Organoids as Model Systems for Gastrointestinal Diseases: Tissue Engineering Meets Genetic Engineering
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- Werner, K., Weitz, J. & Stange, D.E. Curr Pathobiol Rep (2016) 4: 1. doi:10.1007/s40139-016-0100-z
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Organoids are three-dimensional culture systems that resemble their organ of origin, are genetically stable, and can phenocopy diseases. They enable modeling of various cancer entities such as gastric or colorectal cancer, in addition to other gastrointestinal tract diseases such as inflammatory bowel disease. Genetic engineering tools like CRISPR/Cas9 allow their manipulation to repair mutations or unravel gene functions. Individual patient-derived organoids allow to test therapies in vitro before their in vivo application, bringing personalized medicine to a next level. Organoid biobanks can be used to conduct drug screenings and validate biomarkers. Interactions with microbiota can be investigated in realistic in vitro models. Transplantability of genetically engineered organoids opens up new avenues in the tissue engineering research field. Organoid cultures thus represent a versatile system to model diseases and test therapeutic interventions.
KeywordsStem cells Organoids Disease modeling Personalized medicine
Summary of organs of which organoid cultures have been established
References for human and murine organoids derived from
PSCs (ESCs/iPSCs) or embryonic tissue
Tissue-resident AdSCs or progenitors
Modeling Cancer and Personalizing Therapy Using Organoids
Cancer is a leading cause of death worldwide . Despite considerable progress in cancer treatment and extensive knowledge about molecular pathways leading to progression and metastasis, new therapeutical approaches are still in great need. For different cancer entities, histological subtypes have been known for a long time. Modern molecular tools such as next-generation sequencing and expression profiling have unraveled the genetic cause of these differences, but have also enabled the establishment of new molecular subtypes based on the presence of defined genetic alterations . It nevertheless emerges as a general fact that large molecular inter-tumoral differences exist between cancers of the same tissue in individual patients. Even cancers of the same molecular subtype show great variance in their mutational landscape, resulting in difficulties in predicting the response of an individual cancer to a certain therapy. The establishment of organoid cultures from tumor biopsies allows testing of therapies in vitro before a therapeutical intervention in a patient, which could be a way to bring personalized therapy approaches to a next level .
Cultivation protocols for the following cancer entities are established (Table 1): gastric , colorectal [21, 23•], prostate , breast , renal , and pancreatic cancer . In the following, tumors of the gastrointestinal tract and their modeling will be discussed in detail.
The discovery of LGR5 as a marker of intestinal stem cells has allowed the detailed analysis of active pathways in this stem cell population [51, 52]. Combined with detailed knowledge of the biology of the intestine in general , this led to the development of a protocol to grow small intestinal organoids from single Lgr5-positive (Lgr5+) stem cells . Subsequently, organoids from murine and human colon as well as from colorectal cancer (CRC) could be established . The establishment of a CRC cancer biobank was the next logic step, bringing the organoid culture technique on a next level by proofing its usefulness for personalized medicine [23•]. In this study, van de Wetering et al. used surgically resected tissue from untreated CRC patients to establish “paired organoids” from healthy epithelium and tumor. Using next-generation antibiotics, 90 % growth rate was observed and the expanded organoids could be cryopreserved and thawed with a survival rate over 80 %. While Wnt3a and the Lgr5 ligand R-spondin-1 were essential growth factors for organoids from normal tissue, Wnt3a was excluded for selected expansion of tumorous tissue, because more than 90 % of CRC tumors harbor activating mutations within the WNT signaling pathway and grow independently from exogenous Wnt3a supplementation. Of note, the cancer organoids grow as pure epithelial cultures. While this excludes the mesenchymal niche as a potentially important part of the in vivo situation, the analysis of pure epithelial organoid cultures allows sequencing or expression profiling without any contamination of non-transformed mesenchymal tissue, such as fibroblasts, blood vessels, and immune cells. A high concordance of the mutational spectrum of biopsies and corresponding organoids was confirmed by whole-exome sequencing. CRC subtypes of non-hypermutated and hypermutated tumors, as described by recent large genome sequencing projects , were found in patient organoids with the same frequency as in larger clinical cohorts. Somatic copy number alterations were comparable, and common driver and gatekeeper mutations like APC, TP53, KRAS, and SMAD4 were represented and maintained in organoid cultures. Furthermore, the study included a drug screen using 83 compounds, proofing that organoid cancer biobanks are amenable to large-scale drug screens. The detailed molecular analysis of CRC organoids thus allows to first define potentially effective drugs and secondly (and most importantly) allows to test the actual potential of these drugs before administering them to the patient, which enables personalized medicine with an optimized effect–side effect ratio. In addition to drug screenings with standard chemotherapeutics or specific targeted therapies [23•, 54, 55, 56], the response of CRC organoids to ionizing radiation will be a valuable addition to drug screenings , as rectal cancers are routinely pre-treated by a combination of irradiation with chemotherapy before a curative operation. The individual response upon this pre-treatment varies widely, with some patients showing only minor response/no response and some even with progression under treatment . The intrinsic resistance towards chemo- and/or radiotherapy might thus be predicted with the help of patient-derived organoids, which could result in an optimal stratification of patients towards different therapeutic strategies.
Using CRC cancer organoids from patients holds great promises to improve personalized medicine. Nevertheless, the high number of mutations present (median number of non-synonymous mutations: 58 in non-hypermutated and 728 in hypermutated CRC ) combined with the great variance of the mutational landscape of individual CRC organoids makes them an unsuitable tool for functional molecular studies. To circumvent this problem and allow functional studies in a defined genetic background, genetic engineering tools can be used to model CRC starting from normal human or murine organoids. Koo et al. presented Cre recombinase-inducible retroviral vectors for miRNA-induced gene knockdowns or for overexpression of a (mutated) gene of interest. This efficient and fast transduction method can be used for various gain- and loss-of-function studies . Li et al. also used adeno-, retro-, and lentiviral vectors for CRC modeling [22•]. The method is based on an air–liquid interface culture, where gastrointestinal organoids mainly from neonatal mice were grown composed of epithelial and mesenchymal tissue and without supplementation of any exogenous growth factors. Normal colon was stepwise transformed to hyperproliferative adenomas or polyps, culminating in malignant invasive carcinoma. Based on an initial Apc mutation, an activating KrasG12D mutation was introduced and inactivating Smad4 or Tp53 mutations were mimicked by shRNA-conveyed knockdowns. The single Apc mutation and the combination of Apc with one of the other candidate genes induced only minimal dysplasia. Among combinations of three genes, Apc, KrasG12D, and Tp53 mutations together elicited the strongest effect, while the most severe transformation effect was seen in the four-gene module. This module also showed the histological morphology of an invasive adenocarcinoma, and in vivo tumorigenicity could be confirmed by subcutaneous transplantation of organoids into immunodeficient NOG mice.
− Wnt3a and R-spondin
+ EGFR inhibitor gefitinib
+ Nutlin-3 as an inhibitor of MDM2 (degrades TP53)
− Noggin and TGFβ receptor inhibitor A83-01
− EGF or + EGFR inhibitor
+ MEK inhibitor
In a parallel study, Drost et al. analogously introduced an activating mutation of KRAS and knockouts of APC, TP53, and SMAD4 via CRISPR/Cas9 technology in organoids of the human intestine . Quadruple-mutated organoids grew in medium lacking Wnt3a, R-spondin, Noggin, and EGF (Table 2). While triple-mutated organoids (KRASG12D, APCKO, TP53KO) resembled adenomas and showed low proliferation as subcutaneous xenografts in NSG mice, the organoids with four mutations were highly proliferative and displayed an invasive adenocarcinoma in vivo. Chromosomal instability and aneuploidy, both characteristic hallmarks of cancer, were also confirmed in engineered organoids.
In summary, these studies underline the enormous potential of organoids to model malignancies with the help of novel genetic engineering tools. Additional to these genetical analyses, further factors important for tumor progression can be envisioned to be investigated using organoid cultures, such as e.g., inflammation . Besides direct genetic manipulation of inflammatory pathways , activated or manipulated immune system cells might be mixed into organoid cultures to test their oncogenic impact.
Following the identification of the small intestinal stem cell and the subsequent development of an intestinal organoid culture, an AdSC of the pyloric part of the stomach could be unraveled using an Lgr5-CreERT2 knock-in line for lineage tracing experiments . Adapting the small intestinal culture conditions by including factors to activate known stomach signaling pathways, single Lgr5+ pyloric stem cells could be grown out into organoids. The definition of the Lgr5+ small intestinal stem cell signature  set the stage for the identification of several important stem cell maintenance genes [62, 63, 64]. It also allowed the generation of new Cre lines to identify novel stem cell populations . One of these new Cre lines, using the gene Troy as a Cre-driver, revealed a stem cell population in the gastric corpus . This stem cell population, albeit being mostly quiescent in vivo and functioning as a reserve stem cell population, reverts to a vividly proliferating population of stem cells in organoid cultures as well as after damage to the stomach in vivo. Building on the knowledge from mouse stomach organoid cultures, Bartfeld et al. developed a culture protocol for human stomach tissue . Next to AdSC-derived stomach organoids, PSC-derived organoids have also been developed . Whereas McCracken et al. described the development of human gastric organoids resembling the pyloric region , Noguchi et al. developed a protocol to differentiate murine ESCs into corpus resembling organoids . Similar to intestinal organoids, the same conditions as for normal human stomach also enabled Bartfeld et al. to successfully outgrow human stomach cancers into cancer organoids. This technique will enable a detailed molecular characterization of individual gastric cancers followed by in vitro screening for active drugs as already performed for colorectal cancer in the near future.
Regarding gastric cancer modeling, Li et al. used the same air–liquid interface culture system described before for the intestine to successfully grow gastric organoids from neonatal tissue [22•]. In these gastric organoids, the transformation effect of an activating KrasG12D mutation and a Tp53 knockout was tested. The emerging dysplasia was more profound when both alterations were combined. In vivo the mutated organoids caused invasive carcinomas in NOG mice.
Nadauld et al. modeled the metastatic potential of a TGFBR2 bi-allelic mutation in gastric organoids . The authors found this mutation only in an ovarian metastasis, but not in the corresponding primary tumor of a patient with hereditary diffuse gastric cancer (HDGC). In murine organoids with a constitutive Tp53 and Cdh1 knockout, an additional knockdown of Tgfbr2, a receptor within the TGFβ signaling pathway, was achieved by shRNA. These triple-mutated organoids showed features of HDGC histologically. Subcutaneous xenograft experiments in NOG mice exhibited poorly differentiated gastric adenocarcinomas and pulmonary metastasis. This work nicely demonstrates the usefulness of organoid cultures to model gastric cancer and test novel candidate genes by establishing TGFBR2 as an important tumor suppressor gene for HDGC.
Modeling of Gastrointestinal Diseases and Therapeutic Interventions in Organoids
The pathogen Helicobacter pylori colonizes the stomach of around half of the world’s population and can promote gastritis, peptic ulcer disease, and gastric cancer . Rodent models show only mild gastritis without any progression towards ulceration or cancer. Bartfeld and colleagues established microinjection of GFP-expressing H. pylori into the lumen of gastric organoids. An intimate contact of the bacteria with the epithelium was visualized by electron microscopy. Microarray analyses after infection revealed the primary response to the pathogen, including many target genes of the NF-κB pathway, like e.g., IL8 and Sonic Hedgehog [38, 70, 71]. Other studies in organoids showed a phosphorylation of c-Met and a twofold increase in proliferation of epithelial cells upon microinjection of H. pylori . This seems to be dependent on the H. pylori virulence factor CagA, which translocates to epithelial cells and forms a complex with the c-Met receptor. Sigal et al. described a direct colonization of Lgr5+ stem cells by H. pylori . Glands from infected mice formed organoids with a significantly higher capacity and larger sizes. Gastric organoids thus evolved to a new state-of-the-art in vitro tool for H. pylori research.
Research on intestinal microbiota is becoming a quickly evolving research field due to the increasing realization of the impact of the microbiome on various aspects of health and disease. Different gastrointestinal diseases like obesity, type 2 diabetes, inflammatory bowel disease, and CRC are associated to these intestinal microbes (for details refer to ).
To study host–microbiota interactions, organoids of the intestinal epithelium are increasingly recognized as a useful tool. Lukovac et al. incubated mouse ileal organoids with Akkermansia muciniphila,Faecalibacterium prausnitzii and conditioned medium from these bacteria including the typical short-chain fatty acids . These microbes and their metabolites are discussed to decrease the risk of gastrointestinal disorders (e.g., obesity and Crohn’s disease). While F. prausnitzii and its metabolite acetate induced only weak effects on the host, A. muciniphila and propionate or butyrate had an influence on the expression of important transcription factors, cell cycle control-, apoptosis-, lipid metabolism-, and lipolysis-associated genes.
Intestinal organoids are also valuable infection models. iPSC-derived and AdSC-derived human and murine intestinal organoids can be infected with fecal viruses [75, 76]. Infection of host cells as well as viral replication and production of infectious viral particles was shown both for a rhesus rotavirus laboratory strain with wide tropism and clinical rotavirus isolates from stool samples. Interestingly, virus samples had distinct sensitivities to antiviral medications.
Zhang et al. visualized infections of murine intestinal organoids with Salmonella . Morphologic changes and disrupted tight junctions were observed in the host epithelium. An inflammatory response based on NF-κB pathway activation was shown and the number of stem cells was decreased significantly. Extensive studies about Clostridium difficile as a leading cause for nosocomial diarrhea are also available [78, 79, 80, 81]. Using murine organoids among other techniques, the inhibition of Na+/H+-exchanger isoform 3 (NHE3) by C. difficile toxin B was shown, causing an altered environment and region-specific change in bacterial growth. By infection of normal human intestinal organoids with C. difficile or its toxins, the decreased NHE3 expression was confirmed and a disrupted epithelial morphology was observed. Furthermore, the mucus composition of patient-derived organoids was changed regarding MUC1 and MUC2 expression and the oligosaccharide composition. Altogether, the identified molecular changes provide new targets for novel therapeutic applications. Further studies focus on defense mechanisms of the intestinal epithelium, e.g., production of bactericidal defensins and lysozyme, and the function of Toll-like receptors and Nod2 as immune sensors for microbial patterns [82, 83, 84, 85, 86].
Inflammatory Bowel Disease (IBD)
Crohn’s disease and ulcerative colitis, the two main disease entities of inflammatory bowel disease, are characterized by chronic inflammation of the entire or parts of the gastrointestinal tract. Patients with Crohn’s disease show intestinal fibrosis among other pathological findings. Rodansky et al. differentiated human ESCs into intestinal organoids with mesenchymal and epithelial cell types . Under fibrogenic cytokine stimulation with TGFβ, an induction of fibrogenic genes was shown, which could be reversed using spironolactone as an anti-fibrotic drug. Prospectively, organoids established from patient tissue using protocols for co-culturing of epithelial organoids and mesenchymal cell types, as described by Schumacher et al. for gastric organoids , could reveal novel insights. These complex model systems might enable further studies on cell–cell interactions and the establishment of personalized therapies based on drug screenings can be envisioned. In addition, experiments combining IBD patient-derived organoids with IBD-associated gut bacteria open up new avenues of research . To gain better insights into the genetic background of IBD, Mokry et al. sequenced biopsies and organoid cultures established from patient tissue . They confirmed risk loci for IBD, which regulate gene expression in the intestinal epithelium and immune cells. This knowledge is useful for further genetic manipulations of organoids to model these disorders, get a better understanding of affected signaling pathways, and test targeted therapies. For example, TNFα treatment of small intestinal organoids induced an overexpression and activation of PAK1, which modulates the PPARγ/p65 cascade and promotes an inflammatory response .
Organoids might evolve in the future as an ideal source for tissue regeneration in IBD patients. Yui et al. demonstrated that organoids are transplantable and help to repair damaged colon epithelium [47•]. In an in vivo model of inflammation-induced damage, the authors showed that organoids integrated in the single-layer epithelium of the gut, forming normal crypt structures. The Lgr5+ stem cells of the cultivated and transplanted organoids showed self-renewing capacities over 25 weeks. In addition, the transplanted mice showed signs of disease amelioration, regaining their weight faster than non-transplanted mice. Further studies with intestinal organoids confirm their repair capacity [91, 92]. Thus, patients with IBD or other gastrointestinal diseases might benefit from organoid transplantation techniques in the future, especially when the disease-causing genetic alterations can be corrected by gene editing tools.
Further Gastrointestinal Disorders
Multiple intestinal atresia, a rare kind of bowel malformation, is characterized by homogenous calcifications in the abdominal cavity, widespread atresia, and in a subgroup of patients a combined immunodeficiency. Bigorgne et al. showed defects in proliferation, differentiation, and epithelial apicobasal polarity in patient-derived organoids . They demonstrated an increased Rho kinase activity caused by TTC7A mutations and could reverse the phenotype with a ROCK inhibitor.
Mutations of the cystic fibrosis transmembrane conductor receptor (CFTR) gene, encoding for an epithelial chloride channel, which is cAMP- and cGMP-regulated, cause a disrupted chloride and bicarbonate secretion in mucoviscidosis patients. Epithelial-lined organs, especially the lung, but also the gastrointestinal tract, are clogged by viscid mucus. Various drug screenings using intestinal organoids from cystic fibrosis patients have been conducted [94, 95, 96, 97]. Especially, medications targeting the misfolding, retention in the endoplasmic reticulum, and proteasomal degradation of CFTR are under investigation. For these screening purposes, the functionality of CFTR can be tested by a phenotypical assay established by Dekkers et al. : addition of forskolin results in an increase of intracellular cAMP, inducing an osmotic fluid secretion into the lumen of the organoids. This swelling of wild-type organoids is absent in CFTR mutant organoids. Schwank et al. succeeded to reverse this CF-associated phenotype by CRISPR/Cas9-mediated gene repair [46•]. In this study, the most common deletion in exon 11 (F508del) was corrected by homologous recombination in patient-derived organoids of the small and large intestine. Organoids have also been already applied to mucoviscidosis-associated pulmonary problems [99, 100].
An increasing number of tissues can be cultivated as organoids resembling to different degrees their corresponding organ. Depending on the experimental approach, pure epithelial cultures or more complex organoids including mesenchyme can be employed. Since the establishment of Lgr5+ stem cell-derived organoids, gastrointestinal organoid cultures are intensively used in various research fields. This is mainly due to their ease of generation from mouse or human tissue in combination with their accessibility to genetic manipulation. Colorectal cancer patient-derived organoids were established in a “living biobank,” genetically analyzed and subjected to drug screens. This enables to link altered molecular pathways with therapy response upon targeted therapies, previously only achievable in expensive patient-derived xenograft (PDX) mouse models. With genetic engineering tools like viral transduction or CRISPR/Cas9, the functional role of individual genes can be validated. For CRC, alterations in APC, TP53, KRAS, SMAD4, and PIK3CA were confirmed as important driver and gatekeeper mutations that push tumor progression. For gastric cancer, where specific mouse cancer models are largely missing, the importance of KRAS, TP53, and CDH1 mutations could be confirmed using gastric organoids. The culture system also enables functional tests of additional candidate genes from next-generation sequencing efforts, as performed e.g., for TGFBR2. The possibility to study in vitro intimate microbiota–host interactions as well as the impact of certain immune stimulatory signals on the epithelium opens up new research fields. Genome editing tools can be used for autologous gene repair, enabling the restoration of organoids affected by genetic diseases, such as cystic fibrosis. The theoretical possibility to transplant corrected or otherwise genetically modified organoids back into patients is an alluring perspective, substantiated by data confirming the genomic stability of AdSC-derived organoids . Organoids thus have evolved in a short time to a new valuable tool allowing for genetic engineering and tissue engineering to move boundaries in translational research.
|Funder Name||Grant Number||Funding Note|
|European Research Council (BE)|
|Deutsche Krebshilfe (DE)|
|Wilhelm Sander-Stiftung (DE)|