Fig 1 - uploaded by Jason R Spence
Content may be subject to copyright.
Schematic of human pluripotent stem cell-derived intestinal organoids. Human-induced pluripotent (iPSC) or embryonic (hESC) stem cells are differentiated into FOXA2/SOX17 positive endoderm with [85 % efficiency. A small proportion of cells (*2–5 %) also differentiate into Brachyury (T)-positive mesoderm. Induction of the intestinal epithelial transcription factor CDX2 is achieved by activating FGF and WNT signaling for 4 days. The mesenchymal population expands and expresses the intestine–mesenchyme
Source publication
Many significant advances in our understanding of intestine development, intestinal stem cell homeostasis and differentiation have been made in recent years. These advances include novel techniques to culture primary human and mouse intestinal epithelium in three-dimensional matrices, and de novo generation of human intestinal tissue from embryonic...
Similar publications
Intestinal cell models have been widely studied and used to evaluate absorption and metabolism of drugs in the small intestine, constituting valuable tools as a first approach to evaluate the behavior of new drugs. However, such cell models might not be able to fully predict the absorption mechanisms and metabolic pathways of the tested compounds....
Increasing evidence suggests that in many cancer types only a minor proportion of cells, the so-called 'cancer stem cells', is responsible for fostering continuous tumour growth. Similar to the non-malignant stem cells that maintain tissue homeostasis, cancer stem cells are seemingly able to self-renew indefinitely. A recent study from the lab of W...
Citations
... These stem cells have been used to establish the 3-dimensional (3D) multicellular intestinal organoids, also known as the enteroids, which recapitulate intestine epithelial structure and function and have been widely used for studying intestinal physiology, pathophysiology, molecular mechanisms of host-pathogen interactions and intestinal disease in mammals. Enteroids are defined as a mimic of an intestinal organ following 3 main capabilities, exhibiting multiple organ-specific cell types, recapitulating organ-specific functions, and organizing into a structure like an organ in vivo (Finkbeiner and Spence, 2013). ...
This symposium offered up-to-date perspectives on field experiences and the latest research on significant viral and bacterial diseases affecting poultry. A highlight was the discussion on the use of enteroids as advanced in vitro models for exploring disease pathogenesis. Outcomes of this symposium included identifying the urgent need to improve the prevention and control of avian influenza by focusing research on vaccine effectiveness. In this regard, efforts should focus on enhancing the relatedness of vaccine antigen to the field (challenge) virus strain and improving immunogenicity. It was also revealed that gangrenous dermatitis could be controlled through withholding or restricting the administration of ionophores during broiler life cycle, and that administration of microscopic polymer beads (gel) based-live coccidia vaccines to chicks could be used to reduce necrotic enteritis-induced mortality. It was emphasized that effective diagnosis of re-emerging Turkey diseases (such as blackhead, fowl cholera, and coccidiosis) and emerging Turkey diseases such as reoviral hepatitis, reoviral arthritis, Ornithobacterium rhinotracheale infection, and strepticemia require complementarity between investigative research approaches and production Veterinarian field approaches. Lastly, it was determined that the development of a variety of functionally-specific enteroids would expedite the delineation of enteric pathogen mechanisms and the identification of novel vaccine adjuvants.
... We have previously investigated human iPS cell-derived intestinal organoids (HIOs) and reported a method for culturing HIOs with a budding-like structure and pharmacokinetic functions from human iPS cells 10 . HIOs are characterized by the abundance of multiple types of cells that constitute the intestinal tract [13][14][15] . Therefore, they might be more versatile than two-dimensional structures. ...
... Therefore, they are not adequate to evaluate complex cell-cell interactions, interactions with intestinal bacteria, and responses to in ammation. Intestinal organoids contain a variety of cell types that comprise the intestinal tract and might be similar in nature to living organisms [13][14][15] . Nevertheless, their characteristic morphology with luminal structures is not suitable for use in HTS and quantitative evaluation. ...
Reproducing intestinal cells in vitro is important in pharmaceutical research and drug development. Currently, Caco-2 cells and human induced pluripotent stem cell (iPS cell)-derived intestinal epithelial cells are widely used, but there are a few evaluation systems that mimic the three-dimensional structure of the crypt-villus-like structure. Therefore, we attempted to generate intestinal cells mimicking the three-dimensional structure from human iPS cells. After inducing the differentiation of iPS cells into intestinal organoids, they became single-celled and were cultured two-dimensionally. Air–liquid interface culture was used, and CHIR99021, forskolin , and A-83-01 were used as key compounds. In addition to general analyses, the 3D structure of cells in culture was observed using Cell3iMager Estier to quantify cell irregularities and height. Long-term culture was also performed by adding Wnt3a, Noggin, and RSPO1, which are frequently used in organoid culture. For results, the combination of air–liquid interface culture and several compounds successfully induced the formation of a crypt-villus-like structure, which grew rapidly around day 6. The expression of pharmacokinetic genes such as CYP3A4 was also enhanced. Moreover, the addition of Wnt3a, Noggin, and RSPO1 stabilized the expression of genes involved in the maintenance of intestinal stem cells, and the cells were successfully cultured for a long period of 22 days. We were able to construct a crypt-villus-like convexitystructure on a very simple culture platform, viz., cell culture inserts. The characteristics of this structure were extremely similar to those of living intestinal tissues, and it might possess homeostatic mechanisms similar to those of living organisms.
... However, it is unclear whether intestinal organoids represent the cellular heterogeneity observed in vivo. By carrying out unsupervised hierarchical clustering of scRNAseq data, Fujii et al. (2018) and Finkbeiner and Spence (2013) showed that human intestinal organoids (HIOs) most closely resembled the human fetal intestine, in which genes related to digestive function and Paneth cell host defense were expressed at low levels. This study also revealed that the ISCs marker OLFM4 expression was limited in the fetal intestine and HIOs, but robust in adult crypts. ...
The intestinal tract is composed of different cell lineages with distinct functions and gene expression profiles, providing uptake of nutrients and protection against insults to the gut lumen. Changes in or damage to the cellulosity or local environment of the intestinal tract can cause various diseases. Single-cell RNA sequencing (scRNA-seq) is a powerful tool for profiling and analyzing individual cell data, making it possible to resolve rare and intermediate cell states that are hardly observed at the bulk level. In this review, we discuss the application of intestinal tract scRNA-seq in identifying novel cell subtypes and states, targets, and explaining the molecular mechanisms involved in intestinal diseases. Finally, we provide future perspectives on using single-cell techniques to discover molecular and cellular targets and biomarkers as a new approach for developing novel therapeutics for intestinal diseases.
... Human PD-L1 PCR primers: forward, 5'-TCCA CTCAATGCCTCAAT and reverse, 5'-GAAGACCTCACAGACTCAA. Human LgR5 PCR primers were described by other groups [53]: forward, 5'-CA GCGTCTTCACCTCCTACC and reverse, 5'-TGGGAATGTATGTCAGAGCG. The relative expression of the target gene is the averages of triplicates that are normalized against the transcription levels of β-Actin RNA. ...
Cancer stem cells (CSCs) drive tumor initiation, progression, metastasis, and drug resistance. We report here that programmed cell death ligand 1 (PD-L1) is constitutively expressed in cancer cells to maintain and expand CSC through a novel mechanism in addition to promoting cancer cell immune evasion. We discovered that PD-L1 interacts with receptor Frizzled 6 to activate β-catenin signaling and increase β-catenin-targeted gene expression, such as a putative stem cell marker leucine-rich-repeat-containing G-protein-coupled receptor 5. Blockage of PD-L1 function, using a specific small hairpin RNA or a specific antibody, inhibits disease progression by reducing the CSC population in both colorectal and breast tumors. Moreover, β-catenin conversely regulates PD-L1 expression through a β-catenin complex binding site in the PD-L1 promoter. Our discoveries reveal that besides assistant tumor cell immune escaping, PD-L1 and β-catenin signaling form a positive feedback loop to promote cancer progression through CSC maintenance and expansion.
... Three-dimensional (3D) cell culture allows for a clearer understanding of disease mechanisms, e.g., for inflammatory bowel disease or colorectal cancer. It provides more reliable results in drug screening, especially for orally administered drugs and their uptake by the gut [1][2][3][4][5][6][7] due to the presence of mucus and different cells and cell layers. However, it is still not yet frequently used for routine lab work, as producing realistic complex intestinal in vitro models is time-consuming. ...
Three-dimensional (3D) complex in vitro cell systems is well suited to providing meaningful and translatable results in drug screening, toxicity measurements, and biological studies. Reliable complex gastrointestinal in vitro models as a testbed for oral drug administration and toxicity are valuable in achieving predictive results for clinical trials and reducing animal testing. However, producing these models is time-consuming due to the lengthy differentiation of HT29 or other cells into mucus-producing goblet cells or other intestinal cell lineages. In the present work, HT29 cells were grown on an inorganic topographic surface decorated with a periodic pattern of micrometre-sized amorphous SiO2 structures for up to 35 days. HT29 cells on topographic surfaces were compared to undifferentiated HT29 in glucose-containing medium on glass or culture dish and with HT29 cells differentiated for 30 days in the presence of methotrexate (HT29-MTX). The cells were stained with Alcian blue for mucus, antibodies for mucus 2 (goblet cells), villin (enterocytes), lysozyme (Paneth cells), and FITC-labeled lectins to identify different cells, glycomic profiles, and cell features. We observed that HT29 cells on topographic surfaces showed more similarities with the differentiated HT29-MTX than with undifferentiated HT29. They formed islands of cell clusters, as observed for HT29-MTX. Already after 2 days, the first mucus secretion was shown by Alcian blue stain and FITC-wheat germ agglutinin. After 4–6 days, mucus was observed on the cell surface and in the intercellular space. The cell layer was undulated, and in 3D reconstruction, the cells showed a clear polarisation with a strong actin signal to one membrane. The lectins and the antibody-staining confirmed the heterogeneous composition of differentiated HT29 cells on topographic surfaces after 6–8 days, or after 6–8 days following MTX differentiation (30 days).
... 16−18 Human induced pluripotent stem (iPS) cell-derived intestinal organoids include not only intestinal epithelial cells, but also mesenchymal cells, unlike tissue-derived intestinal organoids. 16,19,20 Therefore, human iPS cell-derived intestinal organoids would be a powerful tool for screening therapeutic targets and to study the pathophysiology of diseases in the small intestine. 15,21 Although most intestinal organoids are currently cultured in Matrigel, 16,18,22 large numbers cannot be obtained using this method. ...
Human induced pluripotent stem (iPS) cell-derived intestinal organoids have low invasiveness; however, the current differentiation method does not reflect the crypt-villus-like structure due to structural immaturity. Here, we generated budding-like organoids that formed epithelial tissue-like structures and had the characteristics of the mature small intestine from human iPS cells. They showed a high expression of drug transporters and induced the expression of cytochrome P450 3A4 and P-glycoprotein. When treated with tumor necrosis factor-α and/or transforming growth factor-β, the budding-like organoids replicated the pathogenesis of mucosal damage or intestinal fibrosis. Upon dissociation and seeding on cell culture inserts, the organoids retained intestinal characteristics, forming polarized intestinal folds with approximately 400 Ω × cm² transepithelial electrical resistance. This novel method has great potential for disease modeling and drug screening applications.
... In comparison to organoids obtained from adult stem cells, iHIOs develop crypts and villus-like structure and present the advantages of possessing a mesenchyme surrounding the epithelial layer. 56 Furthermore, during their generation, iHIOs recapitulate the developmental program of the intestine, making this system a model of choice to study human gastrointestinal development. 56 However, iHIOs exhibit immature features, with a more fetal phenotype and require further maturation in vitro 57 or by transplantation in vivo. ...
... 56 Furthermore, during their generation, iHIOs recapitulate the developmental program of the intestine, making this system a model of choice to study human gastrointestinal development. 56 However, iHIOs exhibit immature features, with a more fetal phenotype and require further maturation in vitro 57 or by transplantation in vivo. 58 Overall, we dispose of various cellular models to study the gastrointestinal tract, and the choice of the source of cells will depends on the question and application (Table 1). ...
The intestinal epithelium, the fastest renewing tissue in human, is a complex tissue hosting multiple cell types with a dynamic and multiparametric microenvironment, making it particularly challenging to recreate in vitro. Convergence of recent advances in cellular biology and microfabrication technologies have led to the development of various bioengineered systems to model and study the intestinal epithelium. Theses microfabricated in vitro models may constitute an alternative to current approaches for studying the fundamental mechanisms governing intestinal homeostasis and pathologies, as well as for in vitro drug screening and testing. Herein, we review the recent advances in bioengineered in vitro intestinal models.
... Studies have shown that the use of Caco-2 cells can reproduce the basic structure and function of the intestinal organs, such as the production of villous morphology, drug transport, mucus production, and a certain barrier function [153,154]. While experimenting with Caco-2 cells, the researchers also attempted to compare the characteristics of human pluripotent stem cells and living tissue primary epithelial cells with caco-2 cells [121,155,156] and provided ideas for subsequent intestinal chip research (Table .1). ...
... A. Schematic of human pluripotent stem cell-derived intestinal organoids[155]. B. Schematic of workflow for the incorporation of iPSC-derived HIOs into the Intestine-Chip. C. Western blot analyses and quantification of phosphorylation of STAT1, Quantitative reverse-transcription polymerase chain reaction analyses and IFN-g drug response. ...
Standard two/three dimensional (2D/3D)-cell culture platforms have facilitated the understanding of the communications between various cell types and their microenvironments. However, they are still limited in recapitulating the complex functionalities in vivo, such as tissue formation, tissue-tissue interface, and mechanical/biochemical microenvironments of tissues and organs. Intestine-on-a-chip platforms offer a new way to mimic intestinal behaviors and functionalities by constructing in vitro intestinal models in microfluidic devices. This review summarizes the advances and limitations of the state-of-the-art 2D/3D-cell culture platforms, animal models, intestine chips, and the combined multi-organ chips related with intestines. Their applications to studying intestinal functions, drug testing, and disease modeling are introduced. Different intestinal cell sources are compared in terms of gene expression abilities and the recapitulated intestinal morphologies. Among these cells, cells isolated form human intestinal tissues and derived from pluripotent stem cells appear to be more suitable for in vitro reconstruction of intestinal organs. Key challenges of current intestine-on-a-chip platforms and future directions are also discussed.
... Research on the development of intestinal organoids from stem cells, progenitor cells, or tissue explants has increased rapidly over the past years. 72,99,177 These organoids might be of interest for studying new NDCs and NDC mixtures as they closely resemble the in vivo GIT. Intestinal organoids are epithelial 3D-spheroids that are cultured on extracellular matrixes and represent either intestinal villi or crypts containing a mixture of complex intestinal epithelial cell populations mimicking the intestinal physiology and pathology ( Figure 3a). ...
Human milk (HM) is the gold standard for the nutrition of infants. An important component of HM are human milk oligosaccharides (hMOs), which play an important role in gut microbiota colonization and gut immune barrier establishment, and thereby contribute to the maturation of the immune system in early life. Guiding these processes is important as disturbances have life-long health effects and can lead to the development of allergic diseases. Unfortunately, not all infants can be exclusively fed with HM. These infants are routinely fed with infant formulas that contain hMOs analogs and other non-digestible carbohydrates (NDCs) to mimic the effects of hMOs. Currently, the hMO analogs 2’-fucosyllactose (2’-FL), galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS), and pectins are added to infant formulas, however, these NDCs cannot mimic all hMO functions and therefore new NDCs and NDC mixtures need to become available for specific groups of neonates like pre-term and disease-prone neonates. In this review we discuss human data on the beneficial effects of infant formula supplements such as the specific hMO analog 2’-FL and NDCs as well as mechanism of effects like stimulation of microbiota development, maturation of different parts of the gut immune barrier and anti-pathogenic effects. Insights in the structure-specific mechanisms by which hMOs and NDCs exert their beneficial functions might contribute to the development of new tailored NDCs and NDC mixtures. We also describe the needs for new in vitro systems that can be used for research on hMOs and NDCs. The current data suggest that “tailored infant formula” for infants of different ages and healthy statuses are needed to ensure a healthy development of the microbiota and the gut immune system of infants.
... [37][38][39][40] As shown in Figure 2, the models generated by Barker et al are limited to the expression of intestinal epithelia. 41 These epithelial organoids can be used to study biology, including signaling pathways and molecular inhibitors (Fig. 2). Some researchers have also utilized an air-liquid interface system that increases cell viability due to improved oxygenation. ...
Background:
Colorectal cancer and inflammatory bowel disease account for a large portion of the practice of colorectal surgery. Historical research models have provided insights into the underlying causes of these diseases but come with many limitations.
Objective:
The aim of this study was to systematically review the literature regarding the advantage of organoid models in modeling benign and malignant colorectal pathology DATA SOURCES:: Sources included PubMed, Ovid-Medline, and Ovid Embase STUDY SELECTION:: Two reviewers completed a systematic review of the literature between January 2006 and January of 2020 for studies related to colon and intestinal organoids. Reviews, commentaries, protocols, and studies not performed in humans or mice were excluded.
Results:
A total of 73 articles were included. Organoid models of colorectal disease have been rising in popularity to further elucidate the genetic, transcriptomic and treatment response of these diseases at the individual level. Increasingly complex models utilizing co-culture techniques are being rapidly developed that allow in-vitro recapitulation of the disease microenvironment.
Limitations:
This review is only qualitative and the lack of well utilized nomenclature in the organoid community may have resulted in exclusion of articles.
Conclusions:
Historical disease models including cell lines, patient derived tumor xenografts, and animal models have created a strong foundation for our understanding of colorectal pathology. Recent advances in 3-dimensional cell cultures, in the form of patient-derived epithelial organoids and induced human intestinal organoids have opened a new avenue for high-resolution analysis of pathology at the level of an individual patient. Recent research has shown the potential of organoids as a tool for personalized medicine with their ability to retain patient characteristics including treatment response.
