vivo.

This complex process is regulated by several key signaling pathways, including Wnt, BMP, and Notch [46]. For instance, BMP inhibition has been shown to induce de novo crypt formation and is implicated in juvenile polyposis [47], while BMP signaling governs the functional compartmentalization of human intestinal epithelium [18]. R-spondin1, a potent activator of the Wnt pathway, acts as a high-affinity ligand for LRP6, inducing its phosphorylation and activating β-catenin signaling [48]. Additionally, the culture conditions for organoids vary depending on their tissue of origin. For example, mouse small intestinal organoids require Wnt signaling for growth, whereas colon organoids depend on the addition of Wnt3a and R-spondin1, both activators of the Wnt pathway [49].

HiOs represent a versatile tool in various applications. Derived from embryonic stem cells or induced pluripotent stem cells, HiOs can recapitulate the stages of intestinal development in vitro by incorporating specific growth factors, simulating the progression from the zygote to definitive endoderm, then to mid-hindgut, and finally to intestinal organoids [50]. These induced organoids not only contain epithelial components, such as crypt-villus structures and major intestinal epithelial cell types (intestinal stem cells, Paneth cells, goblet cells, enteroendocrine cells, enterocytes, transit-amplifying cells, tuft cells, and microfold cells), but also include stromal cells derived from the intestine [51]. HiOs can be expanded, passaged, and even cryopreserved in chemically defined media, ensuring a stable and sustainable cell source. Over successive iterations, the culture methods for HiOs have been refined and optimized to achieve high reproducibility and functionality. By co-culturing with enteric neural crest cells, HiOs can exhibit contractile activity, mimicking intestinal peristalsis in a more physiologically relevant environment [52].

Beyond differences in derivation methods, ASCs and HIOs exhibit distinct applications. For instance, ASCs—sourced directly from patient tissues with a shorter induction time—offer greater convenience, making them widely adopted for personalized transplantation therapies. In contrast, HIOs require more time to generate but better recapitulate intestinal developmental processes, rendering them particularly valuable for modeling disease mechanisms and etiological studies. Today, human intestinal organoid models are widely used to recapitulate epithelial cell diversity and the pathophysiological changes observed in IBD [53].The construction and iterative improvement of intestinal organoids have established them as essential tools for studying intestinal diseases.

Pathophysiology of Inflammatory Bowel Disease

IBD is characterized by chronic intestinal inflammation, often accompanied by extra-intestinal manifestation [54]. Histologically, the intestines of IBD patients exhibit diverse pathological changes. CD is typified by transmural inflammation, destruction of intestinal crypts, and the formation of non-caseating granulomas, which are often located in the deeper layers of the intestinal wall. Clinically, it primarily manifests as chronic diarrhea, severe right lower quadrant abdominal pain, and complications such as fistulae and strictures [55]. In contrast, UC primarily affects the mucosal layer, manifesting as persistent ulcers, loss of crypts, and infiltration by non-specific inflammatory cells [56]. Its hallmark clinical features include hematochezia and urgency, frequently accompanied by complications such as toxic megacolon and cholangitis [57].

The pathogenesis of IBD is complex, involving a combination of environmental, genetic, and immune factors (Fig. 1). Disruption of the intestinal barrier and dysbiosis of the gut microbiota play pivotal roles in the onset and progression of IBD. The impaired intestinal barrier leads to microbial dysbiosis and excessive release of inflammatory cytokines, exacerbating disease progression [58]. Therefore, a deeper understanding of these physiological changes and their impact on disease development is critical for identifying effective therapeutic strategies and preventive measures.

Fig. 1.

Possible factors contributing to IBD pathogenesis and pathophysiological manifestations of IBD. Several factors modify the risk of development of IBD. IBD patients may suffer from intestinal and extra-intestinal manifestations of IBD, which results from complicated factors involving both internal and external interactions

Genetic and Epigenetic Alterations in IBD

IBD is a polygenic disorder influenced by both genetic and environmental factors. To date, over 200 genetic loci associated with IBD have been identified. Genetic alterations are considered a significant contributor to the pathogenesis of IBD. Whole-exome sequencing and targeted sequencing have revealed rare variants in the TRIM22 gene in patients with very early-onset IBD (VEO-IBD). TRIM22 variants impair the interaction between TRIM22 protein and NOD2, as well as NOD2-mediated activation of the NF-κB signaling pathway [59]. Proteins encoded by IBD susceptibility genes are involved in a wide range of biological processes contributing to IBD pathogenesis, including pattern recognition (e.g., NOD2), autophagy (e.g., ATG16L1, IRGM), cytokine signaling (e.g., IL23R, IL10), epithelial barrier function (e.g., ECM1), and immune cell function (e.g., CARD9) [59–61]. These genes influence IBD by modulating microbial recognition pathways, cytokine networks, and barrier integrity, underscoring the complex interplay between genetics and disease mechanisms.

DNA methylation, a critical epigenetic modification, is typically associated with gene silencing. Analyses of DNA methylation and transcriptome profiles in CD patients have revealed specific alterations in methylation patterns and transcriptional profiles, which correlate with clinical outcomes [60]. These findings underscore the pivotal role of epigenetic regulation in IBD subtypes and disease prognosis. Histone modifications represent another essential epigenetic mechanism, influencing gene expression and cellular fate. Smillie et al. [1] employed single-cell RNA sequencing to analyze mucosal tissues from ulcerative colitis (UC) patients, identifying diverse cell subpopulations associated with inflammatory responses. The histone modification states of these cells likely modulate their functional roles and contribute to inflammation regulation. Additionally, Oh et al. [62] demonstrated that IL10R mutations are associated with severe pediatric IBD, potentially exacerbating inflammatory responses through interactions with epigenetic mechanisms.

Shared Genetic Susceptibility Across Diseases

Intriguingly, studies have revealed genetic associations between IBD and other diseases, suggesting that multiple autoimmune disorders may share similar susceptibility loci. These loci are also implicated in various immune-mediated diseases [61]. For example, NOD2 is strongly associated with Crohn’s disease, particularly ileal CD, as well as with Blau syndrome [63] and early-onset sarcoidosis [64]. Similarly, IL23R is linked to psoriasis and multiple sclerosis (MS) [65], highlighting the overlapping genetic architecture of immune-related disorders.

Immune Dysregulation in IBD

Recent research has elucidated the critical role of altered immune states in the pathogenesis of IBD. The inflammatory features of IBD include immune cell infiltration and excessive expression of pro-inflammatory cytokines. Immune cells are integral components of the immune system, and in the physiological gut, both specific immune-related cells (e.g., T cells, B cells) and non-specific immune-related cells (e.g., macrophages, dendritic cells) are present in the submucosa and muscularis layers. These cells collectively maintain intestinal homeostasis and mediate immune responses.

Simultaneously, the signaling of intestinal immune cells and their secreted cytokines plays a crucial role in intestinal stem cell regeneration and epithelial cell proliferation. Researchers have extensively explored the interactions between intestinal epithelial cells and immune cells. Czerwinski et al., using single-cell RNA sequencing, revealed that the interaction between CD4 + T helper cells and intestinal stem cells is essential for maintaining intestinal homeostasis and repair processes. In the context of IBD, dysregulation of this interaction may lead to impaired intestinal repair and exacerbated inflammation [66]. Lymphocyte signaling also plays a significant role in the self-repair of the intestinal epithelium. Studies have reported that the secretion of IL-23 and IL-22 promotes epithelial cell proliferation and repair [53]. Additionally, IECs respond to interferons (IFNs), enhancing antiviral defenses. Wright et al. [4] demonstrated that Interferon Regulatory Factor 6 (IRF6) plays a critical role in regulating IEC immune responses, and its deficiency significantly amplifies IFN-mediated antiviral responses. In IBD patients, aberrant changes in immune cells are a major contributor to disease onset and progression. Studies have identified significant immune cell infiltration in the intestines of IBD patients, including T cells, B cells, and macrophages [67]. Notably, immune cell imbalance, particularly the altered ratio of regulatory T cells (Tregs) to Th17 cells, is considered a key mechanism in IBD pathogenesis. Research shows that Treg numbers are markedly reduced in IBD patients, while Th17 cells are significantly increased, driving excessive intestinal inflammation. Mucosal-associated invariant T (MAIT) cells, which is a subset of innate-like lymphocytes with antimicrobial functions, exhibit significant functional alterations in IBD patients. Studies indicate that MAIT cell numbers are reduced in the peripheral blood of IBD patients but are significantly increased in inflamed intestinal tissues. These cells display an activated state in IBD, releasing higher levels of cytokine (such as IL-17 and IL-22, while IFN-γ secretion varies across different IBD subtypes [7].

Among the array of cytokines implicated in IBD, members of the IL-6 family play a pivotal role in disease pathogenesis. Notably, the upregulation of TNF and other pro-inflammatory cytokines is strongly associated with the severity of intestinal inflammation [68]. Genome-wide association studies (GWAS) have identified multiple susceptibility loci linked to IBD. Liu et al. [69] characterized 38 novel susceptibility loci, which exhibit consistent effect directions and strengths across diverse populations. Among these, TNFRSF14/MMEL1 (1p36) and TNFRSF9 (1p36) are functionally related to the TNF signaling pathway. Oncostatin M (OSM), a member of the IL-6 family, is highly expressed in the intestinal tissues of IBD patients and is associated with resistance to anti-TNF therapy, suggesting its potential as a biomarker for IBD [70]. In the context of intestinal immune regulation, mutations in IL10RA are linked to severe cases of early-onset IBD in children. Oh et al. [62] discovered that patients carrying compound heterozygous mutations in IL10RA exhibit early and severe intestinal inflammation. These mutations impair the IL-10 signaling pathway, disrupting immune responses in the gut and highlighting the critical role of IL-10 signaling in IBD pathogenesis. Interferon-γ (IFN-γ) is significantly upregulated in IBD patients, and its excessive release is closely associated with damage to IECs. IFN-γ exacerbates inflammation by activating pro-inflammatory signaling pathways [71]. Additionally, the sustained release of pro-inflammatory cytokines, such as IL-17 and TNF-α, is intricately linked to the pathological progression of IBD. Furthermore, a meta-analysis by CWL et al. [61] identified 47 susceptibility loci in ulcerative colitis, expanding the list of IBD-associated loci. These loci include genes involved in cytokine responses, such as IL1R2, IL8RA/B, and JAK2, underscoring the central role of cytokines in IBD. While GWAS is a powerful tool for identifying genetic associations, it often overlooks non-coding SNPs in haplotypes that influence epigenetic regulation. Epigenetic regulation of inflammatory cells plays a pivotal role in modulating gene expression. Current studies indicate that non-coding RNAs (especially microRNAs, miRNAs) primarily function by binding to the 3‘-untranslated region (3‘-UTR) and 5’-untranslated region (5‘-UTR) of target genes, precisely regulating gene expression at both transcriptional and post-transcriptional levels. Furthermore, these regulatory mechanisms are directly involved in key pathological processes of IBD, including but not limited to T-cell subset differentiation and IL-23/Th17 signaling pathway activation, which contribute to immune dysregulation [72, 73].

Autophagy and its Role in IBD Pathogenesis

Beyond cytokine dysregulation, alterations in autophagy are increasingly recognized as critical contributors to IBD pathogenesis. Insufficient autophagy may impair the ability of intestinal epithelial cells to clear bacteria and other harmful substances, thereby exacerbating inflammatory responses [74]. The ATG16L1 gene encodes a key protein involved in the autophagy process, a cellular mechanism essential for maintaining homeostasis, degrading damaged organelles and proteins, responding to nutrient deprivation, and defending against pathogen infections. Variants in the ATG16L1 gene have been linked to susceptibility to Crohn’s disease, highlighting the importance of autophagy in maintaining intestinal barrier function and modulating inflammatory responses [5].

The Role of the Gut Microbiome in IBD Pathogenesis

The gut microbiome refers to the diverse community of microorganisms, including bacteria, fungi, viruses, and protozoa, residing in the human intestinal tract. These microorganisms play a crucial role in maintaining host health, particularly in regulating the immune system. Research has demonstrated a complex interplay between the gut microbiome and immune responses, where the composition of the microbiome can influence the host’s immune status, thereby either preventing or promoting the development of IBD.The gut microbiome is highly diverse, predominantly comprising members of the Bacteroidetes, Firmicutes, and Proteobacteria phyla, among thousands of other microbial species. These microbes are not only involved in digestion and metabolism but also modulate immune responses through the production of microbial metabolites, such as SCFAs. For instance, certain Clostridium species produce butyrate, which inhibits intestinal inflammation and promotes the secretion of anti-inflammatory cytokines, thereby maintaining intestinal immune homeostasis [75]. In IBD patients, the production of SCFAs is often suppressed [76].Giri et al. [75] identified specific Clostridium strains capable of inhibiting NF-κB activation in host goblet cells and epithelial cells, thereby reducing inflammation. Moreover, certain bacterial strains have been shown to suppress IL-8 secretion, further alleviating inflammatory responses [77]. Recent studies have highlighted the close association between alterations in the gut microbiome, changes in gene expression patterns, and the onset of IBD. The composition of the gut microbiome and its interactions with the host are of significant importance in the pathological mechanisms of IBD. Microbial dysbiosis is recognized as a key factor in IBD pathogenesis, with studies showing a marked reduction in microbial diversity in IBD patients. Imbalances in specific bacterial communities, such as an overabundance of adherent-invasive Escherichia coli (AIEC), which has been closely linked to the pathological features of CD [78], can impair intestinal barrier function and trigger aberrant immune responses, contributing to the onset and progression of IBD [18]. AIEC mediates adhesion via type 1 pili and enhances invasion through flagella activation. Furthermore, both pili and flagella trigger the classical NF-κB pathway, inducing HIF- 1α production, which synergistically regulates IL-8 transcription and pro-angiogenic factor expression, thereby exacerbating inflammation and vascularization [78, 79]. Pathogenic bacteria in the gut can activate immune cells through pro-inflammatory signaling, further exacerbating intestinal barrier damage [80]. The gut microbiome also modulates immune responses through interactions with host immune cells. For example, MAIT cells in IBD patients exhibit an activated state and play a role in regulating intestinal inflammation [7]. Recent advancements have demonstrated that fecal microbiota transplantation (FMT) can alleviate symptoms in IBD patients, underscoring the critical role of the gut microbiome in IBD [81]. Notably, a Lancet-published double-blind trial on FMT for IBD demonstrated that an 8-week regimen—contrasted with historical protocols employing either weekly enemas (over 6 weeks) or nasoduodenal tube delivery (at weeks 1 and 3)—elicited significantly greater endoscopic response rates in the treatment group versus controls, highlighting the requirement for extended therapy to achieve primary endpoints [82]. Consequently, UC patients may necessitate more intensive and sustained FMT regimens to attain clinical remission—a striking divergence from Clostridioides difficile colitis, where a single FMT enema often suffices for cure. Critically, therapeutic efficacy hinges upon multiple factors: transplantation methodology, donor selection, microbial diversity, and recipient disease status [83]. Nevertheless, restoring gut microbial equilibrium represents a promising frontier for IBD treatment. In summary, the gut microbiome plays a pivotal role in the regulation of the immune system, and its dysregulation is closely associated with the development of IBD. Future research should delve deeper into the specific mechanisms by which the gut microbiome influences IBD pathogenesis and explore targeted strategies to modulate the microbiome for disease prevention and treatment. As our understanding of the gut microbiome continues to advance, therapeutic approaches focusing on specific microbial species hold significant promise for improving outcomes in IBD patients.

Alterations in Intestinal Function and Barrier Integrity

Defects in the intestinal barrier represent a critical pathological basis for IBD [17], IECs play a central role in IBD pathogenesis by maintaining gut immune homeostasis through the regulation of mucus production and composition. In IBD patients, both the structure and function of IECs are often compromised, leading to dysregulated intestinal immune responses. The interplay between various cell types, including epithelial cells, goblet cells, and Paneth cells, is essential for maintaining intestinal homeostasis. Epithelial cells form the physical barrier of the gut, while goblet cells secrete mucus to protect the intestinal lining from pathogens. Paneth cells, with their antimicrobial properties, produce a range of antimicrobial peptides that contribute to the gut’s immune defense. Studies have shown significant alterations in the function of intestinal epithelial cells in IBD patients. The number and functionality of goblet cells are impaired, resulting in insufficient mucus secretion and compromised protection of the epithelial layer [84]. This deficiency renders the gut more susceptible to bacterial invasion and other harmful agents, exacerbating inflammatory responses. Additionally, Paneth cells are markedly reduced in IBD patients, potentially weakening the gut’s antimicrobial defenses [50]. The loss of Paneth cells may disrupt the balance of the gut microbiota, further contributing to immune dysregulation and intensified intestinal inflammation. Acho-Fernandez et al. highlighted the role of lymphotoxin-beta receptor (LTbR) signaling in promoting epithelial cell repair by enhancing cell proliferation and survival. Disruption of this signaling pathway may lead to epithelial cell damage and barrier dysfunction, thereby triggering inflammatory responses [85]. In IBD, the combined dysfunction and altered numbers of epithelial cell types collectively contribute to the breakdown of the intestinal barrier and the amplification of inflammation.

The Role of Enteric Neurons in IBD Pathogenesis

The enteric nervous system is a critical component of intestinal functional homeostasis. Damage to enteric neurons can lead to recurrent reductions in intestinal motility during colitis [86], In IBD patients, motility disorders are observed in approximately 30% of cases [87, 88]. Inflammatory cytokines such as TNF-α, IL-1β, and IL-6, which are abundantly secreted during intestinal inflammation, exert toxic effects on neurons, leading to their apoptosis or necrosis and subsequent neuronal damage [89]. Brian D. Gulbransen et al. reported [90] that in colitis, inflammation induces enteric neuron death through the activation of a neuronal signaling complex composed of P2 × 7 receptors (P2 × 7Rs), pannexin- 1 (Panx1) channels, the Asc adapter protein, and caspases. Interestingly, Panx1 levels are reduced in CD but not in UC, suggesting distinct mechanisms and progression of neuronal damage between these two forms of IBD. Further investigation is warranted to elucidate these mechanisms in greater detail.

Application of Intestinal Organoids in IBD Research

Advances in organoid technology have enabled the cultivation of intestinal tissues with complete structural and functional properties in vitro (Fig. 2). The development of organoid models has not only advanced fundamental research in IBD but also provided a novel platform for the exploration of personalized therapeutic strategies [15]. By integrating cutting-edge technologies such as single-cell RNA sequencing (scRNA-seq), gene editing, and microfluidic organ-on-a-chip systems, human-derived organoid models have been developed, offering a powerful tool to investigate the biological mechanisms of IBD and identify potential therapeutic interventions [91, 92].

Fig. 2.

Schematic of the intestinal development and traditional construction of intestinal organoids. (a) The intestinal epithelium consists of columnar epithelial cells that form a crypt-villus structure. ISCs, that located within crypts, self-organize into intestinal organoids, forming 3D structures with crypt-like buds. (b) Intestinal organoids could derived from hPSCs by simulating natural formation of mature intestine

Enhancing the Relevance of Organoids for IBD Research

To better align organoid models with the specific needs of IBD research, efforts have been made to develop diverse types of organoids, enhancing their applicability. The pathological sites of UC and CD differ, with UC predominantly affecting the colorectum and CD involving the entire intestinal tract. To address this, organoids representing different intestinal segments have been generated in vitro through distinct induction methods, thereby more accurately modeling the colon and small intestine and enabling the study of region-specific physiological characteristics [80]. For example, John P. Gleeson et al. utilized iPSC-derived intestinal organoids, including HIO and human colonic organoids (HCO), to demonstrate increased epithelial permeability in IBD-derived organoids under immune factor stimulation, effectively modeling the impaired barrier function observed in IBD patients [93]. The formation of intestinal organoids primarily relies on the differentiation and proliferation of intestinal stem cells and their microenvironmental influences. Organoids generated under 2D air-liquid interface (ALI) cultures exhibit functional and morphological differences compared to their 3D counterparts but provide additional research opportunities, such as improved co-culture conditions with microbes or viruses [94, 95]. Hou et al. [45] demonstrated that human and mouse-derived organoids can be maintained without exogenous growth factors and used to form tissue-engineered small intestine (TESI) in vivo. Fluorescence imaging revealed that TESI contains epithelial, mesodermal, and neural cell populations, closely resembling the composition of native intestinal tissue. Furthermore, Workman et al. [96] generated organoids with enhanced enteric neuron colonization by co-culturing them with neural crest cells, providing a model to study enteric neuronal pathology in IBD. Additionally, to eliminate the confounding effects of cytokines present in Matrigel, non-adhesive alginate hydrogels with simpler compositions have been increasingly utilized for organoid culture [97], Kraiczy et al. optimized the differentiation of iPSC-derived intestinal organoids using small molecule compounds, thereby improving their biological similarity [98]. ASC and HIO represent an almost inexhaustible cell source, offering a robust platform for modeling rare diseases and validating novel therapeutic approaches (Fig. 3).

Fig. 3.

Multiple applications of intestinal organoid in IBD. (a) Application of intestinal organoids in regenerative medicine; (b) Application of intestinal organoids in genetic engineering; (c) Application of intestinal organoids in host microbial interactions

Intestinal Organoids as Models for Investigating IBD Pathogenesis

The use of iPSCs to generate intestinal organoids in vitro has facilitated the study of pathological changes in intestinal epithelial cells. Interactions between epithelial cells, goblet cells, and Paneth cells play a critical role in maintaining intestinal homeostasis, although the underlying mechanisms remain incompletely understood. Organoid models enable in-depth investigation into the pathological mechanisms of IBD in individual patients. In IBD, epithelial cell damage is a hallmark, with disease severity often linked to an imbalance between injury and repair. Thus, exploring epithelial self-repair mechanisms using IBD models is of particular importance.The proliferative state of intestinal stem cells is frequently disrupted in IBD, though the specific functional alterations remain unclear. Studies using adult stem cell-derived organoids (ASO) have provided insights into these changes. It has been reported that ASO derived from IBD patients exhibit fewer monolayered classical organoids and a higher frequency of pseudostratified structures compared to controls [99], suggesting impaired proliferative states and altered stem cell homeostasis as potential contributors to IBD pathogenesis [100]. Sarvestani et al. [91] established an iPSC-derived organoid model from UC patients (iHUCO), which recapitulated key pathological features of UC, including impaired proliferation, reduced goblet cells, and disrupted epithelial barrier integrity. The study identified excessive activation of the CXCL8/CXCR1 signaling pathway in iHUCO. Notably, mice lack a homolog for human CXCL8, underscoring the utility of iHUCO for modeling gene knockdown or overexpression phenotypes to elucidate the mechanisms of epithelial injury in IBD.

IBD is a complex, multi-gene regulated disorder. Among its subtypes, very early-onset IBD (VEO-IBD), defined as IBD onset before the age of six, represents a distinct clinical entity. Whole-genome sequencing has revealed a novel hemizygous defect in the NOX1 gene, which encodes NADPH oxidase 1, in a patient with ulcerative colitis-like VEO-IBD [101]. This discovery was further investigated using primary organoids derived from the patient, highlighting the role of the NOX1 pN122H mutation in reducing reactive oxygen species (ROS) production. This finding underscores the contribution of NOX1 to early epithelial-luminal microbial interactions in VEO-IBD. However, the progression of VEO-IBD remains poorly understood. Given the ability of iPSC-derived HIO to model developmental processes, HIOs hold significant potential for elucidating the pathological mechanisms and progression of VEO-IBD. Moreover, iPSC-derived organoids are not only valuable for studying gene-related etiologies of adult IBD but also for exploring gene functions during intestinal development. Yu-Han et al. [102] demonstrated this by differentiating iPSCs into small intestinal spheroids, which recapitulated various stages of intestinal embryogenesis. Using cutting-edge genomic technologies and chromatin run-on sequencing (ChRO-seq), they delineated the dynamic landscape of active promoters, enhancers, and gene bodies across different developmental stages and segments of early human small intestine. This work provides a foundational biological map of early intestinal development and offers insights into how developmental genes may influence IBD susceptibility. Beyond immune-related genes, enteric developmental genes may also contribute to IBD susceptibility. Defects in genes regulating enteric neural crest cell development can impair intestinal innervation, leading to failure of neural colonization in affected bowel segments - a hallmark of Hirschsprung disease HSCR. Notably, epidemiological studies [103] have demonstrated a higher incidence of IBD in HSCR populations, suggesting a potential role of developmental genes in IBD pathogenesis. However, the exact mechanistic link remains elusive. IHOs offer a powerful platform for investigating this unresolved question, providing unprecedented opportunities to dissect the developmental-immune axis in IBD etiology.

Immune homeostasis disruption is a critical factor in IBD pathogenesis and is closely linked to various components of the intestinal microenvironment. Cytokines such as TNF-α and IL-6 significantly impact the development and function of intestinal organoids [104]. These cytokines not only induce cell death in organoids but also disrupt intercellular junctional proteins, exacerbating intestinal barrier damage [105]. iPSC-derived intestinal organoids have been widely used to replicate pathological features of IBD. For instance, Gleeson et al. [93] developed an in vitro model using iPSC-derived human intestinal organoids to study barrier function. They observed that inflammatory cytokines like TNF-α and IFN-γ markedly increased organoid permeability, mirroring the characteristics of IBD patients. Similarly, Onozato et al. [84] demonstrated that iPSC-derived organoids treated with TNF-α and TGF-β effectively modeled mucosal injury and fibrosis associated with IBD. These findings highlight the consistency across studies in using iPSC-derived organoids to investigate IBD and suggest that cytokine dysregulation may be a key mechanism underlying IBD pathogenesis.

The onset and progression of IBD are closely tied to alterations in gut bacterial abundance and diversity. Intestinal organoids serve as powerful tools for studying the interactions between gut bacteria and the intestinal epithelium [106]. For example, Leslie Jhansi et al. [107] injected Clostridium difficile into human intestinal organoids and observed disruption of epithelial barrier function and structure within 12 h. Beyond direct bacterial-mucosal interactions, bacterial metabolites play a crucial role in maintaining intestinal homeostasis. Sorrentino et al. [106] utilized HIOs to investigate the effects of bile acids on intestinal stem cells, revealing that bile acids activate the G protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5) on epithelial cells, promoting stem cell proliferation and facilitating intestinal repair post-injury. Similarly, Giri et al. [77] used organoid models to screen for bacteria and metabolites that inhibit NF-κB activation in IBD, identifying strains that ameliorate colitis. These studies underscore the value of intestinal organoids and organoid culture systems in elucidating host-microbe interactions, offering new avenues for IBD drug development [108].

In addition to luminal microbiota, intestinal epithelial cells are regulated by stromal cells located in the submucosa or lamina propria [109]. Beyond podocytes and myofibroblasts, single-cell sequencing has identified four fibroblast subpopulations that are activated in the context of intestinal inflammation. Research has also shown that Crohn’s disease (CD) inflamed biopsies contain inflammatory fibroblasts expressing markers such as Thy1, PDPN (podoplanin), CTHRC1, and CHI3L1 [99]. Collectively, these findings reveal that colonic mesenchyme remodels to drive inflammation and barrier dysfunction in IBD [110]. Collectively, these findings reveal that colonic mesenchyme remodels to drive inflammation and barrier dysfunction in IBD [111]. The specific roles of stromal cells can be validated using organoid models. For instance, although adult stem cell-derived organoids (ASO) lack stromal components, co-culture with exogenous stromal cells promotes their growth, mediated by TGFB1 [112]. In contrast, iPSC-derived HIOs contain both mesenchymal and epithelial cells, making them ideal for studying the role of stromal cells in disease progression. For example, Daichi Onozato et al. demonstrated that in iPSC-derived organoids, TNF-α-induced IBD pathology led to increased expression of fibrosis-related and epithelial-mesenchymal transition genes, highlighting the functional changes in stromal cells [84]. These findings emphasize the importance of investigating stromal cell alterations in IBD pathogenesis and provide insights into the underlying pathological mechanisms.

Intestinal Organoids as Models for IBD Therapy Development

IBD is a chronic autoimmune disorder for which treatment strategies primarily include pharmacological interventions and surgical procedures. Pharmacological treatments encompass anti-tumor necrosis factor (TNF) antibodies, immunomodulators, and corticosteroids. Corticosteroids are widely used during acute flare-ups but long-term use may lead to adverse effects such as osteoporosis, diabetes, and increased infection risk [113]. Anti-TNF agents like infliximab and adalimumab have demonstrated significant clinical remission rates and mucosal healing in IBD patients. However, approximately one-third of patients do not respond to these therapies, and among initial responders, about 40% lose responsiveness within one year [114, 115]. Additionally, these drugs are associated with severe adverse events, including opportunistic infections and malignancy risks [116]. In cases where pharmacological treatments fail or complications arise, surgery may be necessary. Nevertheless, surgery does not cure the disease and may lead to postoperative complications and prolonged recovery periods. While surgeries such as intestinal stricture resection or perforation repair can provide temporary relief, postoperative recurrence remains a concern, and some patients may develop functional bowel disorders [117]. Mesenchymal stem cell therapy has emerged as a promising strategy to improve long-term outcomes for IBD patients. However, limitations such as small sample sizes and variability across studies persist [118–120]. Therefore, there is an urgent need to explore novel drugs and therapeutic approaches.

Organoid technology has proven to be a powerful tool for studying human intestinal development and advancing drug screening and regenerative medicine [18]. HIOs are increasingly recognized for their utility in identifying and validating therapeutic targets for IBD. Kozuka et al. [121] developed a monolayer platform derived from mouse and human small and large intestinal epithelial cells, screening over 2,000 bioactive compounds. By applying specific cytokines, researchers successfully recapitulated IBD pathological features, such as epithelial injury and fibrosis, providing an effective in vitro model for drug development [84]. For instance, iPSC-derived organoids accurately model the complexity of ulcerative colitis (UC) tissues, particularly the overexpression of the CXCL8/CXCR1 axis in UC. Inhibition of this pathway with Repertaxin has been shown to attenuate UC phenotypes [91]. Onozato et al. [84] utilized hiPSC-derived intestinal organoids to construct models of mucosal injury and fibrosis in IBD, validating the effects of IFX and SB431542 in these models. These studies provide a theoretical foundation for clinical drug selection in IBD.

HIOs not only enable the replication of IBD pathological states but also facilitate high-throughput drug screening through direct interaction with therapeutic agents. For example, Workman et al. evaluated the efficacy of multiple drugs using iPSC-derived organoids, identifying compounds that significantly improved organoid function and structure, highlighting their potential for IBD treatment [122]. The advantage of using iPSC-derived HIOs for drug screening lies in their ability to identify effective therapies and assess drug suitability and efficacy across different individuals.

HIOs, rich in intestinal stem cells and differentiated epithelial cells, can serve as a cellular source for therapeutic interventions. Research has demonstrated the potential of human induced pluripotent stem cell (iPSC)-derived HIOs in repairing damaged intestinal epithelium. Nakanishi et al. transplanted developed HIOs into colitis mouse models, demonstrating superior engraftment and repair capabilities compared to controls, with significant improvements in weight and clinical scores [80].

IBD pathogenesis involves complex interactions between multiple genes and environmental factors [123]. iPSCs, combined with CRISPR-Cas9 technology, offer a robust platform for gene editing to investigate disease mechanisms and identify therapeutic targets. Sens et al. [124] utilized an sgRNA-CRISPR-Cas9-expressing lentiviral system to generate iPSC lines with knockout mutations in IL-10RA, IL-10RB, and downstream targets STAT1 and STAT3. These iPSC-derived macrophages were used to model IL-10 signaling-associated KO and VEO-IBD. Similarly, organoid models carrying specific gene mutations or knockouts can be constructed. Takeda et al. [125] applied this approach in colon cancer organoids, using big data to screen disease-associated genes and validating their findings with CRISPR-Cas9-edited organoids. Schwank et al. [126] cultured intestinal stem cells from cystic fibrosis patients, editing the CFTR gene in small intestinal organoids, which restored forskolin-induced swelling phenotypes post-editing. However, research leveraging gene-edited organoids for IBD therapy development remains limited, warranting further exploration.

Intestinal organoids can model host-microbe interactions to deepen our understanding of IBD and develop feasible treatments. Co-culture of organoids with pathogens such as enterohemorrhagic Escherichia coli, norovirus, and Clostridium difficile has been extensively reported [94, 107, 127]. Gut microbiota can also modulate intestinal function through various pathways, suggesting that microbiota or their metabolites may mitigate inflammation. Giri et al. [77] employed organoid-based in vitro methods to screen bacteria and metabolites that inhibit NF-κB activation, thereby modulating intestinal inflammation. Zhao et al. [128] highlighted the role of oxidative stress and elevated reactive oxygen species (ROS) in IBD pathogenesis, which are closely linked to gut microbiota. Meiyu Bao demonstrated that delivering ROS-inhibiting nanodrugs could increase beneficial bacteria while reducing harmful ones [129]. The impact of microbiota changes on mucosal function remains unclear. Norkin et al. proposed a novel high-throughput screening platform to systematically analyze complex biological systems in drug discovery [108], enabling the study of interactions between microbiota, their metabolites, and intestinal inflammation. In this context, future studies may develop microbiota-targeted therapeutic strategies to improve the health outcomes of IBD patients by culturing and analyzing patient-specific samples [84].

Challenges to Existing Engineered Intestinal Tissues

Despite the broad applicability of iPSC-derived intestinal organoids in studying intestinal biology and related diseases, several limitations persist. While human iPSC-derived HIOs can recapitulate certain key features of the intestine, they often lack critical cell types such as neurons, endothelial cells, and smooth muscle [130], Additionally, organoids exhibit morphological and gene expression differences compared to primary intestinal epithelial cells, indicating that they may not fully replicate the complex structure and functionality of the in vivo intestine [131]. This incomplete mimicry limits the ability of organoids to fully emulate the functional and environmental complexity of the native intestine. Optimization of differentiation protocols remains a pressing challenge. Although current organoids mostly resemble fetal-stage intestines, some advances have been made. For instance, transplantation of organoids in vivo can induce a more mature phenotype, characterized by the expression of adult intestinal stem cell markers such as OLFM4 [92], Furthermore, neural crest-colonized organoids exhibit peristaltic phenotypes in response to electrical stimulation, highlighting their potential for functional modeling.

At the biological level, source cells for intestinal organoids may undergo pathological changes during in vitro culture, potentially compromising their suitability for IBD research [80, 132]. For example, stem cells isolated from patient tissues may accumulate mutations or phenotypic alterations during culture, resulting in organoids that do not fully match the biological characteristics of the original lesions.

Current organoid models are limited by the absence of immune cells and microbiota, which prevents them from fully replicating the complexity of the intestinal environment [108]. This single-cell-type culture system restricts in-depth investigations into the intestinal microecology and its interactions with the host. Moreover, interactions between intestinal organoids and other tissues, such as the liver, remain underexplored, limiting their application in multi-organ models [133]. Although iPSC-derived organoids can be used to study changes in gut microbiota, effectively reproducing the in vivo microbial ecology and host-microbe interactions remains a significant challenge [134]. Therefore, future research should focus on optimizing organoid culture and expansion techniques to better model the intricate environment of the human intestine.

Summary

The application of stem cells and organoids in IBD research and therapy holds tremendous promise. HIOs and HCOs, combined with multi-omics technologies such as transcriptomics and metabolomics, enable comprehensive analyses that better simulate intestinal barrier function and pathological states. These advancements facilitate a deeper understanding of IBD pathogenesis. Through interdisciplinary collaborations, such as the integration of micro-engineered chips, researchers can enhance the complementary strengths of different models, advancing the development of research and therapeutic strategies in the field of IBD. This approach offers the potential for more precise and personalized treatments at the cellular and molecular levels. Despite the existing limitations of iPSC-derived HIOs, they lay a foundational framework for future clinical applications, offering hope for improving the quality of life for IBD patients. Continued innovation and refinement of organoid technologies will be critical to overcoming current challenges and unlocking their full potential in IBD research and therapy.

Author Contributions

Qi Zhao: wrote original draft; Miaoli Shao, Lisha Ma: participated in the discussion; Renfang Zhou provided advice and supervised the manuscript.

Funding

This work was supported by the Natural Science foundation of Zhejiang Province (LQN25H200007) and Wenling City Social Development Technology Project (2023S00201).

Data Availability

None declared.

Code Availability

Figure was created with https://app.biorender.com/.

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The authors declare that they have no competing interests.

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The authors declare that they have no competing interests.