2.1 Characteristics of the intestine under homeostasis

The small intestine is the fastest renewing tissue in the adult body. The small intestine contains a significant number of immune cells and serves as a crucial immunological barrier in the body. In response to internal and external environmental stimuli, the intestinal epithelium is continuously resupplied by self-renewal and migratory differentiation of stem cells to maintain intestinal homeostasis.^{1, 7, 8} The intestinal epithelium is lined by finger-like structures called villus (Figure 1), which contain absorptive and secretory cells.⁹ Absorptive cells are responsible for nutrient and water uptake, while secretory cells include goblet cells, which secrete mucus; enteric endocrine cells, which produce hormones; and Paneth cells, which release antimicrobial peptides.¹⁰ These cells play a role in regulating intestinal immunity and other physiological functions.^{1, 2, 9} Lieberkühn crypts reside at the bottom of the villus, there are wedge-shaped crypt base columnar (CBC) cells, which are also referred to as ISCs (Figure 1). Self-renewal and differentiation of leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5⁺) ISCs are critical for maintaining a stable turnover of intestinal epithelial cells.^10-12 Lgr5⁺ ISCs located at the base of the crypts are widely regarded as the primary agents of intestinal epithelial renewal. There is also a type of reserve stem cells at the +4 position of the crypt, and these cells take over stem cell functions after the loss of Lgr5⁺ ISCs.¹³ Recent studies have shown that the main source of Lgr5⁺ ISC is in the upper crypts. Researchers have found a type of progenitor cell that expresses fibroblast growth factor-binding protein 1 (Fgfbp1) in the upper crypts. These Fgfbp1⁺ cells, distinct from the previously identified +4 cells, possess the capability to proliferate bidirectionally. Under homeostatic conditions, Fgfbp1⁺ cells can migrate upward to differentiate into mature cells or migrate downward to form Lgr5⁺ ISCs.¹⁴ Intestinal homeostasis is coregulated by multiple signaling pathways. The wingless-related integration site (WNT)/β-catenin signaling is highly enriched in crypts and drives ISC self-renewal. The bone morphogenetic protein (BMP) signaling pathway promotes differentiation of ISCs into mature cells. When intestinal epithelial cells are damaged, the Yes-associated protein (YAP) signaling pathway induces intestinal cell reprogramming, enabling rapid repair.^15-19 Small intestinal organoids have emerged in recent years as powerful tools for studying intestinal development and disease. Intestinal organoids are three-dimensional (3D) mini-intestines with intact crypts and villus structural domains. These organoids can be cultured from a crypt or a single Lgr5⁺ ISC.²⁰

The intestine initially arises from endodermal cells.²¹ At days 8–9.5 of embryonic development (E8–E9.5), cells of the endodermal lineage form a closed intestinal tube and give rise to the regions of the anterior, middle, and posterior intestine.²² From E9.5 to E14.5, the intestinal tube becomes a pseudostratified layer and the intestinal epithelium and mesenchyme proliferate rapidly.²³ Lineage tracing shows that Lgr5⁺ ISC first appears at E12.5.²⁴ Around E16.5, epithelial cells differentiate into absorptive and secretory intestinal epithelial cells. By 14 days after birth (P14), the intestinal crypts and villus structure resemble those of the adult mouse.²³ The fetal intestinal organoids derived from the early fetal intestine, fetal organoids have a greater area for proliferation, evenly dispersed proliferating cells on the surface and present a spherical structure without budding.²⁵ The proliferation of adult intestinal organoids is restricted to the crypt structural domains, and the crypt domains extend outward to form branching structures known as budding (Figure 1). Fetal intestinal organoids and adult intestinal organoids display notable differences in their transcriptomes.²⁵ But when intestinal epithelial cells sustain injury, adult intestinal organoids undergo transient reprogramming to generate fetal-like organoids that proliferate rapidly to promote damage repair.^{16, 25, 26} We have effectively restored the damaged intestine by transplanting and colonizing intestinal organoids into the injured intestinal mucosa.³

2.2 Characteristics of the intestine during injury

The intestinal epithelium acts as a barrier against the harsh environment. It hosts a diverse and complex community of microorganisms, chemicals, and metabolites, some of which possess pathogenic and toxic properties.²⁷ The intestinal tract is very susceptible to injury from various external factors, including mechanical stress, radiation, chemotherapy agents, pathogenic bacteria, and intestinal diseases. Such insults cause damage to the intestinal epithelium.¹ However, the epithelium possesses a remarkable capacity for rapid self-repair, efficiently restoring its structural integrity and thereby mitigating the risk of infection.²⁸ Intestinal surgery, irritable bowel syndrome, intestinal obstruction, and Crohn's disease (CD) result in elevated intraluminal pressure, leading to deformation of the intestinal mucosa. This deformation can cause mucosal atrophy, which negatively impacts both intestinal physiology and the healing processes.²⁹ In mice, mechanical stress resulting from simulated colon tumor expansion activates WNT target genes in adjacent normal tissue. This process accompanied by crypt enlargement during the formation of early tumorous aberrant crypt foci.³⁰ Irradiation and 5-fluorouracil (5-FU) are widely used anticancer treatments in clinical practice.^{31, 32} However, the intestine is particularly sensitive to these treatments. After irradiation, the apoptotic factors such as p53, poly ADP-ribose polymerase 1 (PARP-1), and caspase-3 being highly expressed in crypt cells.³³ High doses of radiation can lead to the loss of Lgr5⁺ ISCs and the elimination of proliferating progenitor cells, resulting in crypt atrophy.³⁴ After irradiation, endothelial cells generate significant amounts of reactive oxygen species (ROS) and secrete ceramide and acidic sphingomyelinase, leading to increased vascular permeability and tissue hypoxia.^{35, 36} Immune cells, such as M1 macrophages and mast cells are activated, triggering a long-term inflammatory response and inhibiting crypt regeneration.^{37, 38} Acute inflammation and IBD can also cause the loss of Lgr5⁺ ISCs.³⁹ Irradiation can also disrupt the balance of the gut microbiota, leading to an increased abundance of pathogenic bacteria, such as Helicobacter and Clostridium. While concurrently reducing the relative abundance of beneficial phyla like Bacteroidetes and Firmicutes.⁴⁰ Bacterial, viral, or parasitic infections can cause extensive damage to the intestinal tract, inducing the formation of wound-associated epithelial (WAE) cells. These cells rapidly cover the damaged regions, facilitating the swift repair of the intestinal epithelium.^{41, 42}

However, earlier studies have demonstrated that Lgr5⁺ ISC are not essential for intestinal regeneration. Even when Lgr5⁺ ISC are depleted following radiation-induced damage, the intestinal epithelium retains the ability to recover.⁴³ Studies show that differentiated cells can undergo dedifferentiation upon injury, giving rise to stem cells with self-renewal capacity.^44-46 Additionally, dormant reserve stem cells located at position +4 are activated after damage to perform stem cell functions.⁴⁷ Current findings suggest isthmus progenitors within the crypts are now considered the primary contributors to intestinal homeostasis and regeneration.⁴⁸ Following ISC loss after injury, differentiated epithelial cells can revert to a stem cell state with self-renewal capabilities. Secretory lineage cells such as delta-like canonical Notch ligand 1 (Dll1) and atonal basic helix-loop-helix (bHLH) transcription factor 1 (Atoh1) secretory progenitors, as well as lysozyme 1 (Lyz1⁺) Paneth cells and goblet cells, have the potential to generate fully functional ISCs.^{44-46, 49} After the depletion of Lgr5⁺ ISCs, cells marked by Bmi1, mouse telomerase reverse transcriptase (mTert), homeodomain only protein (Hopx), or leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1) are activated at the +4 position above the crypts. These cells take on stem cell functions and contribute to epithelial repair.^{13, 47, 50-56} A population of cells highly expressing clusterin (Clu) has also been found in the intestinal epithelium after irradiation. These cells are known as the revival stem cell.²⁶ After irradiation, Lgr5⁺ ISC ablation mediated by diphtheria toxin (DT) or dextran sulfate sodium salt (DSS) injury, these cells undergo transient expansion, reviving the homeostatic stem cell compartment and replenishing the damaged intestinal epithelium. During the repair process, the damaged epithelial cells undergo transient reprogramming, resulting in the upregulation of fetal and regeneration markers, while the markers of adult stem cell and differentiation are suppressed.^{16, 26} When cultured in vitro, parasite-infected crypts can develop a spheroid with a fetal-like transcriptome, known as fetal-like organoid.⁵⁷ The WNT, Notch, and YAP/transcriptional coactivator with PDZ-binding motif (TAZ) signaling pathways are currently recognized as key contributors to intestinal damage repair.^{3, 16, 58, 59} The WNT pathway is essential for maintaining stem cell homeostasis, and its inhibition impairs crypt repair following irradiation.⁵⁸ Upon intestinal injury, Notch pathway target genes such as Hes family bHLH transcription factor 1 (Hes1) and the Notch intracellular domain (NICD) are upregulated.⁴⁶ The YAP/TAZ pathway plays a critical role in damage repair.⁶⁰ YAP expression increases rapidly after irradiation and DSS injury, and blocking YAP/TAZ activity hinders the repair process.¹⁶ Additionally, inhibiting YAP/TAZ can prevent the overactivation of the WNT pathway during injury and restrain stem cells from excessively differentiating into Paneth cells.⁶¹ The genes regulated downstream of YAP are markedly enriched in small intestinal organoids derived from embryonic mice. Furthermore, YAP overexpression in adult organoids significantly enhances organoid formation efficiency and induces fetal-like organoids.^{62, 63} However, the mechanisms by which these pathways interact to coordinate tissue repair remain poorly understood.

2.3 Characteristics of ISC and the intestine during aging

Aging is accompanied by progressive changes in the intestines, which are linked to a higher incidence of intestinal diseases, including malnutrition, chronic constipation, and colorectal cancer (CRC).^64-67 These age-related alterations in the intestines encompass loss of stem cells, downregulation of cellular metabolism, dysfunction of the intestinal barrier, and dysbiosis of the gut microbiota.⁶⁸ The aging process of the intestine is characterized by notable morphological alterations. In aged mice, the villus are significantly elongated and twisted compared the young.⁶⁹ Additionally, ISCs tend to differentiation of secretory lineage. A decrease in Notch1 expression was observed in the aged intestinal epithelium, which in turn leads to a higher abundance of Paneth cells and goblet cells.⁷⁰ Mechanistically, the excessive accumulation of proinflammatory cells in the lamina propria of the aging intestine leads to elevated levels of interferon gamma (IFNγ). This increased IFNγ activates signal transducer and activator of transcription 1 (Stat1) in ISCs, resulting in the overexpression of secretory lineage marker genes and causing ISC to preferentially differentiate into secretory cells.⁷¹ The number of apoptotic cells increases in senescent crypts, while the number of ISCs decreases.⁷⁰ This is accompanied by a functional decline. Compared to young mice, 17–24-month-old mice exhibited a notable 62% reduction in Lgr5-GFP^High ISCs,⁷² accompanied by a decline in the ISC marker, olfactomedin 4 (OLFM4).^{69, 70, 73} After two rounds of irradiation (10 Gy + 10 Gy), the rate of crypts regeneration is significantly lower in aged mice (18–22 months).⁷⁰ In vitro, crypts isolated from aged mice (24 months) demonstrate a markedly reduced ability to form organoids. Crypts from young mice can produce organoids that can be passed down through successive generations. While organoids from senescent crypts form fewer colonies after three to four passages. That indicates a decline in ISC function with aging.^74-76 Several signaling pathways are involved in regulating the downregulation of senescent ISC function, such as perturbation of WNT, mammalian target of rapamycin (mTOR) signaling, and reduction of fatty acid oxidation (FAO). In senescent ISCs, Paneth cells, and mesenchymal cells, the key Wnts such as Wnt3 are downregulated.⁷⁰ Meanwhile, the extracellular WNT inhibitor Notum is significantly upregulated in senescent Paneth cells, leading to reduced WNT signaling and a consequent WNT deficiency in aged ISCs.⁷⁴ In aged mice (17.5 months), mechanistic target of rapamycin complex 1 (mTORC1) is hyperactivated in ISCs and progenitor cells. Hyperactivated mTORC1 drives villus aging by inhibiting ISC/progenitor cell proliferation through amplifying the MKK6-p38-p53 stress response pathway.⁷⁷ Aged ISCs have impaired FAO and reduced lipid utilization, while FAO agonists and short-term fasting can activate FAO and restore and improve the activity of ISCs and progenitor cells.⁷³ In addition, the N-terminal domain of protein tyrosine kinase 7 (PTK7) released by senescent fibroblasts is a senescence-associated secretory phenotype (SASP). PTK7 activates WNT/Ca²⁺ signaling, which in turn triggers nuclear translocation of YAP, reduces stem cell activity and differentiation, and ultimately impairs crypts formation.⁷⁸ It is evident that ISC function declines with age, and is accompanied by a compromised intestinal barrier and immune function.^79-82 These declines may be a key factor contributing to the development of age-related intestinal diseases and possibly even intestinal cancer.

3.1 Regulation of intestinal homeostasis by epithelium

The maintenance of intestinal homeostasis relies on the self-regulation on ISCs, which is closely tied to stem cell fate determination. ISCs undergo self-renewal to produce new ISCs while also differentiating into progenitor cells with proliferative capacity. These progenitor cells continue to differentiate, eventually giving rise to mature epithelial cells.^{90, 91} The processes of ISC self-renewal and differentiation are maintained in a dynamic balance, and any disruption to this balance can compromise intestinal homeostasis.^{92, 93} Numerous transcription factors play a critical role in determining ISC fate. In Beumer and Clevers’ review, the key transcription factors and classical signaling pathways that regulate the differentiation of ISCs are described in detail.¹ Recently, more key genes and transcription factors have been identified as crucial regulators of intestinal development and ISC fate determination. Our laboratory has identified hepatocyte nuclear factor 4 (HNF4) as a key transcription factor that regulates intestinal development and maintains intestinal function.^{83, 94-96} Embryonic ablation of HNF4 induces extensive cytoskeletal reorganization across multiple organs, including the intestine, kidneys, and yolk sacs, culminating in the absence of brush borders.⁹⁵ Moreover, Hnf4αγ^DKO at E18.5 results in pronounced intestinal dysplasia, characterized by a translucent and distended intestinal lumen, as well as a loss of villus differentiation.⁸⁵ In the intestine of adult mice, the HNF4–SMAD4 signaling pathway plays a crucial role in regulating the fate of enterocytes. The deletion of HNF4 promotes ISC differentiation into progenitor and secretory cell lineages.⁹⁶ Additionally, HNF4 is instrumental in the activation of intestinal FAO and supports ISC self-renewal.⁸³ Similarly, deletion of Krüppel-like factor 5 (KLF5) results in the loss of ISC identity, while the Lgr5^ΔKlf5 causes accelerated ISCs proliferation but a loss of self-renewal capacity. This imbalance leads to excessive progenitor cell proliferation within the crypts, eventually exhausting ISCs and triggering premature differentiation of enterocyte.⁸⁶ Caudal type homeobox 2 (CDX2) and TATA-box-binding protein-associated factor 4 (TAF4) are both critical transcription factors in establishing the gut. CDX2 is essential for the interplay between intestinal epithelium and mesenchyme, and its ectopic expression in the gastric epithelium can induce a transformation into intestinal epithelium.⁹⁷ When deletion of Cdx2 during embryonic development, the transcriptome of the mutant ileum is highly similar to the esophagus, with intestinal epithelial cells in the distal small intestine being replaced by keratinocytes.⁹⁸ Similarly, loss of Taf4 in mice at E18.5 results in a villus-deficient intestine with a flat epithelium. In adult mice, the ablation of Taf4 in the epithelium leads to a highly fragile intestine, with the cecum becoming filled with gas.⁹⁹ Dachsous cadherin-related 1 (Dchs1) and FAT atypical cadherin 4 (Fat4) are essential for the formation of villus and mesenchymal clusters. In E15.5 mice, Dchs1^CKO and Fat4^CKO led to abnormally flat villus, with mesenchymal cells beneath the villus failing to cluster properly. Instead, these disorganized mesenchymal cells prevent the formation of fully functional villus.¹⁰⁰ Special AT-rich sequence-binding protein 2 (SATB2) and metastasis-associated 1 family member 2 (MTA2) are crucial transcription factors involved in regulating the fate of colon stem cells and colon formation. In adult mice, deletion of Satb2 causes colon stem cells to transform into ileum-like stem cells, resulting in the conversion from colon cells to small intestinal cells.⁸⁴ While the small intestine is responsible for absorbing nutrients such as lipids and carbohydrates, the colon primarily absorbs electrolytes. Loss of Mta2 in the colon of adult mice leads to the activation of Hnf4α, which upregulates the expression of lipid transporter fatty acid-binding protein 6 (FABP6) and microsomal triglyceride transfer protein (MTTP), consequently increasing lipid uptake in the colon.¹⁰¹ Furthermore, the forkhead box A (FOXA) shapes the intestinal microbiota in mice by controlling the glycosylation of intestinal epithelial cells. When Foxa1 and Foxa2 are knocked out in intestinal epithelial cells, the glycosylase network is disrupted, leading to drastic changes in microbial composition and spontaneous IBD.¹⁰²

3.2 Regulation of intestinal homeostasis by mesenchymal cells

The maintenance of intestinal homeostasis is not only dependent on the regulation of epithelial cells. The tissue microenvironment also plays a crucial role (Table 1). Intestinal mesenchymal cells work synergistically to regulate intestinal homeostasis through interactions with epithelial cells and immune cell.¹⁰³ Mesenchymal cells are a highly heterogeneous population of cell types (Figure 2), including fibroblasts, myofibroblasts, pericytes, smooth muscle cells, and mesenchymal stem cells.⁸⁸ Crypts myofibroblasts located at the base of crypts, and they produce WNT ligands that maintain ISC growth.¹⁰⁴ Platelet-derived growth factor receptor alpha (Pdgfrα⁺) mesenchymal cells express high levels of R-spondin3, which amplifies WNT signaling.¹⁰⁵ Villus fibroblasts (VF) are a major source of BMP ligands.^{106, 107} Pdgfrα-high VF progenitors secrete the epidermal growth factor (EGF) family ligand neuregulin 1 (NRG1), which induces ISC differentiation toward to the secretory cell lineage.¹⁰⁸ NGR1 was strongly upregulated in macrophages and Pdgfrα⁺ stromal cells during injury, and activation of the mitogen-activated protein kinase (MAPK) pathway and phosphatidylinositol 3-kinase (PI3K)/AKT, also known as protein kinase B (PKB) signaling supported proliferation of stem and progenitor cells during repair.⁸⁷ Irradiation and DSS resulted in a large expansion of fibroblasts near the injury site and expression of large amounts of R-spondin3 to promote intestinal repair.^{49, 109} There is a subset of MAP3K2-regulated intestinal stromal cells (MRISC) in the basal part of colonic crypts that upregulate R-spondin1 expression through the ROS-MAP3K2-Extracellular signal-regulated kinase 5 (ERK5)-KLF2 pathway and enhance WNT signaling. It maintains the function of Lgr5⁺ ISC, and resists DSS-induced colitis.¹⁰³ Smooth muscle cells in the mucosal muscularis propria, located near the base of colonic crypts, serve as a source of BMP antagonists while also enhancing the YAP signaling pathway. These cells express the matrix metallopeptidase 17 (MMP17), which remodels the extracellular matrix and plays a crucial role in tissue repair.⁸⁹ Beneath the crypts lies a rare class of prostaglandin-endoperoxide synthase 2 (Ptgs2⁺) fibroblasts that secrete prostaglandin E2 (PGE2), which activates YAP and promotes the expansion of the lymphocyte antigen 6 family member S (Ly6a) cells population, resembling a reserve stem cell pool.⁸⁸

3.3 Regulation of intestinal homeostasis by immune cells

During intestinal injury, the immune system activates (Figure 2) and leads to the recruitment of a substantial number of immune cells to the site of damage.^{118, 119} Our study found that on day 2 after irradiation, macrophages accumulate at the base of crypts. These cells secrete high levels of transforming growth factor beta 1 (TGFB1) and activates the regenerative transcription factors YAP-SRY-Box transcription factor 9 (SOX9) circuit. It induces the epithelium to undergo reprogramming, and generates fetal-like cells to promote intestinal repair.³ Additionally, bone marrow-derived mesenchymal stem cells secrete C–C motif chemokine ligand 2 (CCL2) and C–X–C motif chemokine ligand 12 (CXCL12), which promote the polarization of M2 macrophages and activate interleukin-10 (IL-10⁺) T cells and IL-10⁺ B cells within the intestinal tract, exerting an inhibitory effect on colitis.¹¹⁰ Recent findings highlight the critical role of innate lymphocyte type 3 (ILC3) and interleukin-22 (IL-22) in the process of intestinal regeneration. After injury, ILC3 activates and secretes IL-22. It induces signal transducer and activator of transcription 3 (STAT3) phosphorylation and promotes ISC proliferation.¹²⁰ Additionally, ILC3 detects lysophosphatidylserine (LysoPS) released from apoptotic neutrophils through its membrane receptor G-protein-coupled receptor 34 (GPR34), which triggers the PI3K-AKT and RAS-ERK pathways, leading to the release of IL-22 and initiation of tissue repair.¹¹¹ Furthermore, both adrenalines secreted by intestinal nerve cells and viral infections can activate ILC3, drive IL-22 production and enhance the regeneration of intestinal epithelium.^{121, 122}

3.4 Regulation of intestinal homeostasis by gut microbiota

The intestinal lumen harbors a vast and diverse community of gut microorganisms, which play a crucial role in maintaining gut health and function.^{123, 124} These gut microbiotas are essential for the establishment of a mature immune system during early embryonic development.^{125, 126} The gut microbiota stimulates the formation of the intestinal capillary network.¹²⁷ Chorionic capillaries in germ-free (GF) mice are underdeveloped after weaning and remain so into adulthood.¹²⁷ Additionally, early gut microbiota induces the secretion of antimicrobial peptides by Paneth cells, shaping the innate immune response in the intestinal epithelium.¹²⁸ Microbes during the early stages of intestinal development also activate the WNT pathway, enhance the expansion of Lgr5⁺ ISC. They promote the growth of intestinal organoids and facilitate regeneration of crypts after injury.¹²⁹ The intricate interactions between specific gut microbiota an ISC niches have been increasingly recognized as essential for maintaining intestinal homeostasis.^{130, 131} Two excellent reviews offer comprehensive insights into the intricate crosstalk between ISCs and the gut microbiota, elucidating the specific molecular mechanisms involved in this dynamic interaction.^{130, 131} Here, we have added the latest discoveries about the gut microbiota (Figure 2). The intestinal probiotic Lactobacillus reuteri has been shown to stimulate the secretion of IL-22 from lamina propria lymphocytes (LPLs), leading to the activation of STAT3 and subsequent acceleration of intestinal epithelial cell proliferation. Additionally, Lactobacillus reuteri promotes the proliferation of intestinal organoids by inducing the expression of R-spondins under homeostatic conditions, thereby sustaining the activation of the WNT/β-catenin pathway.¹¹² This probiotic also reduces the secretion of proinflammatory cytokines in the intestine, lowers concentrations of serum lipopolysaccharides (LPS), enhances the expression of antimicrobial peptides, and inhibits the colonization of Citrobacter rodentium.¹¹² Furthermore, a recent study revealed that Lactobacillus reuteri can promote oxytocin secretion from human intestinal tissues and organoids via proinsulin signaling.¹¹³ Lactic acid produced by Bifidobacterium and Lactobacillus has been shown to stimulate Paneth and stromal cells via G-protein-coupled receptor 81 (GPR81), thereby promoting epithelial regeneration. Furthermore, preadministration of lactic acid has demonstrated protective effects against irradiation-induced damage.¹¹⁴ While Bacteroides thetaiotaomicron produces tryptamine, which enhances intestinal transit after colonization of GF mice.¹¹⁵ Bacteroides fragilis—a species known to reinforce the intestinal epithelial barrier. It can derivative 3-phenylpropionic acid and promotes the integrity of the intestinal epithelial barrier by activating aryl hydrocarbon receptor (AhR) signaling.¹¹⁶ Additionally, various specialized gut microbiota is implicated in host metabolism. The Clostridium bifermentans and its metabolites increase the expression of the diacylglycerol O-acyltransferase 2 (Dgat2), which is associated with lipid absorption in the small intestine. It can enhance oleic acid uptake and regulate lipid absorption.¹¹⁷ In GF mice, the absence of intestinal microbiota impairs glucose uptake and storage capacity in skeletal muscle. It leads to an increase in brown adipogenesis, which prevents obesity. However, this metabolic alteration is accompanied by reduced ATP production, resulting in diminished exercise capacity. The specific microbial strains responsible for regulating this process remain unidentified.¹³²

4.1 Regulation of ISC by nutrient metabolism

4.1.3 Glucose metabolism

The glycolytic metabolism is the primary source of energy for organismal activity, and its intermediates serve as ample precursors for the synthesis of amino acids and nucleotides. While most cancer cells adhere to the “Warburg” metabolism, ISCs predominantly rely on aerobic mitochondrial metabolism, owing to their higher mitochondrial activity. The Paneth cells utilize anaerobic glycolysis to produce large amounts of lactate to fuel ISCs.¹⁶² Organoid formation was impaired with treatment of sodium dichloroacetate (DCA; glycolysis inhibitor) or OXPHOS inhibitor.¹⁶² Glycolysis is also closely linked to ISC fate decisions (Figure 3). In the intestinal epithelium, deletion HK2, an enzyme that controls the speed of glycolysis, resulted in reduced ISC self-renewal and increased secretory cells. Mechanistically, HK2 regulates secretory cell differentiation through increased ATOH1 expression and activation of p38 MAPK.¹⁴⁰ Interestingly, lactate was able to rescue the phenotype caused by HK2 deletion.¹⁴⁰ Under aerobic conditions, pyruvate produced from glycolysis is transported into the mitochondria through the mitochondrial pyruvate carrier 1 (MPC1), where it enters the tricarboxylic acid (TCA) cycle and undergoes aerobic catabolism. MPC1 is lowly expressed in the Lgr5⁺ ISC, but its expression significantly rises in the villus as ISC differentiation progresses. Deletion of MPC1 in the Lgr5⁺ ISC leads to an increase in the number of ISCs, but the number of mature differentiated cells (like goblet cells) remains unchanged. MPC1-deficient organoids derived from Lgr5-EGFP mice maintain a larger proportion of GFP-positive stem cells,¹⁴¹ suggesting that inhibition of MPC1-mediated pyruvate metabolism drives ISC self-renewal. But histone acetylation is reduced, and pyruvate metabolism is increased after MPC1 deletion. However, the mechanism by which MPC regulates ISC has not been determined.¹⁴¹

4.2 Metabolites regulate ISC function

Metabolites are intricately linked to the renewal of ISCs. As mentioned previously, Paneth cells produce large amounts of lactate, which enhances ISC OXPHOS to support stem cell function.¹⁶² Lactate stimulated the growth of intestinal organoids, and prompted a notable upregulation of Wnt3 and Wnt2b in stromal cells.¹¹⁴ Thus, coculture of stromal cells and lactate with intestinal organoids induced larger spherical organoids.¹¹⁴ Fatty acids play a critical role in maintaining ISCs, with exposure to palmitic or oleic acid resulting in increased Lgr5⁺ ISCs in the organoids. Palmitic and oleic acid also enhanced generation of organoids. Mechanistically, fatty acids activate PPAR-δ, which drives the organoid self-renewal process.¹⁵⁸ The β-OHB is key product of ketone body metabolism, supplementation of exogenous β-OHB increased Lgr5⁺ ISCs and improved the integrity and survival of crypts after injury.¹⁴³ Exogenous supplementation β-OHB has been shown to upregulate Oct4 and lamin B1 in vascular smooth muscle and endothelial cell, thereby mitigating aortic senescence in mice.¹⁸⁰

Amino acids serve not only as building blocks for proteins but also as precursors for the synthesis of a variety of signaling molecules and hormones. Gln is the most abundant nonessential amino acid in the body. In early weaned mice, the self-renewal process of ISCs was impaired, but exogenous Gln activated the WNT signaling pathway and promoted ISC self-renewal.¹⁸¹ Short-term Gln deprivation led to a reduction in the crypt domains of the organoids, while long-term Gln deprivation resulted in organoid atrophy.¹⁸¹ Gln also activated the YAP pathway, significantly attenuated intestinal damage in burned mice, and promoted crypt repair and regeneration.¹⁸² Cysteine (Cys), an essential amino acid, and its derivative—taurine are strongly associated with the aging process. The number of ISCs recovered after taurine supplementation in aging mice.¹⁸³ Methionine (Met) is one of the essential amino acids, and its metabolite, S-adenosylmethionine (SAM), is critical for ISCs. Met suppressed stem cell proliferation and promoted intestinal organoid differentiation. Additionally, deprivation of Met caused a reduced number of ISCs in Drosophila.^{139, 184} Tryptophan (Trp) is one of the essential amino acids and a precursor of hormones and neurotransmitters. Serotonin (5-HT) is derived from Trp, it serves as a neurotransmitter produced and secreted by enterochromaffin cells in the intestine. 5-HT plays a crucial role in driving self-renewal of ISCs. Valeric acid, a metabolite produced by gut microbes, stimulates the production of 5-HT in the intestine. Inhibition of 5-HT production led to reduced numbers of ISCs, as well as atrophy of crypt and villus. Additionally, it attenuated crypt regeneration after irradiation. Refeeding 5-HT significantly restored the number of ISCs. Mechanistically, 5-HT activated macrophages to produce PGE2, which in turn promoted ISC self-renewal via WNT/β-catenin.¹⁸⁵

4.3 Metabolic intermediates against pathological stimulation in the intestine

Metabolic intermediates play an important regulatory role in maintaining intestinal homeostasis against pathological stimulation. For example, lactate and pyruvate contribute to increasing intestinal resistance to Salmonella infection, potentially enhancing intestinal immunity.¹⁸⁶ The α-KG and malate, key products of the TCA cycle, have recently been shown to both activate macrophages and promote damage repair in the intestinal mucosa.¹⁸⁷ Supplementation with α-KG leads to a reduction in the abundance of pathogenic bacteria, including Ehrlichia and Enterococcus. It also enhances intestinal barrier function and facilitates the restoration of colon damage induced by DSS.¹⁸⁸ Gln also plays an important role in maintaining the intestinal barrier during intestinal injury. Gln supplementation increases blood glutathione (GSH) levels, reduces intestinal permeability, and restores small intestinal barrier function after ischemia/reperfusion injury.¹⁸⁹ Deprivation of Gln or inhibition of Gln synthetase in Caco-2 cells significantly diminishes the expression of tight junction proteins.¹⁹⁰ However, the specific mechanism by which Gln regulates intestinal barrier function is poorly understood. Additionally, the abundance of the Trp metabolites, xanthurenic acid (XANA) and kynurenic acid (KYNA), is negatively correlated with IBD. Supplementation with XANA and KYNA increases mitochondrial respiration, promotes intestinal epithelial cell proliferation, and facilitates repair in a DSS-induced colitis model. XANA and KYNA also facilitate the differentiation of Th17 cells, which strengthens the tight junctions of the intestinal epithelium to maintain the intestinal barrier.¹⁹¹

Metabolites also play roles in colorectal tumors. For example, α-KG promotes DNA and histone H3K4me3 hypomethylation, inhibits WNT signaling, and promotes cellular differentiation. Moreover, it inhibits CRC tumor growth and shows promising anticancer effects.¹⁹² Oleic acid reduces tumors in APC-Min mice,¹⁷⁰ while arachidonic acid (AA) promotes tumorigenesis. Mechanistically, feeding AA enriches gram-negative bacteria, which promotes the conversion of AA to PGE2 and ultimately promotes CRC development.¹⁹³ Gamma amino butyric acid (GABA) is an inhibitory neurotransmitter expressed in enteric nerves, immune cells, and endocrine-like cells.^{194, 195} GABA is involved in the regulation of gastrointestinal motility.¹⁹⁶ Activation of GABA receptors inhibits autophagy and enhances the immune response to prevent bacterial infection.¹⁹⁷ Additionally, GABA receptors have been found to be upregulated in the intestinal epithelium after treatment with 5-FU, irradiation, and DSS.^{194, 198} Both endogenous and exogenous GABA exacerbate intestinal injury by activating GABA receptors.¹⁹⁴ GABA regulates macrophages and participates in immune regulation. Supplementation with exogenous GABA promotes colon tumor growth. Mechanistically, GABA may facilitate the differentiation of monocytes into anti-inflammatory macrophages that secrete IL-10, consequently inhibiting the cytotoxic function of CD8 T cells.¹⁹⁹

4.4 The role of microbial metabolite in gut homeostasis

Metabolites produced by the gut microbiota are key molecular mediators in the crosstalk between the microbiota and the intestine (Table 3). These microbial metabolites can be broadly classified into two categories. The first category includes compounds produced by bacteria that may induce toxic responses in the host, such as LPS and bacterial endotoxins. The second category consists of two subgroups of metabolites associated with the gut microbiota. The first subgroup comprises metabolites generated by intestinal bacteria from dietary components, including short-chain fatty acids (SCFAs), tryptophan, and indole derivatives. The second subgroup involves metabolites initially produced by the host and subsequently modified by intestinal bacteria, such as bile acids.²⁰⁰ LPS enhances apoptosis through the modulation of Toll-like receptor 4 (TLR4), leading to a reduction in the proliferation of stem and progenitor cells.^{201, 202} Clostridioides difficile exerts its deleterious effects on colonic stem cells via the exotoxin TcdB, impairing the colon's capacity to repair damaged.²⁰³ SCFAs, such as acetate, butyrate, and propionate, are metabolites produced through microbial fermentation in the gut.²⁰⁴ SCFAs are capable of activating GPR43 in immune cells, thereby playing a significant role in the resolution of inflammation.²⁰⁵ Butyrate suppresses the proliferation of stem cells exposed to the intestinal lumen by acetylating histones and modulating the activity of FOXO3.²⁰⁶ Tryptophan and its metabolites play a crucial role in regulating intestinal homeostasis, particularly under pathological conditions. Elevated concentrations of tryptophan activate AhR, thereby inhibiting intestinal tumorigenesis through the regulation of E3 ubiquitin ligases ring finger protein 43 (RNF43) and zinc and ring finger 3 (ZNRF3), which suppress WNT/β-catenin signaling to prevent the overproliferation of ISCs.²⁰⁷ Additionally, indole-3-aldehyde stimulates lamina propria LPLs to secrete IL-22 via AhR activation, which subsequently induces STAT3 phosphorylation, accelerating the proliferation of intestinal epithelial cells and promoting the restoration of damaged intestinal mucosa.²⁰⁸ Indoleacrylic acid (IA) and indolepropionic acid (IPA) contribute to the protection of the intestinal epithelial barrier by enhancing macrophage function and modulating the inflammatory response, partially through pregnane X receptor (PXR) signaling.^{209, 210} Polyamines are small molecules derived from the metabolism of L-arginine, and the addition of spermidine (SPMD) resulted in the transformation of naive T cells from differentiation into Th17.²¹¹ The intestine is rich in bile acids, and bile is involved in various physiological processes through various receptors such as farnesoid X receptor (FXR), PXR, and vitamin D receptor (VDR).²¹² Bile extract or lithocholic acid (LCA) promotes intestinal organoid growth by activating G-protein-coupled bile acid receptor (TGR5) in the ISC, and LCA inhibits nuclear factor kappa B (NF-κB) signaling and activates SIRT1/Nrf2 by activating VDR, thereby increasing tight junction proteins and strengthening the epithelial barrier.^{213, 214} However, due to the vast variety of metabolites, many remain unknown and unidentified. In the future, advanced approaches in metabolic research are expected to identify more metabolites involved in regulating intestinal homeostasis.

5.1 Untargeted and targeted metabolomics

The maintenance of intestinal homeostasis is inextricably linked to the dynamic metabolic processes of enterocytes and the intestinal microbiota.¹³⁹ Dietary intake includes approximately 8000 non-nutritive compounds, such as dietary fiber, and polyphenols.²¹⁶ Most of these compounds cannot be digested by human digestive enzymes and are instead catabolized by gut microbiota, leading to the production of a wide array of metabolites.²¹⁷ The metabolic processes and pathways involved are highly dynamic. Metabolomics, the comprehensive analysis of metabolites in tissues and cells, allows for the detection of subtle changes in biological pathways. This analysis provides critical insights into the mechanisms underlying various physiological conditions and pathological processes. Moreover, metabolomics can be utilized to track metabolites produced by both the host and gut microbes, thereby enhancing our understanding of the complex metabolic interactions between gut microbiota and their hosts.²¹⁸ Metabolites are predominantly characterized using both untargeted and targeted mass spectrometry (MS)-based metabolomics approaches. Untargeted metabolomics aims to detect the dynamic changes of metabolites within a sample, followed by biostatistical analysis to identify differential metabolites. Targeted metabolomic focuses on the qualitative and quantitative analysis of a specific class of metabolites of interest. Most analyses of fecal metabolomics can be conducted with targeted metabolomics approaches.^{219, 220} Liquid chromatography (LC) and gas chromatography (GC) coupled with MS are among the most widely employed platforms in metabolomics research.^221-224 LC–MS and GC–MS have different application scenarios. LC–MS is highly sensitive and excels in the separation of thousands of small molecules, including polar metabolites such as organic acids, organic amines, nucleosides, nucleotides, and polyamines. GC–MS is particularly effective for fractionating less polar small molecules, such as alkyl derivatives, AAs, essential oils, esters, fragrances, terpenes, waxes, volatiles, carotenoids, flavonoids, and lipids.²²¹ In addition, GC–MS usually requires the derivatization of certain substances through alkylation, acylation, or silylation reactions to enable analysis.

5.2 Advances in metabolomics technologies

In fact, there are some challenges and advances in the application of metabolomics. First, both untargeted and targeted metabolomics do not provide information on intracellular metabolic rates and the relative activity of metabolic pathways. The most effective method for elucidating the dynamic metabolic status is metabolic flux analysis (MFA), which involves the use of stable isotopes, such as carbon-13 (¹³C), nitrogen-15 (¹⁵N), or deuterium (²H), for tracing purposes.²²⁵ Isotopic labeling of specific compounds, including nutrients or substrates, allows for the detailed tracking of their metabolic fate within the body.^{226, 227} Using ¹³C₁₆-palmitate and ¹³C₂-acetate, we followed the metabolic flux and found that loss of HNF4 in small intestinal organoids resulted in impaired TCA cycle but increased fatty acid synthesis.⁸³ Second, some metabolites may remain undetectable due to their low abundance or poor ionization efficiency. Derivatization can alter the properties of metabolites and enhance detection sensitivity. It can also facilitate the design of subsequent enrichment steps. For example, ketones and aldehydes are particularly challenging to detect and identify with LC–MS. Conway et al. developed a chemoselective probe immobilized on magnetic beads to improve the metabolites concentration and ionization efficiency. They successfully identified 112 ketone and aldehyde metabolites, and elucidated their exact chemical structures.²²⁸ Third, the identification of metabolites typically depends on database searches, which can result in the neglect of many unknown metabolites. The focus of microbiome research is increasingly shifting from the gut microbiota to their small molecules.²²⁹ However, a large number of microbiome-derived metabolites are not recorded in databases. To address this, researchers have proposed a new approach to metabolomics research—reverse metabolomics. It first requires newly synthesized compounds, followed by LC–MS/MS to obtain the mass spectra of the compounds. Then researchers utilized the Mass Spectrometry Search Tool (MASST) program to search the obtained MS spectra within the existing MS datasets and explored associations between metabolites and various phenotypes, species, and sample types. Using this approach, researchers have identified that bile acids bound to amino acids, such as glutamate, isoleucine/leucine, phenylalanine, threonine, tryptophan, and tyrosine, are elevated in the feces of patients with CD. Additionally, they discovered that cholestatic amide compounds produce by Bifidobacterium, Clostridium, and Enterococcus may contribute to intestinal inflammation by modulating IFNγ production and activating the PXR in T cells.²³⁰

5.3 Future directions in metabolomics research: Single-cell and spatial levels of metabolomics

Currently, using untargeted and targeted metabolomics techniques can obtain information at the bulk tissue level. However, these methods are limited in their ability to track dynamic metabolism at the single-cell and spatial levels. For example, stem and progenitor cells within the intestinal epithelium are relatively scarce, but understanding their metabolic characteristics at the single-cell level is crucial for studying intestinal homeostasis or disease pathogenesis. Metabolomics analysis typically requires millions of cells.²³¹ Consequently, the use of small-scale metabolomics to analyze metabolic changes within cellular subpopulations and achieve spatially resolved metabolic profiling represents a critical direction for future research in metabolomics. Recently, researchers have employed flow cytometry in conjunction with hydrophilic interaction liquid chromatography (HILIC) and high-sensitivity Orbitrap MS. A total of 160 metabolites were identified using this advanced analytical technique in 10,000 cells.²³² Additionally, hyperpolarized micromagnetic resonance spectroscopy (HMRS) technology has been developed by integrating nuclear magnetic resonance (NMR) spectroscopy and imaging with hyperpolarization of nuclear spins. This method allows for real-time metabolic analysis in intact cells or organs and requires only approximately 10,000 cells.²³² Furthermore, a class of metabolite-specific biosensors has been developed, which can be combined with single-cell techniques to dynamically monitor changes in specific metabolites at the single-cell level. Wu et al. developed a lactate biosensor. When paired with single-cell imaging, it enables the measurement of glycolytic metabolites. The lactate, glucose, pyruvate, and ATP can be detected in individual endothelial cells.²³³ Similarly, Tao et al. designed a genetically encoded fluorescent indicator for nicotinamide adenine dinucleotide phosphate (NADPH). It is capable of quantifying cytosolic and mitochondrial NADPH pools in a single living cell.²³⁴

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) enables spatial localization of target metabolites and proteins within tissues.^{229, 235} Recently, researchers have developed SpaceM, a technique based on MALDI imaging MS combined with optical microscopy. This method utilizes optical microscopy for image segmentation and calibration, followed by MALDI imaging to analyze metabolites. SpaceM offers nontargeted metabolic profiles, fluorescence intensity measurements, and spatial morphological characteristics at the single-cell level.²³⁶ For example, MALDI-MSI has been employed to detect and spatially map 42 lipid species produced by Bacteroides thetaiotaomicron in the mouse colon.²³⁷ Furthermore, combining MALDI-MSI with stable isotope tracing allows for the assessment and visualization of region-specific metabolism. ¹³C-labeled glycerol and glucose were used to directly visualize glycolytic and gluconeogenic activities in distinct regions of the mouse kidney.²³⁵ Additionally, to study metabolic differences within organelles, techniques such as organelle precipitation enrichment have been developed. Mitochondrial immunopurification (MITO IP) enables the quantification of metabolites specifically within mitochondria,²³⁸ and this approach has now been extended to lysosomes and peroxisomes.^{239, 240} In summary, single-cell and spatial levels of metabolomics show tantalizing promise, but large-scale applications have yet to be realized.