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Adult Stem Cell Behavior Follows a Caudal to Rostral Pattern (A) Schematic diagram of the Proximal, Mid, and Distal parts of the small intestine and the representative images of cultures derived at P2. (B) Relative proportion of FEnS and organoids in the different sections of the small intestine. (C) Expression analysis in material isolated from Proximal, Mid, and Distal regions. Data represent the mean, and the error bars, the SEM (n = 3). Data are expressed relative to Proximal, on a Log 2 scale.
Source publication
Regeneration and homeostasis in the adult intestinal epithelium is driven by proliferative resident stem cells, whose functional properties during organismal development are largely unknown. Here, we show that human and mouse fetal intestine contains proliferative, immature progenitors, which can be expanded in vitro as Fetal Enterospheres (FEnS)....
Contexts in source publication
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... overall morphology and growth of FEnS as spheres are reminis- cent of that reported for organoids that form as a result of augmented Wnt signaling following loss of APC ( Sato et al., 2011b). However, expression analysis demonstrates distinct expression patterns between FEnS and APCnull organoids (Fig- ure S3B). In particular, it is clear that loss of APC causes increased levels of adult stem cell markers, whereas these are generally reduced in the fetal state ( Figure S3B). ...
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... expression analysis demonstrates distinct expression patterns between FEnS and APCnull organoids (Fig- ure S3B). In particular, it is clear that loss of APC causes increased levels of adult stem cell markers, whereas these are generally reduced in the fetal state ( Figure S3B). In summary, this demonstrates that progenitors within the fetal small intestine have a unique behavior that sets them aside from both normal and cancerous adult stem cells. ...
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... maturation in vivo has been proposed to follow a wave from proximal to distal sites ( Spence et al., 2011a). To assess the positional effect along the length of the small intestine, we analyzed the regional differences in in vitro growth potential at postnatal day 2 ( Figure 3A). Contrary to expectations, FEnS formed from proximal tissue, whereas more distal tissues formed organoids ( Figures 3A and 3B). ...
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... assess the positional effect along the length of the small intestine, we analyzed the regional differences in in vitro growth potential at postnatal day 2 ( Figure 3A). Contrary to expectations, FEnS formed from proximal tissue, whereas more distal tissues formed organoids ( Figures 3A and 3B). Gene expression analysis showed that the ability to form organoids correlates with increased levels of Lgr5 and Axin2 ( Figure 3C). ...
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... to expectations, FEnS formed from proximal tissue, whereas more distal tissues formed organoids ( Figures 3A and 3B). Gene expression analysis showed that the ability to form organoids correlates with increased levels of Lgr5 and Axin2 ( Figure 3C). Analysis of the cultured material from the proximal and mid regions of the small intestine shows variable but comparable expression of Wnt target genes, suggesting that FEnS can respond to Wnt stimula- tion and that this represents a transitory and dynamic cellular state ( Figure 3D). ...
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... expression analysis showed that the ability to form organoids correlates with increased levels of Lgr5 and Axin2 ( Figure 3C). Analysis of the cultured material from the proximal and mid regions of the small intestine shows variable but comparable expression of Wnt target genes, suggesting that FEnS can respond to Wnt stimula- tion and that this represents a transitory and dynamic cellular state ( Figure 3D). In line with the observed adult stem cell behavior, the distal part of the small intestine expresses higher levels of secretory lineage markers, which are characteristic of the adult small intestine, and contains a greater number of Ulex europaeus agglutinin I (UEA-I) reactive secretory cells (Figures 3C, 3E-3E 00 , and 3F). ...
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... Lgr5 and Axin2 are both Wnt target genes, and given the dynamic regional expression correlating with organoid formation ( Figure 3C), we investigated whether Wnt3a can induce intestinal maturation in vitro. Stimulation of cells from E16 proximal intes- tine, which normally only form FEnS, promoted the transition into budding organoids in a proportion of the forming structures (Fig- ure S4D). ...
Similar publications
Human intestinal organoids (HIOs) are a tissue culture model in which small intestine-like tissue is generated from pluripotent stem cells. By carrying out unsupervised hierarchical clustering of RNA-sequencing data, we demonstrate that HIOs most closely resemble human fetal intestine. We observed that genes involved in digestive tract development...
Citations
... Upon intestinal damage, the loss of ISCs and reversion to a fetal-like state significantly reduces OLFM4 expression in the intestine [74]. In organoids, a higher OLFM4 expression level correlates with maturation and the ability to bud and differentiate into different lineages [75,76]. Therefore, enhancing adult ISC function could be a key therapeutic goal in NEC treatment, as it facilitates the regeneration and maturation of the injured premature intestine. ...
Background
Necrotizing enterocolitis (NEC) is a critical gastrointestinal disease in preterm infants, for which no specific treatment is established. We previously demonstrated that thrombin-preconditioned mesenchymal stromal cell-derived extracellular vesicles (thMSC-EVs) enhance protection against other neonatal tissue injuries. Therefore, this study aimed to evaluate the therapeutic potential of thMSC-EVs in modified in vitro, in vivo, and organoid models of NEC.
Methods
In vitro, the effects of thMSC-EVs and naïveMSC-EVs were compared in hyperosmotic, ischemic, and hypothermic (HIT)-stressed IEC-6 cells and LPS-treated peritoneal macrophages. In vivo, NEC was induced in P4 mouse pups by three cycles of formula feeding, oral LPS administration, hypoxia, and hypothermia, followed by overnight dam care. 2 × 10⁹ thMSC-EVs were intraperitoneally administered daily for three days, and the therapeutic effects were assessed macroscopically, histologically, and biochemically. NEC mouse-derived organoids were established to evaluate the thMSC-EVs’ effect in mature enterocytes. LC-MS/MS was performed to analyze the EV proteomics.
Results
In vitro, compared with naïveMSC-EVs, thMSC-EVs significantly improved cellular viability in HIT-induced IEC-6 cells and reduced pro-inflammatory (IL-1α, IL-1β, TNF-α) but increased anti-inflammatory (TGF-b) cytokine levels in LPS-treated peritoneal macrophages. In vivo, thMSC-EVs significantly attenuated clinical symptoms, reduced intestinal damage, and retained intestinal stem cell markers, showing more significant localization in NEC-induced intestines than in healthy intestines. In NEC mouse-derived organoids, thMSC-EVs significantly increased OLFM4 and claudin-4 expression and reduced stress-related markers such as sucrase-isomaltase, defensin, and chromogranin A. Proteomic analysis revealed that thMSC-EVs were greater enriched in anti-apoptotic, anti-inflammatory, cell adhesion, and Wnt signaling pathways than naïveMSC-EVs.
Conclusion
thMSC-EVs improved cellular viability, reduced apoptosis, attenuated inflammation, and upregulated key intestinal stem cell markers, collectively suggesting their tissue-protective effects and highlighting their potential as a treatment for NEC.
... For example, researchers have demonstrated that mouse colon organoids can be effectively expanded in vitro and successfully transplanted into damaged mice 145 , where they remodel functional crypt units. Similarly, fetal progenitor-derived small intestinal organoids have exhibited the ability to integrate and function in vivo 146 . When intestinal organoids derived from PSCs were implanted under the kidney capsule of mice, they displayed good permeability and peptide uptake, highlighting their potential in treating conditions like short bowel syndrome 147 . ...
Organoids are three-dimensional (3D) cell cultures derived from human pluripotent stem cells or adult stem cells that recapitulate the cellular heterogeneity, structure, and function of human organs. These microstructures are invaluable for biomedical research due to their ability to closely mimic the complexity of native tissues while retaining human genetic material. This fidelity to native organ systems positions organoids as a powerful tool for advancing our understanding of human biology and for enhancing preclinical drug testing. Recent advancements have led to the successful development of a variety of organoid types, reflecting a broad range of human organs and tissues. This progress has expanded their application across several domains, including regenerative medicine, where organoids offer potential for tissue replacement and repair; disease modeling, which allows for the study of disease mechanisms and progression in a controlled environment; drug discovery and evaluation, where organoids provide a more accurate platform for testing drug efficacy and safety; and microecological research, where they contribute to understanding the interactions between microbes and host tissues. This review provides a comprehensive overview of the historical development of organoid technology, highlights the key achievements and ongoing challenges in the field, and discusses the current and emerging applications of organoids in both laboratory research and clinical practice.
... Gene expression analysis revealed that pluripotent stem cell-derived IECs are more similar to fetal IECs than adult IECs [36]. To confirm whether the in vitro replication of GII.2 HuNoV is specific to adult tissue-derived IECs, we prepared IECs from several other human iPSC lines: 1383D6 (to IEC#25), TkPP7 (to IEC#29), TkC01 (to IEC#33), and TkD2 (to IEC#34), and infected them with GII.2 HuNoV. ...
Background
Human norovirus (HuNoV) is a major cause of enteric infectious gastroenteritis and is classified into several genotypes based on its capsid protein amino acid sequence and nucleotide sequence of the polymerase gene. Among these, GII.4 is the major genotype worldwide. Epidemiological studies have highlighted the prevalence of GII.2. Although recent advances using human tissue- and induced pluripotent stem cell (iPSC)-derived intestinal epithelial cells (IECs) have enabled in vitro replication of multiple HuNoV genotypes can replicate, GII.2 HuNoV could only replicate in tissue-derived IECs, but not in iPSC-derived IECs.
Methods
We investigated the factors influencing GII.2 HuNoV replication in IECs, focusing on histo-blood group antigens. Additionally, we assessed the immunogenicity of GII.2 virus-like particles (VLPs) and their ability to induce neutralizing antibodies. Antibody cross-reactivity was tested to determine if GII.2 VLPs could neutralize other HuNoV genotypes, including GII.4, GII.3, GII.6, and GII.17.
Results
Our findings indicated that GII.2 HuNoV replication in vitro requires the presence of both H and B antigens. Moreover, GII.2 VLPs generated neutralizing antibodies effective against both GII.2 and GII.4 but not against GII.3, GII.6, or GII.17. Comparatively, GII.2 and GII.17 VLPs induced broader neutralizing responses than GII.4 VLPs.
Conclusions
This study suggests that GII.2 and GII.17 VLPs may be advantageous as HuNoV vaccine candidates because they elicit neutralizing antibodies against the predominant GII.4 genotype, which could be particularly beneficial for infants without prior HuNoV exposure. These insights will contribute to the development of effective HuNoV vaccines.
... 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. ...
... 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. ...
... 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 ...
Metabolism serves not only as the organism's energy source but also yields metabolites crucial for maintaining tissue homeostasis and overall health. Intestinal stem cells (ISCs) maintain intestinal homeostasis through continuous self‐renewal and differentiation divisions. The intricate relationship between metabolic pathways and intestinal homeostasis underscores their crucial interplay. Metabolic pathways have been shown to directly regulate ISC self‐renewal and influence ISC fate decisions under homeostatic conditions, but the cellular and molecular mechanisms remain incompletely understood. Understanding the intricate involvement of various pathways in maintaining intestinal homeostasis holds promise for devising innovative strategies to address intestinal diseases. Here, we provide a comprehensive review of recent advances in the regulation of intestinal homeostasis. We describe the regulation of intestinal homeostasis from multiple perspectives, including the regulation of intestinal epithelial cells, the regulation of the tissue microenvironment, and the key role of nutrient metabolism. We highlight the regulation of intestinal homeostasis and ISC by nutrient metabolism. This review provides a multifaceted perspective on how intestinal homeostasis is regulated and provides ideas for intestinal diseases and repair of intestinal damage.
... In an animal study of experimental colitis, the successful transplantation of intestinal stem cells (ISCs), which are in charge of tissue homeostasis and damage response, has shown that they stick to and integrate with the epithelium, enhancing mucosal repair. ISCs are found near the base of the intestinal crypts, where they promote mucosal regeneration and replace the epithelium by differentiating into many epithelial progenies (Fordham et al., 2013;Fukuda et al., 2014;Yui et al., 2012). Organoids, three-dimensional self-organizing structures can be created in vitro by cultivating ISCs. ...
Human body structure and reaction to environmental stimuli are
subject to change in tandem with changing living situations. Besides
genetically transmitted conditions a sedentary lifestyle and the intake of
processed foods can lead to organ dysfunctions, particularly in the digestive
and absorption systems. Is it possible that the observed diseases are a result
of the current morphology of our digestive organs in comparison to other
mammalian species?
Crohn's disease and ulcerative colitis are examples of inflammatory
bowel diseases (IBD), which result in inflammation and gastrointestinal
issues. Geographical location, an improper diet, heredity, and an inadequate
immune response are just a few of the many contributing factors causing
IBD. Intestinal tissue cells are essential for digestive function and nutrition
uptake. As a recent development, stem cell and organoid technology enables
the manipulation and efficient expansion of intestinal epithelial tissue by the
laboratory in vitro method and is very important in medical research and the
progress of new treatments. Here, a current discussion of stem cell
technology in the management of IBD is provided in the context of the
literature.
... Enteroids have been used to replace damaged intestinal epithelium in animal models. [3][4][5][6][7][8][9][10][11][12] Furthermore, an enteroid-based therapeutic is currently being clinically evaluated by Tokyo Medical and Dental University in Japan (Trial UMIN000013524). In the trial, biopsies from healthy gut regions are obtained from patients with inflammatory bowel disease (IBD) with refractory ulcers and used to generate enteroids. ...
... 3 Several similar reports have also demonstrated the ability of enteroids to contribute to intestinal and colonic epithelial healing; however, efficiencies were rarely reported. [4][5][6][7][8][9][10][11][12] Consistent with our data on retained regional identity following engraftment, Sugimoto et al. demonstrated sites of ileal enteroid engraftment retained their small bowel identity within the mouse colon. 10 To our knowledge, this is the first report of using multi-lineage seeding material, epithelial and mesenchymal, to achieve in vivo transmural intestinal repair as a cell therapy without loop/segment removal from the host. ...
... The expression level of Cdx2 was increased and those of Alb and Vim were decreased after the formation and passages of SOs (Fig. 5b). These SOs were composed of actively proliferating, polarized intestinal epithelial cells that express E-cad basolaterally, Villin (also known as Vil1) apically, and nuclear Cdx2 and Sox9 ( Fig. 5c and Supplementary Fig. 6a), similar to FIPC-derived SOs 28,29 . Also, dediHep-derived SOs were formed by cells expressing CK19 and Hnf4α but not Alb, indicating that these SOs were not biliary organoids that express CK19 but not Hnf4α and Alb ( Supplementary Fig. 6b). ...
... In addition to our in vitro analyses, we sought to assess whether dediHep-derived intestinal epithalial cells could contribute to tissue reconstitution in vivo. It is known that FIPCs contribute to regeneration of adult colonic epithelium after transplantation into a chemicallyinduced colonic injury model 29 . Thus, we examined whether dediHepderived intestinal epithelial cells behaved similarly. ...
... Thus, we examined whether dediHepderived intestinal epithelial cells behaved similarly. To this end, dediHep-derived SOs were transplanted into the colons of immunodeficient NOD/SCID/gamma (NSG) mice with dextran sulfate sodium (DSS)-induced acute colitis 29,30 . In mice transplanted with dediHepderived SOs, donor cell clusters were macroscopically observed in colonic tissues 3 months after transplantation (Fig. 5f). ...
Hepatocytes play important roles in the liver, but in culture, they immediately lose function and dedifferentiate into progenitor-like cells. Although this unique feature is well-known, the dynamics and mechanisms of hepatocyte dedifferentiation and the differentiation potential of dedifferentiated hepatocytes (dediHeps) require further investigation. Here, we employ a culture system specifically established for hepatic progenitor cells to study hepatocyte dedifferentiation. We found that hepatocytes dedifferentiate with a hybrid epithelial/mesenchymal phenotype, which is required for the induction and maintenance of dediHeps, and exhibit Vimentin-dependent propagation, upon inhibition of the Hippo signaling pathway. The dediHeps re-differentiate into mature hepatocytes by forming aggregates, enabling reconstitution of hepatic tissues in vivo. Moreover, dediHeps have an unexpected differentiation potential into intestinal epithelial cells that can form organoids in three-dimensional culture and reconstitute colonic epithelia after transplantation. This remarkable plasticity will be useful in the study and treatment of intestinal metaplasia and related diseases in the liver.
... Successful transplantation of organoids for regenerative purposes has been demonstrated in mouse models of intestinal and salivary gland injury. [159][160][161] Transplantation of organoids from these two tissues is currently in human clinical trials to test for functional organ recovery after cell therapy. WNT mimetics may potentially provide an abundant and sustainable source of cells for therapeutic purposes. ...
Wingless-related integration site or Wingless and Int-1 or Wingless-Int (WNT) signaling is crucial for embryonic development, and adult tissue homeostasis and regeneration, through its essential roles in cell fate, patterning, and stem cell regulation. The biophysical characteristics of WNT ligands have hindered efforts to interrogate ligand activity in vivo and prevented their development as therapeutics. Recent breakthroughs have enabled the generation of synthetic WNT signaling molecules that possess characteristics of natural ligands and potently activate the pathway, while also providing distinct advantages for therapeutic development and manufacturing. This review provides a detailed discussion of the protein engineering of these molecular platforms for WNT signaling agonism. We discuss the importance of WNT signaling in several organs and share insights from the initial application of these new classes of molecules in vitro and in vivo. These molecules offer a unique opportunity to enhance our understanding of how WNT signaling agonism promotes tissue repair, enabling targeted development of tailored therapeutics.
... Moreover, intestinal organoids derived from fetal mice demonstrated cell differentiation identical to that of surrounding recipient tissues. 30 Thus, both fetal and adult intestinal epithelial organoids may serve as sources of cells for generating a functional epithelium in the gastrointestinal tract. ...
... We performed motif analysis in open chromatin regions identified by ATAC-seq and found increased chromatin accessibility for transcription factors of the SOX family (SOX2, SOX3, SOX4, SOX6, SOX9, SOX10, SOX15 and SOX17; P < 10 −14 ) in primary AKP organoids (Supplementary Table 1), consistent with a study that demonstrated increased chromatin accessibility to SOX family members during human CRC tumour evolution 17 . Of note, gene set enrichment analysis (GSEA) of RNA-seq data also revealed an enhancement of a fetal intestinal gene expression signature [18][19][20] in primary tumour-derived AKP organoids (Fig. 1c), indicating reactivation of developmental genes in the primary tumour-derived AKP organoids. To identify transcription factors that regulate the transcriptomic and epigenetic alterations in primary tumour-derived AKP organoids, we integrated and compared RNA-seq DGE and ATAC-seq open promoter peak and motif enrichment analyses. ...
A hallmark of cancer is the avoidance of immune destruction. This process has been primarily investigated in locally advanced or metastatic cancer1–3; however, much less is known about how pre-malignant or early invasive tumours evade immune detection. Here, to understand this process in early colorectal cancers (CRCs), we investigated how naive colon cancer organoids that were engineered in vitro to harbour Apc-null, KrasG12D and Trp53-null (AKP) mutations adapted to the in vivo native colonic environment. Comprehensive transcriptomic and chromatin analyses revealed that the endoderm-specifying transcription factor SOX17 became strongly upregulated in vivo. Notably, whereas SOX17 loss did not affect AKP organoid propagation in vitro, its loss markedly reduced the ability of AKP tumours to persist in vivo. The small fraction of SOX17-null tumours that grew displayed notable interferon-γ (IFNγ)-producing effector-like CD8⁺ T cell infiltrates in contrast to the immune-suppressive microenvironment in wild-type counterparts. Mechanistically, in both endogenous Apc-null pre-malignant adenomas and transplanted organoid-derived AKP CRCs, SOX17 suppresses the ability of tumour cells to sense and respond to IFNγ, preventing anti-tumour T cell responses. Finally, SOX17 engages a fetal intestinal programme that drives differentiation away from LGR5⁺ tumour cells to produce immune-evasive LGR5⁻ tumour cells with lower expression of major histocompatibility complex class I (MHC-I). We propose that SOX17 is a transcription factor that is engaged during the early steps of colon cancer to orchestrate an immune-evasive programme that permits CRC initiation and progression.
