Use of organoids to study regenerative responses to intestinal damage

Abstract

Intestinal organoid cultures provide an in vitro model system for studying pathways and mechanisms involved in epithelial damage and repair. Derived from either embryonic or induced pluripotent stem cells or adult intestinal stem cells or tissues, these self-organizing, multicellular structures contain polarized mature cells that recapitulate both the physiology and heterogeneity of the intestinal epithelium. These cultures provide a cutting-edge technology for defining regenerative pathways that are induced following radiation or chemical damage, which directly target the cycling intestinal stem cell, or damage resulting from viral, bacterial, or parasitic infection of the epithelium. Novel signaling pathways or biological mechanisms identified from organoid studies that mediate regeneration of the epithelium following damage are likely to be important targets of preventive or therapeutic modalities to mitigate intestinal injury. The evolution of these cultures to include more components of the intestinal wall and the ability to genetically modify them are key components for defining the mechanisms that modulate epithelial regeneration.

INTRODUCTION

The mammalian intestine supports a myriad of functions, including digestion, secretion, absorption, barrier function, movement/elimination of waste, immune responses, and provision of a home for the microbiome. Consisting of a tube that is open at both ends to the outside environment, the lumen is lined by an epithelium that is at the core of the gastrointestinal tract’s many functions. Homeostasis of this epithelium is associated with rapid turnover that results in complete renewal of cells approximately every 3–7 days. Disruptions in homeostasis of the epithelium can occur through a variety of means, including radiation- or chemotherapy-based therapeutic treatments, infection with pathogenic organisms, and inflammation. Repair of the damaged epithelium and restoration of epithelial homeostasis is dependent on the intestinal stem cell (ISC) and its associated environment, referred to as the ISC niche. Although both Drosophila melanogaster and mice have provided valuable insight into epithelial regeneration, they are limited in the ability to fully predict the human ISC response due to differences in intestinal anatomy, physiology, genetics, microbiome, environment, and diet. Transformed human cell lines have provided an in vitro system in which many fundamental biological questions have been addressed. However, their inability to accurately replicate the complex physiology and biology of the normal human epithelium has been an important drawback. Organ cultures of human intestine provide a more biologically relevant model but have limitations due to rapid cell death and the inability to renew cells. In the last 10 years, the development of new in vitro models of the human intestinal epithelium, termed organoids, has significantly advanced understanding of the biology of the human intestine by providing an in vitro model that mirrors the complexity of in vivo epithelium. Recently, these models have come to the forefront as key tools to understand how the epithelium undergoes repair after damage. Here, we review the use of organoid cultures to study intestinal regeneration following injury due to radiation and chemical treatment, as well as damage that results from pathogenic infections.

ORIGIN OF INTESTINAL ORGANOID CULTURES

In vitro models of the intestinal epithelium can be derived from human and animal stem cells (50, 52, 56). These stem cell-derived cultures develop into three-dimensional organoids with a spherical shape that surrounds a lumen (Fig. 1). The organoid cultures are genetically stable and grow indefinitely (52), in contrast to primary intestinal cells or tissue explant models, which only offer short-term culture capabilities. After differentiation by manipulation of various growth factors, the cultures contain polarized differentiated cells of the intestinal epithelium. These self-organizing, multicellular structures recapitulate many properties of the intestine, including the heterogeneity of the cellular composition, appropriate physiology, and region-specific features of the intestine. Additionally, these cultures allow for the study of the human intestinal epithelium in the context of individual genetic variability, disease status, and other demographic factors including age, gender, and ethnicity, and they are expected to become a key component for developing personalized approaches to medical treatments for intestinal diseases.

Fig. 1.

The origin of human intestinal organoid cultures. Intestinal organoid cultures can be derived from either human embryonic stem cells or induced human pluripotent stem cells or adult stem cells derived from the intestinal tissue itself. These stem cells are propagated in vitro and form three-dimensional structures grown in matrigel matrix in a tissue culture dish. Figure was created with Biorender.com.

Human Pluripotent Stem Cell-Derived Organoids

Intestinal organoid cultures are derived from either human embryonic or human-induced pluripotent stem cells (PSC) (Fig. 1). Based on gene expression profiling (13), both embryonic and human-induced, PSC-derived organoids contain a more fetal-like epithelium when compared with native adult intestinal epithelium (13). Consisting of an intestinal epithelial layer surrounded by mesenchyme, limited regional specificity (proximal small intestine and colon) can be controlled by growth factor exposure during derivation (35, 60). A more mature phenotype, including formation of crypts and villi as well as underlying stroma and muscle layers, can be induced by transplantation of the PSC organoid into heavily vascularized areas such as below the kidney capsule or in the omentum of immunocompromised mice (7, 66). These organoids have particular advantages in studying epithelial-mesenchymal interactions and intestinal development and can be used to study how these components aid in repair of the epithelium following damage resulting from radiation, chemotherapy, and intestinal infections.

Adult Stem Cell-Derived Organoids

Adult stem cell (ASC)-derived organoids, referred to as enteroids (57), are derived from either the intestinal crypts (50) or isolated leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)+ ISCs (52, 73) from human or animal intestinal tissue (Fig. 1). These primary cells are cultured with various growth factors necessary for their growth and propagation (23, 52). Removal of these growth factors promotes epithelial differentiation resulting in the presence of mature intestinal epithelial cells (goblet, enteroendocrine, tuft, enterocyte, Paneth) in addition to the ISCs. These differentiated cells are present in the appropriate ratios in the cultures, and their locations within the organoid somewhat replicate the cellular organization in vivo (17, 51). Unlike the PSC-derived organoids, the ASC-derived organoids lack a mesenchymal component but more closely model adult epithelium and retain region-specific functions that are useful for studying mature human epithelial biology. Most importantly, the ASC-organoids contain the fully developed human adult ISC population, which previously could not be generated or propagated in vitro. Thus, they offer a new system in which the biology of stem cells can be more closely studied in the context of epithelial damage following radiation, chemotherapy, and intestinal infections.

USING ORGANOID CULTURES TO STUDY INTESTINAL INJURY AND REGENERATION RESULTING FROM RADIATION AND CHEMOTHERAPY

Radiation and Chemical Damage of the Epithelium

Radiation and chemotherapy, which are used to treat cancer and other conditions, are toxic to proliferating ISCs and dramatically affect the regenerative capacity of the intestinal epithelium. Side effects from these treatments, including nausea, vomiting, dyspepsia, abdominal pain, and ulcerations that occur as a result of decreased stem cell reserves and ability to repair cell damage, cause both short- and long-term complications in the gastrointestinal tract (4, 54). Currently, there is no approved agent to treat radiation- or chemical-induced GI injury or complications. In vivo models of radiation and chemotherapy have been used extensively to interrogate the mechanisms of regeneration of the proliferative ISC (1, 9, 30, 34, 43, 47, 48, 55, 71, 72, 74). Several hypotheses have been proposed to explain how the epithelium recovers from toxic damage. These include crypt fission, in which surviving crypts expand to replace crypts lost due to stem cell depletion (68), the existence of quiescent stem cells that repopulate the cycling ISC (2, 34, 42, 58), and the capacity of differentiated epithelial cells that exhibit cell plasticity and revert into ISCs (1, 5, 22, 59, 62, 71, 72, 74). The signaling pathways that mediate the regeneration of the ISC after damage are important avenues for understanding how ISC and niche homeostasis is regained. Both chemical and radiation damage can be applied to intestinal organoids, and the response of the epithelium in these models will help identify the upstream regulators of the signaling pathways that are modulating epithelial repair.

Intestinal Organoids and Radiation Damage

Currently, organoid cultures are being utilized in two different approaches to examine recovery of the epithelium after radiation damage: 1) irradiation is performed in vivo in the mouse, and subsequently intestinal organoid formation is assessed in vitro (organoid-forming assay (63, 70) and 2) organoids are established from either human or mouse intestine and the organoids themselves are exposed to radiation in vitro (70). The organoid-forming assay has been used primarily to dissect key pathways in regeneration following irradiation of reporter and knockout mice in which specific components of the pathways are deleted or altered. In a recent study, the organoid-forming assay demonstrated that the myeloid translocation gene MTG16 plays a role in negatively regulating regeneration (41). This assay was also used to demonstrate that SRY-box transcription factor 9 (SOX9)+ epithelial cells can survive and expand to form organoids after exposure to radiation in vivo (63). Organoid-forming assays were used to demonstrate that the lack of expression of blood vessel epicardial substance, which negatively regulates WNT, resulted in increased organoid plating efficiency, spheroid formation, and proliferation and expression of stem cell markers following radiation exposure in vivo (45). A radio-resistant cell that expresses kertain 19 (KRT19) has been identified in murine colonic organoid-forming assays that seems to render the LGR5+ ISC dispensable for recovery from radiation (1). Radiation treatment of established organoids has resulted in identification of potential mitigators of radiation-induced gastrointestinal syndrome. Using human colonic organoids, an antineoplastic small molecule, BCN057, was shown to induce regeneration and mitigate toxicity of radiation exposure (3). Regeneration was quantified by increased organoid budding and transcriptional expression of stem cell and proliferative markers, such as LGR5 and KI67, respectively. Wnt-β catenin signaling, which is an important component of the ISC niche (8), was induced by BCN057 and accelerated the repair pathway in the organoids. In similar studies (36), human colonic organoids were used to demonstrate that Auranofin, an anti-inflammatory agent, was protective against radiation-induced damage and improved survival in the cultures via activation of p53/p21 cell cycle arrest that was reversible (36). Addition of CHIR9901, a GSK-3 inhibitor, to human intestinal cultures significantly improved survival and growth after radiation (65) via selective blockade of p53-induced apoptosis in the ISC. These in vitro organoid studies suggest that p53 regulation may be important to radioprotective responses. Using a high-throughput format to identify molecules associated with increased murine small intestinal-organoid recovery following irradiation, a library of 2,534 compounds was screened and 2 compounds were identified that increased recovery from gamma irradiation. Two compounds were identified that improved the viability of the organoid cultures following radiation exposure via mechanisms that had not been previously linked to radiation protection: CGP052608, a synthetic thiosemicarbaszone that acts through retinoic acid receptors, and G023850, which inhibits methionine amnopetidases (29). These organoid studies suggest potential new target pathways that could be modulated to mediate the effects of radiation. Although mouse organoids were used in the screen, this approach seems easily transferable to human organoids. Automation will be a key component in the evolution of using organoids in a high-throughput format to screen for compounds that mitigate radiation injury. An automated organoid morphometry platform has been developed to assess growth survival and regeneration of intestinal organoids following irradiation (46). Platforms such as these are currently facilitating drug screens to identify candidates to counteract the side effects of radiation exposure in the intestine.

Intestinal Organoids and Chemical Damage

Chemotherapeutic agents, similar to radiation, have detrimental side effects on the ISC and cause significant complications. The effect of these agents on epithelial regeneration has begun to be explored using intestinal organoid cultures. Human intestinal organoid cultures were used to understand the dual role of p53 pathways in the damage resulting from treatment with the chemotherapeutic agent, irinotecan (CPT-11), as well as the regenerative response of the epithelium following the damage (27). A small molecule inhibitor of PUMA, a transcriptional target of p53 induction that is important in radiation-induced apoptosis, enhanced preservation of the LGR5+ ISC and promoted organoid growth and epithelial regeneration following CPT-11 damage in both mouse and human colonic organoids. Human organoid cultures have demonstrated the plasticity of Paneth cells to revert to LGR5+ ISC following doxorubicin damage (6). This work provides strong support for a role of the Paneth cell in regeneration when the crypt ISC is damaged by chemical agents. The effect of various inhibitors and growth factors on cell growth and Paneth cell reversion revealed a role for R-spondin, phosphatase and tensin homolog (PTEN), and AKT signaling in the regenerative response. These studies illustrate the capacity of using intestinal organoids to discover new pathways and mechanisms that are potential targets to mitigate chemotherapy-induced intestinal injury.

USING ORGANOID CULTURES TO STUDY REGENERATION FOLLOWING INTESTINAL INFECTIONS

Gastrointestinal Infections and Epithelial Damage and Repair

More than 76 million people in the United States suffer from gastrointestinal infections (viral, bacterial, parasitic) each year (49). In contrast to radiation or chemical damage, which directly target the cycling LGR5+ ISC, pathogenic infections of the intestine predominantly cause damage to the differentiated epithelium that, in many cases, results in villus blunting or atrophy. Because of the clinical importance of these diseases, there is increased interest in understanding the effect of pathogen-induced damage on the ISC response and comparing it to the effects of radiation/chemotherapy. In vitro transformed human cell lines or human explant tissue models have been limited in their use to study regeneration after infection due to the lack of cellular complexity in modeling the intestinal epithelium, the absence of stem cells, and the short-term viability in culture that does not allow infections to be examined. In vivo systems, including studies in mice, better model the complexity of infections and have been used widely; however, these models are not susceptible to many human pathogens or do not exhibit comparable pathogenesis, limiting their use in understanding how the epithelium repairs itself after infections. Despite their limitations, mouse models have revealed that intestinal infections can result in effects on the ISC and its niche. Some pathogenic bacterial products, such as Salmonella AvrA and type III secretion components, appear to increase ISC proliferation through expansion of Paneth cells and upregulation of Wnt signaling in vivo (32, 33). Rotavirus, a common viral infection of the mature enterocytes, stimulates Paneth cell production of WNT and Wnt signaling in the crypt that results in increased proliferation and migration of the epithelial cells in mice (80). In contrast to uninfected tissue where the mesenchyme appears to be the primary modulator of ISC activity (61), epithelial components such as the Paneth cell seem to be a major factor in the regenerative response to infection. Thus, intestinal organoid cultures, which maintain the complexity and physiology of the intestinal epithelium, provide an ideal system to dissect the molecular pathways of the epithelial response and allow for the study of many human intestinal pathogens that previously were unable to be studied outside the human host. Most pathogens initially contact the intestinal epithelium on the apical or luminal side. Modeling this contact interaction using three-dimensional organoids (where the apical side of the epithelium faces the lumen of the organoid) has been somewhat problematic, requiring either labor-intensive microinjection techniques (67) or that the organoids be broken open (53) (Fig. 2). Establishment of culture conditions to grow organoid cultures in a monolayer format that exposes the apical surface (64) makes delivery of infectious organisms much less labor intensive (Fig. 2), and infections using this format have already been demonstrated (12). Pathways identified from these studies will provide a more complete picture of the numerous signals to which the ISC can respond and potentially provide new targets to direct therapeutics to modulate epithelial regeneration.

Fig. 2.

Formats of organoid culturing. Organoids can be cultured as three-dimensional (3D) structures enclosed within a matrigel plug. 3D organoids typically have the apical side of the epithelium facing the inside lumen of the 3D structure, making it difficult to easily access. 3D organoids can be dispersed and grown as a monolayer either in a 96-well plate or on a transwell membrane. In either format, there is easy access to the apical side of the epithelium. Figure was created with Biorender.com.

Organoids and Viral Infections

Despite the role of viral infections as one of the leading causes of acute gastroenteritis worldwide, the effect of viral damage on epithelial regeneration of the human intestinal tract has not been well characterized, in part due to a lack of in vitro models in which the pathogenesis of human viruses recapitulates the in vivo properties. Several human intestinal viruses, including rotavirus, norovirus, enterovirus, adenovirus, and coronavirus, have now been demonstrated to infect human intestinal organoid cultures (10, 12, 14, 19, 53, 79). Human rotavirus infects human organoid cultures much more efficiently than animal rotavirus (53), recapitulating in vivo epidemiology findings. Organoid cultures are able to functionally and physiologically recapitulate some of the damage aspects of in vivo human rotavirus infection, including cytotoxicity and fluid flux characteristic of secretory diarrheas (53). The organoid model has also enabled study of the interactions between human norovirus and the epithelium. Ettayebi et al. (12) demonstrated human noroviruses isolated from stool replicate in human organoid cultures with evidence of cell rounding, destruction of the epithelium, and an increase in dead cells. Additionally, these studies found that the enterocyte is most likely the site for human norovirus replication. In a similar fashion, human adenoviruses were shown to have tropism for goblet cells in organoid cultures, which was not previously known (19). Human enterovirus also appears to replicate in the enterocyte population in organoid cultures and, like rotavirus and norovirus, induces significant cytotoxic effects accompanied by disruption of tight junctions that results in loss of barrier function and cell necrosis (10). Although the organoid cultures are now being widely used to study pathogenesis, studies examining regenerative responses of the epithelium following viral damage are in their infancy. Although spread of avian influenza strains by the fecal oral route is well documented and makes disease hard to prevent and control, little is known about its pathogenesis in the intestine. Human intestinal organoids were used to demonstrate that an avian influenza strain that infects the intestinal tract also impairs ISC proliferation and differentiation (20). This virus appears to damage the Paneth cells in the organoids presumably causing indirect effects on ISCs. The use of organoid cultures to study regeneration of the epithelium following viral damage has the potential to provide insight on epithelial renewal in situations where the differentiated cells are damaged but the crypt remains relatively intact.

Organoids and Bacterial Infections

Intestinal bacterial pathogens also cause a large percentage of acute gastroenteritis worldwide. Organoid cultures have created new models of human bacterial infections in which epithelial regeneration after bacteria damage can be explored. The intestinal organoid culture recapitulates in vivo pathologies of several human pathogenic intestinal bacteria, including Salmonella enterica and typhimurium; Enteropathogenic, Enterotoxigenic, Enterohemorrhagic, and Enteroaggregative Escherichia coli; and Clostridium difficile (11, 15, 21, 24, 28, 44). In particular, some bacterial infections, such as C. difficile (28) and Salmonella (78), recapitulate the tight junction damage that is often associated with reduced barrier function. As with viral infections, regeneration of the intestinal epithelium following bacterial infections is only beginning to be explored via organoid culture models. Recent work using both human and mouse organoids has suggested that the levels of cytokines that are induced by bacterial infections have a significant effect on the ISC niche by modulating proliferation, cell fate, architecture, antimicrobial secretion, and crypt cell hierarchy (31). With the establishment of several models of human bacterial infections in the organoid cultures, scientists are now poised to begin to understand intestinal epithelial regeneration following bacterial injury.

A recent effort is the utilization of organoid cultures to understand the effects of commensal intestinal bacteria on epithelial homeostasis and repair (40). New work has shown that the ISC expresses nucleotide-binding oligomerization domain-containing protein 2 (NOD2), which is stimulated by a peptidoglycan motif that is expressed on most bacterial organisms, suggesting putative pathways that might be conduits for communication between the microbiome and the ISC niche (38). Treatment of organoid cultures with ligands for NOD2 resulted in increased numbers and larger-sized organoids, indicating that these ligands induce epithelial proliferation. These ligands also exhibited cytoprotective effects when organoids were treated with doxorubicin. Support for the concept of a role for bacterial species in the ISC niche comes from studies in mice where a crypt-specific microbiome has been identified that associates with homeostatic proliferation in the colonic crypt. This finding led to organoid studies that demonstrated that modulation of the colonic epithelial balance between proliferation and differentiation occurred through a TLR4-dependent pathway that was triggered by bacterial-derived LPS (37). Other work using the organoid-forming assay has shown that colonic crypts from mice that are devoid of the microbiota lose their regenerative capacity, as assessed by the ability to form organoids (76). The regenerative capacity was recovered by fecal microbiota transplantation that restored the crypt microbial communities (76). Recently, lactate derived from bacteria was shown to mediate small intestinal epithelial proliferation through stimulation of the LGR5+ ISC in murine organoid cultures (26), suggesting there may be specific bacterial-derived factors that interact with the host cells to modulate the ISC response. These initial findings provide strong rationale for exploring the role of the microbiome in homeostasis of the human ISC niche. A role for the microbiome in repair after injury still remains to be defined. The organoid system is just beginning to be used extensively to study whether commensal microbial organisms and their products control crypt homeostasis or contribute to epithelial repair.

Organoids and Parasitic Infections

Although parasites are a significant cause of morbidity and mortality from diarrheal disease, the use of organoids to better understand the pathogenesis of these organisms has lagged behind that of the viral and bacterial fields. Recently, parasitic infections such as Cryptosporidium, Nippostrongylus brasiliensis, and Toxiplasma gondii have been established in organoid cultures (16, 18, 25) and appear to accurately model in vivo infectivity and pathogenesis, surmounting a major barrier in studying these pathogens. Mouse organoids infected with Cryptosporidium demonstrated decreased organoid budding (a marker of proliferation) and LGR5+ ISC marker expression (77), mimicking in vivo findings. Organoid-forming assays performed on crypts isolated from Cryptosporidium-infected mice showed a similar lack of budding compared with uninfected controls (77). The Wnt pathway, a key factor in ISC function, appeared to be inhibited by the pathogen in the organoid culture. Because Cryptosporidium infection is limited to enterocytes, there may be unique signals that arise from the epithelium that modulate the ISC through regulation of the niche. More extensive studies will be necessary to illuminate the mechanisms of epithelial repair following parasitic infection.

SUMMARY

The gap between in vitro human-transformed cell lines and in vivo animal models has now been filled by the establishment of human intestinal organoid culture, in which the human nontransformed epithelium can be cultured and the biology dissected. These cultures allow exquisite control of experimental conditions surrounding epithelial damage and regeneration in vitro. The capability of organoids to be damaged either through irradiation, chemical treatment, or as a result of infection by pathogenic organisms provides an elegant model for defining signals that restore homeostasis after damage occurs. Organoids can also be used to expolore the role of commensal organisms that also may play a role in both controlling epithelial homeostasis and restoring homeostasis following injury. Several pathways are emerging as important in regeneration, including Notch, EGF, bone morphogenetic protein (BMP), phosphatidylinositol 3-kinase (PI3K)/AKT, MAPK, Wnt, and Hippo signaling, which are proposed to induce events such as dedifferentiation of mature epithelial cells, stem cell plasticity, and transduction of inflammatory cues. These pathways can now be explored and validated in the organoid culture. The ability to genetically modify organoids using CRISPR/Cas-9 editing will allow a deeper dissection of these pathways and their individual contributions to epithelial repair. The intestinal organoid cultures are continuing to evolve with improved culture conditions and the incorporation of vasculature, components of the enteric nervous system, immune components, and the incorporation of physical forces that mimic luminal and blood flow as well as peristalsis (39, 69), all of which may contribute to or affect the epithelial repair process. The influence of these components on the molecular mechanisms induced by injury in organoid cultures will be important to fully define the mechanisms that modulate epithelial regeneration.

FUTURE QUESTIONS

Intestinal organoids will make a significant contribution to developing preventive and therapeutic modalities to treat gastrointestinal disorders and will complement the current in vivo models. The cultures hold great potential to provide inroads to understanding the plasticity of intestinal epithelial cell populations and defining under what conditions of damage and by what mechansims they are induced to repopulate the cycling ISC compartment. The molecular pathways that are induced by epithelial damage still remain to be linked to specific responses within the ISC niche and most likely will differ based on type of injury, and upstream regulators that are induced by injury remain poorly defined. Epithelial factors induced by damage can be easily defined using organoids; however, the role of stromal factors cannot be ignored. The evolution of PCS-derived organoids and the incorporation of mesenchymal cells into ASC-derived organoids will be essential to defining the role of the stroma. The interplay of epithelial and mesenchymal factors in repair and regeneration needs to be fully characterized. There has been much discussion about the therapeutic potential of transplanting organoid cultures to repair damaged intestinal epithelium. Initial experiments in mice in which ASC-derived organoids were transplanted to repair chemical damage of the epithelium showed engraftment and proper differentiation of the organoids to the damaged areas (75). More work is necessary to determine if this is a viable therapeutic approach for human gastrointestinal diseases. The organoid models hold the promise of new discoveries that will help promote overall intestinal health and hasten recovery from or prevent intestinal epithelial damage.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.E.B. prepared figures; S.E.B., O.D.K., M.D., N.S., C.G., and M.K.E. drafted manuscript; S.E.B., M.D., N.S., C.G., and M.K.E. edited and revised manuscript; S.E.B., M.D., N.S., C.G., and M.K.E. approved final version of manuscript.