Human organoid cultures: transformative new tools for human virus studies
Introduction
Studies of infectious agents have traditionally focused on in vitro culture systems using transformed cell lines and animal models. While these systems have enabled historic progress in the understanding of microbial pathogenesis and host–pathogen interactions, and facilitated the development of vaccines and therapeutics, the physiological relevance of these models for human pathogens and diseases can be limited. Routinely used cell lines are immortalized and cancer-derived and may not adequately reflect responses of normal human cells [1, 2, 3]. Many signaling pathways are altered in cancer cells and have profound effects on the core metabolism in cancer-derived lines [4]. Furthermore, cell lines are typically comprised of a single cell type and do not mimic the architecture, environmental complexity and functionality of tissues which are comprised of many different cell types. While some of these drawbacks are overcome in animal models, these have other restrictions for studying human infectious diseases. Many human pathogens display unique human specificity that precludes their study in animals while in other cases, animal models fail to reproduce human pathophysiology. Further, findings and effects seen in animal models do not always translate to humans, as has been observed in many different fields [5, 6, 7].
The development of in vitro organoid cultures offer remarkable new model systems to study infectious agents and disease pathogenesis [8]. While organotypic cultures comprised of three dimensional (3D) cell aggregates have been in existence for decades, the term organoids now largely refers to self-organizing, propagatable 3D cultures derived from stem cells that recapitulate the organization, functionality, and genetic signature of the specific tissue or organ and host from which they are derived. Organoids can be derived from embryonic and pluripotent stem cells (ESCs and PSCs, respectively) as well as stem cells isolated from specific human tissues. Through new understanding of defined developmental cues and growth factors, stem cells can be directed to grow ex vivo into organ-like structures. Unlike transformed cells lines and animal models, organoid cultures are multicellular, reflect the cellular heterogeneity of specific human organs and are physiologically active; thus, they are increasingly being validated as relevant models for studies of infectious disease, particularly of human-specific pathogens. Organoids have been established for multiple organs including the intestine, stomach, esophagus, liver, kidneys, lungs, brain, prostate, pancreas, retina and ovary [8]. Although the use of this new technology is in its infancy, paradigm shifting results and unexpected discoveries have been made with several human viruses (Figure 1) and select data are summarized below. Other recent articles address aspects of organoids for disease studies that are not covered in this review [8, 9, 10, 11, 12, 13].
Section snippets
Human intestinal organoids and enteroids
The human gastrointestinal tract is a complex organ with a polarized epithelial layer that contains different cell types including enterocytes, enteroendocrine cells, tuft cells, goblet cells, Paneth cells and stem cells. Distinct regions of the intestine (duodenum, jejunum and ileum, proximal and distal colon) perform unique functions and demonstrate segment-specificity in terms of transport, protein expression and interactions with pathogens [14, 15]. The breakthrough in intestinal organoid
Cerebral organoids
Given the complex organization of the developing cortex with different progenitor and neuronal layers, the generation of 3D brain organoids from human PSCs that mimic human brain development is a significant breakthrough in neuroscience [42•]. Brain organoids have been used extensively in recent times for studies with Zika virus (ZIKV), a mosquito-borne Flavivirus. In 2016, the WHO declared a global health emergency due to the link between the ZIKV outbreak and the increased incidence of
Lung organoids
Lung bud organoids containing the mesoderm and pulmonary endoderm were generated from human PSCs and develop into branching airway and early alveolar structures after in vivo transplantation (xenotransplantation) under the mouse kidney capsule as well as in Matrigel 3D cultures [55••]. Resembling lung buds at the second trimester of gestation, these organoids can be infected with respiratory syncytial virus (RSV), the most common cause of bronchiolitis and pneumonia in children younger than 1
Challenges and future directions
While organoids have several advantages over transformed cell lines and animal models, key challenges remain to be addressed in order to improve their utility for studying viruses (Box 1). One limitation is that current organoid models are devoid of components of the normal host microenvironment such as immune cells and blood vessels. Co-culture models incorporating other cell types including endothelial cells and immune cells is an area of ongoing development [56, 57, 58]. In the case of
Conclusions
The development of organoids representing different sites of human infection is an exciting advance that can facilitate studies and new discoveries about host–virus interactions including molecular pathogenesis of viral infections, novel cell tropisms, new insights into host innate immune responses and characterizing interactions between pathogens (Figure 2). Importantly, human organoids/enteroids provide a functional and physiologically relevant model for pre-clinical evaluation of vaccines,
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
The authors would like to acknowledge support from grants U18-TR000552, UH3-TR00003, U19-AI116497, R01-AI080656, R01-AI105101, P30-DK56338 from the National Institutes of Health and the Food Research Initiative Competitive grant 2011-68003-30395 from the US Department of Agriculture, National Institute of Food and Agriculture.
References (62)
- et al.
Comparative proteomic phenotyping of cell lines and primary cells to assess preservation of cell type-specific functions
Mol Cell Proteomics
(2009) - et al.
Disease modeling in stem cell-derived 3D organoid systems
Trends Mol Med
(2017) - et al.
Engineered human gastrointestinal cultures to study the microbiome and infectious diseases
Cell Mol Gastroenterol Hepatol
(2018) - et al.
Organoid culture systems to study host–pathogen interactions
Curr Opin Immunol
(2017) - et al.
Gastrointestinal organoids: understanding the molecular basis of the host–microbe interface
Cell Mol Gastroenterol Hepatol
(2017) - et al.
Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays
Gut
(2015) - et al.
Effectiveness of rotavirus vaccination: a systematic review of the first decade of global postlicensure data, 2006–2016
Clin Infect Dis
(2017) - et al.
Norovirus and medically attended gastroenteritis in U.S. children
N Engl J Med
(2013) - et al.
Enter at your own risk: how enteroviruses navigate the dangerous world of pattern recognition receptor signaling
Cytokine
(2013) - et al.
E2F/Rb family proteins mediate interferon induced repression of adenovirus immediate early transcription to promote persistent viral infection
PLoS Pathog
(2016)
Zika virus impairs growth in human neurospheres and brain organoids
Science
Replication of Zika virus in human prostate cells: a potential source of sexually transmitted virus
J Infect Dis
Development of a primary human small intestine-on-a-chip using biopsy-derived organoids
Sci Rep
In vitro enteroid-derived three-dimensional tissue model of human small intestinal epithelium with innate immune responses
PLOS ONE
Transcriptome-wide analysis reveals hallmarks of human intestine development and maturation in vitro and in vivo
Stem Cell Rep
Differential protein profiling of primary versus immortalized human RPE cells identifies expression patterns associated with cytoskeletal remodeling and cell survival
J Proteome Res
Comparison of human duodenum and Caco-2 gene expression profiles for 12,000 gene sequences tags and correlation with permeability of 26 drugs
Pharm Res
Regulation of cancer cell metabolism
Nat Rev Cancer
Lost in translation: animal models and clinical trials in cancer treatment
Am J Transl Res
Can animal data predict human outcome? Problems and pitfalls of translational animal research
Eur J Nucl Med Mol Imaging
Mice are not men
Proc Natl Acad Sci U S A
Organoids as an in vitro model of human development and disease
Nat Cell Biol
Stem cell-derived models of viral infections in the gastrointestinal tract
Viruses
Human enteroids as a model of upper small intestinal ion transport physiology and pathophysiology
Gastroenterology
Novel segment- and host-specific patterns of enteroaggregative Escherichia coli adherence to human intestinal enteroids
MBio
Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche
Nature
Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro
Nature
Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium
Gastroenterology
A nomenclature for intestinal in vitro cultures
Am J Physiol Gastrointest Liver Physiol
Human enteroids/colonoids and intestinal organoids functionally recapitulate normal intestinal physiology and pathophysiology
J Biol Chem
Global, regional, and national estimates of rotavirus mortality in children <5 years of age, 2000–2013
Clin Infect Dis
Cited by (62)
Heat stability of foodborne viruses – Findings, methodological challenges and current developments
2024, International Journal of Food MicrobiologyModeling Innate Antiviral Immunity in Physiological Context
2022, Journal of Molecular BiologyCitation Excerpt :Since many of these systems are susceptible and permissive for viral infection, they provide a more robust platform to model physiologically-relevant antiviral responses. Detailed descriptions of these model systems, including organoids of the brain,168,169 airway,170,171 gut,172,173 placenta,174,175 and liver,176,177 have been reviewed elsewhere178–180; thus, in this section, we limit our discussion to the application of these models to studying innate antiviral responses. As noted above, pseudostratified and organotypic models of the airway epithelium and intestinal mucosa recapitulate the cell type-specific expression and polarized distribution of PRRs and IFN receptors observed in vivo.91,92
Rotavirus research: 2014–2020
2021, Virus ResearchCitation Excerpt :Rotaviruses can be propagated in IEs, in particular non-cell culture-adapted human RV isolates in human IEs (HIEs), including in enteroendocrine cells (Saxena et al., 2016; Fig. 5d, 5e). They are highly suitable for studies of the pathophysiology of RVAs including Ca2+ metabolism, Na+ and Cl− secretion, and RV RNA assortment and packaging (Foulke-Abel et al., 2014; Ramani et al., 2018). IEs are long-lived; they can be derived from different regions of the small intestine of various mammalian hosts including humans, be passaged and frozen ad libitum.