Abstract The capacity of the 3D-organoid cultures to resemble a near-physiological tissue organization and to mimic – to a certain degree – organ functionality, make organoids an excellent model for applications spanning from basic developmental/stem cell research to personalized medicine. Here, we review key findings achieved through organoid technology, and we discuss applications such as disease – and tumour modelling, correction of genetic mutations and understanding gene – and cell functions. Finally, we discuss future developments in the field. Introduction Stem cells are defined based on their ability to self-renew and generate differentiated cells, through which they are able to generate all tissues of the developing embryo and to maintain tissue homeostasis in adults. In the past decade, these key stem cell features have been exploited to develop ‘mini-organs’ in vitro, the so-called organoids. Organoids are stem cell-derived and self-organizing 3D cultures that phenocopy cell-type composition, architecture and, to a certain extent, functionality of different tissues. Essential for organoid development and growth is a media composition that recapitulates the in vivo stem cell niche signalling pathways, able to sustain stem cell function and drive their expansion and eventually their differentiation (1). Two kinds of stem cells exist, the embryonic pluripotent stem cells (ESCs), responsible for organ embryonic development, and adult somatic stem cells (ASCs), organ-specific resident stem cells important for mature organ homeostasis and regeneration. These more restricted adult stem cells have shown their ability to grow into in vitro organoids, once embedded in the proper extracellular matrix and provided with the correct molecular clues. Since the initial generation of mouse intestinal organoids from isolated Lgr5+ ASCs (2), there has been a surge of organoid culturing systems from a broad range of mouse and human organs (3–12). Similarly, ESCs and the embryonic stem cell state like induced pluripotent stem cells (iPSCs) have been successfully guided to differentiate and form a variety of tissue-specific organoids (13–22), including the brain, eye and inner ear (23–26). Specific characteristics including advantages and limitations of ASCs and ESCs/iPSCs-derived organoids have been reviewed elsewhere (1,27–29). In this review, we will discuss the possible uses of the organoid technology and highlight studies that employ organoids both in a more translational-oriented context and investigate basic cell development and functions along with gene roles. We aim at providing an overall picture of where the organoid field currently stands, with an emphasis on future applications. Organoids as a Model to Study Diseases Infectious diseases The promise of the organoids to model diseases is steadily being fulfiled. As a major advantage, human-derived organoids are not hampered by interspecies differences when used for disease modelling, which is one of the caveats of the use of animal models. In addition, organoids typically contain multiple different cell types present in the tissue of origin, while media composition can be modulated in order to further skew the differentiation of the organoids into specific cell lineages of interest. These features facilitate their use as a model to study infectious diseases and host–pathogen interaction. Probably one of best examples is represented by studies on the ZIKA virus. The ZIKA virus has a strong teratogenic effect: Infection in pregnant women is associated with microcephaly in newborns (30–32). The use of brain organoids, by mimicking human fetal brain development (25,33,34), has allowed to recapitulate the malformation caused by ZIKA infection (35–37). This causally linked viral infection with impaired neural progenitor differentiation and depletion and provided mechanistic insights into virus entry and its pathogenic activities (38–40). Not limited to that, recent works have also exploited brain organoid technology in developing drugs to prevent or treat ZIKA infection (41–43). Another study has shed light on the diverse cellular effects that can be induced by infecting organoids with different viral strains (38), underlining the importance to scrutinize by high-throughput organoid screening the response of the different common ZIKA strains to drugs. In addition to brain organoids, intestinal organoids have been used to model host–pathogen interactions for human enteric viruses, such as Rotavirus (44,45), and represent the first reproducible in vitro cultivation system to study Noroviruses (46). Our group developed an infection approach to study the interaction between gastric epithelium with bacterial microorganisms (9). Helicobacter pylori can be injected directly into the lumen of gastric organoids in order to model the natural host–pathogen interaction that happens at the apical side of the stomach epithelium (9). This system has allowed us to address the primary response of the epithelium to bacterial infection by microarray analysis and has shown that most of the upregulated genes were targets of the nuclear factor κB pathway. In addition, by modulating media composition, it was possible to direct cellular differentiation into different stomach lineages, and this was important to clarify that the gastric gland lineage is showing the highest inflammatory response to H. pylori infection (9). A further study employing gastric organoids also elucidated that H. pylori senses the urea secreted by the gastric epithelium through its chemoreceptor TlpB, allowing persistence of the infection (47). These findings underline the usefulness of organoids as a system to mimic the infection of pathogens to human epithelia and may stimulate further research aimed at understanding additional pathogen infection mechanisms. In this line of research, a very recent study from our laboratory took advantage of the organoids to elucidate the interaction of a human protozoan parasite, Cryptosporidium, with intestinal and lung epithelia, the two main sites of infection. Studies of many human parasites, including the development of effective treatments, are hampered by the lack of a robust in vitro system that recapitulates the life cycle in the human host. After injection of Cryptosporidium oocysts into the organoid lumen, the Cryptosporidium goes through its asexual and sexual stages to complete its life cycle (48). Importantly, this model allowed us to monitor the transcriptomic changes happening during the course of infection both in the (human) epithelial cells and in the parasite and underscored the importance of interferon I signalling in response to Crypto infection (Heo et al., Nat. Microb., in press). Beside elucidating important aspects of host–pathogen interaction, organoids were also used as a model to investigate the relation between the microbiota and the intestinal epithelium, and key results have been recently reviewed elsewhere (49,50). Genetic diseases In addition to providing a natural environment to promote and study microorganism infection, organoids are increasingly being used to model human genetic diseases. Generally, two types of approaches have been employed: (1) organoids have been established from patient-derived biopsies and (2) specific genetic mutations have been introduced in wild-type organoids. The latter strategy greatly benefits from the advent of clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9)/CRISPR technology (51–55), which allows the easy targeting of any genomic locus that can be recognized by a specific RNA guide. The marriage of organoid- and CRISPR technology appears to represent a winning approach to study the effect of mutations that are lethal during development or in early life and for which obtaining patient-derived biopsies constitutes an almost absolute hurdle. Probably, the best example for patient-derived organoids modelling genetic diseases is represented by the cystic fibrosis (CF) case. Human intestinal organoids were derived from CF patients carrying the common CFTR ΔF508 mutation, and a forskolin-based swelling assay was established to assess CFTR functionality (56) using wild-type and CF patient organoids. The approach was exploited to test the response to different drugs on CF organoids isolated from patients harboring different mutations, including rare variants. This proved the organoids to represent excellent predictors of therapy outcome in a personalized manner (57). As additional examples, intestinal organoids harboring TTC7A deficiency have been derived from multiple intestinal atresia patients (58). These organoids showed how TTC7A deficiency leads to an impaired apical basal polarity in the intestinal epithelium that can be rescued by inhibition of the Rho kinase signalling (58). In addition, organoids were derived from two patients affected by microvillus inclusion disease carrying truncation mutations in the STX3 gene and revealed how the brush border was retained and degraded inside the patients’ organoid cells (59). These studies show not just how organoids can faithfully recapitulate the disease characteristics in a human in vitro model but are a valid resource both for basic research and for development of therapeutics. The intestine is not the only organ from which patient-derived organoids were isolated. For instance, ductal cell-liver organoids established from Alagille syndrome and A1AT-deficiency patients (10) have reproduced the defects observed in vivo (60). Organoids established from patient-derived human iPSC (hiPSC) were also used to model diseases involving several organs, as recently reviewed (61), including brain organoid models of microcephaly (25) and autism (34). Several studies have introduced genetic mutations in hiPSC-derived organoids that recapitulate features of diseases in different tissues. In hiPSC-derived kidney organoids, CRISPR-driven loss-of-function mutations in PODXL (62) and PKD genes (63,64) not only induced defects that mimic nephrotic syndrome and polycystic kidney disease, respectively, but also helped in understanding the gene function in the pathogenesis context. Engineered iPSC-derived liver organoids also elucidated the effect that different mutations in the JAG1 gene, an NOTCH ligand, can have in the formation of bile ducts and development of the Alagille syndrome. This study found that the JAG1 mutation C829X causes profound alterations, while the G274D does not alter organoid properties (65). Specific gene mutations in TREX-1, an antiviral enzyme, are responsible of the Aicardi Goutierès syndrome, an autoimmune disease associated with, among others, intellectual problems. By engineering these mutations in brain organoids, Thomas and colleagues showed that TREX-1 deficiency increases neural death and results in reduced organoid size but also that treatments can be effective in counteracting the mutation-induced defects (66). Tumour modelling and biobanking The organoid technology has led to the development of the so-called ‘tumouroids’, patient-derived 3D cultures of cells isolated from tumour biopsies. Tumouroid lines have been successfully established from multiple cancer types, including colorectal, pancreatic, liver, breast, prostate, brain and bladder cancers, from both primary tumours and metastasis (67–77). Established tumouroid lines can be indefinitely passaged and propagated in vitro, allowing their use for multiple downstream applications. This type of culture has many advantages over the most commonly used 2D cancer-derived cell lines or the patient-derived xenografts (PDXs) (78). The tumouroid culturing has proven efficient in allowing the collection of cancer subtypes from large numbers of individual patients, referred to as a ‘living biobank’ (68,70,72,75,76). Such biobanks comprise a series of tumouroid lines that represent the full histopathological diversity of the disease and can be expanded over time. Importantly, genomic analyses such as whole-genome or exome sequencing has consistently revealed well-maintained mutational profiles between the tumouroid lines and the matching tumours from which they are derived, both at early and at late passages. Typically, the spectrum of mutations found in the tumouroid biobanks reflect the common mutational alterations typical of a certain cancer type. This highly conserved genomic landscape is particularly crucial, as it makes the organoids a trustworthy and expandable source of material recapitulating the features of the parental tumour to perform genotype–phenotype correlation analysis, functional tests and assess drug response. Medium-throughput drug screening has been reported in most of the recent tumouroid biobank efforts (67,68,76), as it is clearly an essential asset to evaluate the importance of the tumouroids as a preclinical model. In some cases, sensitivity to treatment can be correlated with a defined mutational profile. Importantly also, it has been shown that drug resistance in vitro mirrors the patient treatment response, underscoring the potential of tumouroids as a predictive tool for treatment efficacy (79). Xenotransplantation of human organoid cancer lines in mice and treatment with drugs also showed similar response to what observed for the organoid lines in vitro (67,68,75,76,80). Recent evidence has pointed out that tumour evolution and genetic composition of human tumours transplanted to (immunodeficient) mice, the so-called PDXs, substantially diverge from the parental tumour (81). This has so far not been observed in tumouroids (see for instance (71)). This latter consideration is also important for studies of tumour clonal evolution, as the organoid cultures could constitute a preferred model over others. In a study by Lee and colleagues (76), phylogenetic analyses of bladder cancers and the derived tumouroids, tumouroid xenografts and xenograft-derived organoids, it was shown that patterns of tumour evolution resemble those described for bladder cancer in vivo. Also, organoids can be derived and expanded from single cancer cells and therefore be used to mimic intratumour diversification in culture. This in turn allows to capture tumour heterogeneity and determine phylogenetic relations, mutational signatures, transcriptomic and epigenomic status and drug treatment response virtually at single cell level (82). In addition to generating organoids from patient-derived biopsies, carcinogenesis can be modelled by mutating specific cancer genes in wild-type organoids. The first study has used lentiviral-based expression or silencing of cancer genes such as KRAS or TP53 in hiPSC-derived pancreatic organoids (74). Yet, this approach has greatly benefitted from the advent of CRISPR/Cas9 genome editing. Specifically, two groups have shown that sequential mutation of APC, TP53, KRAS and SMAD4 by CRISPR in intestinal organoids reproduces colorectal cancer progression in vitro (83,84). Orthotopic transplantations of intestinal organoids carrying different combinations of those gene mutations allowed to evaluate the specific weight of each individual gene in the metastatic process (85). In a similar manner, mutations were introduced in genes responsible for DNA repair, such as MLH1 and NHTL1 (86). After the initial editing and single cell cloning of the mutated organoids, the mutant organoids were subcloned after a fixed period of 2–3 months. The subclones were expanded, and whole-genome sequencing of clones and subclones revealed the accumulation of mutational changes over time. This allowed us to determine the consequent gene mutational rate and signatures (86). Therefore, in addition to tumour modelling, targeted gene editing holds the potential of dissecting specific gene roles in the carcinogenic process. In conclusion, organoid culture has revealed itself as a valuable resource for cancer research (87). Recent works also aimed at integrating 3D culturing of epithelial organoids with non-epithelial cells, such as stromal and immune cells, in order to obtain a cancer modelling that takes into account parts of the tumour microenvironment (88,89). These studies however are still at an early stage yet could constitute an interesting future development of the field. Correction of Genetic Defects in Human Organoids The capacity of ASC-derived organoids to be isolated from many human tissues and expanded in vitro while maintaining genomic stability (3,10) offers an attractive resource for potential autologous cell therapy. Combined with CRISPR/Cas9 technology, organoids can be isolated from an individual patient affected by a certain genetic monogenic disease, and the organoids can be genome edited to correct the causing mutation. The first proof-of-principle demonstration of correction of a genetic defect by CRISPR/Cas9 genome editing in stem cells of human patients with a genetic disease was provided in 2013. Cas9-mediated-homologous recombination with a wild-type version of the CFTR gene was used to rescue the ΔF508 mutation in intestinal organoids isolated from two different CF patients. The corrected organoids showed restoration of CFTR function in the swelling assay (90). This approach could be clinically relevant, especially when combined with organoid engraftment in the appropriate site, already possible for intestinal organoids in murine hosts (91,92). Congenital dyskeratosis is a disease caused by mutations in the DKC1 gene, which results in impaired maintenance of telomeres length. HiPSC-derived intestinal organoids from patients with this disease have also been used for Cas9-mediated recombination, and mutation correction resulted in a phenotypic rescue (93). Very recently, correction of the RPGR gene mutation in hiPSC-derived retinal organoids from a retinitis pigmentosa patient has reverted several functional defects, including photoreceptor loss (94). Although very promising, there are still only few examples of phenotypic rescue by genome engineering in human organoids. The rapid improvements in the Cas9 technology, including the recent development of Cas9 nickases able to lead to targeted C to T and A to T base substitutions (95–97) without the need of homologous recombination will further widen the ease of use and safety of the technology. Clearly, transplantation techniques of organoids will have to be developed to allow the application of genome-edited, patient-derived human organoids into the clinic. Organoids as a Tool to Study Gene Function and Cell Development Organoid technology can also address fundamental biological questions, such as investigating gene roles and studying cell functions and development. One of the more commonly employed approaches for such studies involves the genetic engineering of mice. Organoids can be easily derived from mice and can combine the power of mouse genetics with the versatility of the 3D culture system. For instance, Tetteh and colleagues were able to show the dedifferentiation of Alpi+ enterocyte progenitors upon diphtheria toxin-mediated ablation of Lgr5+ stem cells and their capacity to repopulate the Lgr5 crypt population (98), therefore showing their high degree of plasticity. A similar approach was taken to eliminate Reg4+ cells, representing deep crypt secretory cells in the colon, and showed that their loss results in Lgr5+ stem cells depletion. This demonstrated their niche function similar to the role covered by Paneth cells in the small intestine (99). Organoids derived from genetically engineered mice has also helped to visualize, through an epitope-tagged Wnt3, the transfer of Wnt3 from Wnt-producing Paneth cells to the neighboring Lgr5+ stem cells and to determine how the Wnt gradient is established in the crypts (100). Another important advantage of the organoid culture is the ease of manipulating the stem cell niche components in order to understand the role of signalling pathways in stem cell maintenance or differentiation. As an example, concomitant blockade of the Notch, Wnt and EGFR or MAPK signalling in intestinal organoids was identified as the most optimal differentiation path for stem cells into enteroendocrine cells (101), an otherwise rare cell type in vivo. As mentioned, the organoid cultures were established for many human tissues and as such hold a great potential to translate findings from mice to human. Genomic engineering of human organoids is obviously performed after the culture is established. As an example of this approach, by employing a large number of different reporter lines engineered from human colorectal cancer organoids, Shimokawa and colleagues were able to visualize and trace human LGR5+ cells in organoid xenotransplants, showing that they represent the cancer stem cell population (102). In addition, after specific ablation of LGR5+ cells by expression of iCaspase, the differentiated traced KRT20+ cells were able to revert into LGR5+ cells and became capable of organoid formation (102). Along similar lines, the use of an ASCL2 reporter allowed to mark stem cells in normal human colorectal organoids and to follow their behaviour during the adenoma carcinoma cancer progression (103). Conclusions In this review, we have attempted to summarize recent key findings that have been obtained through organoid technology (see Fig. 1 for a schematic representation). Although the field has rapidly expanded in the recent years, still many of the organoid possibilities are just shown at a proof-of-principle level and are not yet widely applied. In addition, while the use of intestinal ASC-derived organoids has been explored for almost all discussed applications, the application of organoids generated from other tissues is still in its infancy (Fig. 1). Generation and handling of organoids is more tedious than cells grown in 2D, and the essential growth factors are often expensive and not specifically tested for use in organoid culture. Thus, growth factor preparations often have to be made in-house. This may be changing now as several commercial entities have started to market reagents tailored to organoid culture. Combination of organoids with constantly improving genome editing techniques supports the ease of organoid manipulation and therefore their versatility as model system. We envisage the rapidly growing implementation of organoid technology in both basic and clinical research in the coming years. Figure 1. View largeDownload slide Applications of human-derived organoids. Schematic diagram summarizing the organoid cultures that have been established from the different human organs, either from ASCs or ESCs/iPSCs, as indicated. In the colored boxes we report the various applications for which the tissue-specific organoids have been used, as discussed in the main text. Figure 1. View largeDownload slide Applications of human-derived organoids. Schematic diagram summarizing the organoid cultures that have been established from the different human organs, either from ASCs or ESCs/iPSCs, as indicated. In the colored boxes we report the various applications for which the tissue-specific organoids have been used, as discussed in the main text. Acknowledgements The authors wish to thank Dr. Oded Kopper and Joep Beumer for critical reading of the manuscript and apologize to authors of excellent studies that could not be discussed for lack of space. H.C. is the inventor of several patents concerning ASC-derived organoids. Conflict of Interest statement. None declared. Funding BA was supported by a FEBS_Long Term fellowship and the NWO/VENI (863.15.015). References 1 McCauley H.A. , Wells J.M. ( 2017 ) Pluripotent stem cell-derived organoids: using principles of developmental biology to grow human tissues in a dish . Development , 144 , 958 – 962 . Google Scholar CrossRef Search ADS PubMed 2 Sato T. , Vries R.G. , Snippert H.J. , van de Wetering M. , Barker N. , Stange D.E. , van Es J.H. , Abo A. , Kujala P. , Peters P.J. et al. ( 2009 ) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche . Nature , 459 , 262 – 265 . 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Human Molecular Genetics – Oxford University Press
Published: May 23, 2018
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