Review of studies of severe acute respiratory syndrome related coronavirus–2 pathogenesis in human organoid models

1 INTRODUCTION

Severe acute respiratory syndrome related coronavirus-2 (SARS-CoV-2) is a member of the Coronaviridae family and is a single-stranded positive-sense enveloped RNA virus.1, 2 Human coronaviruses are one of the leading causes of the common cold and tend to result in mild respiratory tract symptoms, although recent data suggest they can cause more severe disease.3 Novel human coronaviruses have emerged over the last two decades including severe acute respiratory syndrome related coronavirus (SARS-CoV), the Middle Eastern respiratory syndrome related coronavirus (MERS-CoV), and recently SARS-CoV-2.2, 4, 5 Covid-19 is caused by SARS-CoV-2 infection and was classified as a pandemic by the World Health Organisation (WHO) in March 2020.6 The symptoms of Covid-19 range widely, presenting as asymptomatic in many individuals or fever, myalgia, cough, chest tightness, and in severe cases pneumonia, occasionally resulting in death.7

The current pandemic has developed globally rapidly, meaning there is urgency around studies of the pathogenetic mechanisms of SARS-CoV-2 infection to inform development of antiviral drugs and vaccines. A key method of investigating pathogenesis of infection, assisting design and development of therapeutics, is the employment of three-dimensional, multi-cell-type culture models known as organoids. These allow (i) investigation in a human, biologically relevant model, (ii) utilisation of multicellular models to demonstrate the effects of infection and treatment on different cell lines, and (iii) dissection of the molecular response of individual cells within a human organ.

1.1 Overview of traditional models for disease pathogenesis research

Two-dimensional cell monocultures and animal models are frequently used to model human viral infections. Monocultures are the primary model used in the isolation and propagation of viruses, including the first culture of SARS-CoV-2 in the Vero/hSLAM cell line.8 However, monocultures do not accurately represent the complexity of in vivo tissues. Different cell types in organs, such as the brain, have specific gene regulation patterns which would otherwise not be taken into account in single-cell culture models.9, 10 This has been further confirmed by reported alterations in cell signalling networks between two- and three-dimensional culture systems.11, 12 In order to overcome these limitations, researchers have begun utilising alternative three-dimensional disease modelling systems, such as organoids. These more closely mimic the complex multi-cell-type composition and structure of human organs.13, 14

Animal models are multi-cell-type, multi-organ models that facilitate the translation from basic research to clinical trials. Animal models are essential in informing the development of antiviral treatments and vaccines and have recently been used to evaluate Covid-19 vaccine candidates in models such as Rhesus macaques.15-17 However, the differing developmental patterns between non-human animals and humans18 have necessitated the development of alternative human organ models such as organoids. Furthermore, the ethical considerations involved in animal research are monitored through the 3Rs principle (replacement, reduction and refinement of animal research) in an effort to maximise high-quality data while minimising harm to animals during research.19 This principle dictates the replacement of animal models where possible through the use of alternative culture systems such as human organoids.20

1.2 Organoid generation and use in modelling viral infection

Organoids are organ-like tissue models from pluripotent stem cells or progenitor cells through differentiation.13 During the differentiation process, multiple cell types arise and self-organize to form cellular organisation and tissue morphology resembling in vivo human organs, which can be used to model infection.13, 14

Organoids have successfully been used to model neurological infections such as with Zika virus (ZIKV).21 Congenital ZIKV infection has been associated with the development of foetal neural malformation such as microcephaly.22 Researchers have utilised cerebral organoids to study pathogenesis of ZIKV infection, including brain-region-specific forebrain organoids.21 In this model, ZIKV induced increased cell death and reduced cellular proliferation, subsequently decreasing neuronal volume, inducing microcephaly-like pathophysiology,21 suggesting a mechanism for ZIKV-induced neural malformation.

Different organoids have been utilised in SARS-CoV-2 mechanistic investigations. The respiratory tract is the major organ system affected by Covid-19 disease, although injury to other organs has been observed including acute kidney injury, cardiovascular disease and neurological disease including encephalopathy, encephalitis, Guillain–Barre syndrome and acute stroke.23, 24 The wide array of organs affected in Covid-19 disease necessitates the use of a variety of organoid models to extrapolate the mechanisms of SARS-CoV-2 infection in different systems. Specific growth and differentiation factors that facilitate the development and generation of a variety of organoids have been used to produce organoids simulating different human organs, allowing a biologically relevant, three-dimensional model for disease.13, 14 In this review, we detail the conclusions drawn from 16 studies investigating SARS-CoV-2 infection of different human organoid models.

2 SARS-CoV-2 AND ORGANOIDS 2.1 Expression of SARS-CoV-2 receptors in organoids

The SARS-CoV-2 spike (S) protein, which is structurally similar to that of SARS-CoV, mediates viral attachment and cellular entry through the binding of the N-terminal S1 subunit to the metallopeptidase, angiotensin I converting enzyme 2 (ACE2) receptor (Figure 1).25-27 Interestingly, SARS-CoV-2 binds to the ACE2 receptor with a higher affinity than does SARS-CoV.25 Hoffmann et al. suggest the SARS-CoV-2 S protein is primed by the transmembrane serine protease 2 (TMPRSS2), inducing virus-host cell fusion, similar to SARS-CoV (Figure 1).26 Using single-cell RNA sequencing datasets, several studies have determined the ACE2 and TMPRSS2 receptors are expressed in the human lung, eye, heart, oesophagus, ileum, kidney, colon, liver, gallbladder and testis (pre-print).28-30 Investigating the presence of these host cell receptors in various organoid models is an important first step in determining their ability to support viral infection. The detection of these receptors in various organoids is summarised in Figure 1.

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Severe acute respiratory syndrome related coronavirus-2 (SARS-CoV-2) and host cell receptors. (a) Viral attachment and cellular entry is mediated by the SARS-CoV-2 spike (S) protein. The N-terminal subunit, S1 binds to the receptor, angiotensin I converting enzyme 2 (ACE2).25-27 Hoffmann et al. suggest the S protein is primed by the transmembrane serine protease 2 (TMPRSS2), inducing host cell fusion.26 (b) Using single cell RNA sequencing datasets, various human organs have been identified as expressing the target receptors for SARS-CoV-2 infection, ACE2 and TMPRSS2.28-30 Subsequently, numerous organoid models have been utilised to detect ACE2 and TMPRSS2.31-43

In organoids, the ACE2 receptor was detected in lung (pre-print),31, 32 bronchial (pre-print),33 the choroid plexus,34 hippocampus,34 brain,35 self-formed ectodermal autonomous multi-zone (SEAM) eye (pre-print),36 intestinal enteroids,37, 38 colonic,32 kidney39, 40 and liver41, 42 organoids, as well as brain spheres.43 Interestingly, ACE2 was also expressed in multipotent neural progenitor cells (NPCs), from which brain spheres are generated through differentiation.43 During foetal cerebral organogenesis, NPCs are a crucial population of cells, with the capability of differentiating into oligodendrocytes, astrocytes and neurons that populate the developed adult brain.44, 45 The presence of the ACE2 receptor in this population of cells suggests SARS-CoV-2 tropism, and further research is required to determine the effects of infection on foetal brain development. However, most guidelines suggest effects on the foetus are unlikely (UK guidelines).24, 46

The TMPRSS2 receptor was expressed in lung (pre-print),31, 32 bronchial (pre-print),33 choroid plexus,34 hippocampal,34 SEAM eye (pre-print),36 intestinal enteroid,37 colonic32 and liver41 organoids. However, TMPRSS2 receptor expression was reportedly below the limit of detection in brain spheres.43

Expression of these target cellular receptors localised in certain cell populations in organoids. In lung organoids, ACE2 was detected predominantly in club31 and alveolar type II (AT2)-like cells (pre-print).31, 32 Interestingly, in alveolar type I (AT1) and AT2-like cultures, SARS-CoV-2 preferentially infected AT2 cells over AT1 cells,47 which may be explained by the reported predominant detection of ACE231, 32 in the AT2 population of cells. Enrichment of TMPRSS2 was reported in AT2-like31, 32 and club31 cells in lung organoids (pre-print). Furthermore, results from Suzuki et al. suggested both ACE2 and TMPRSS2 are expressed in the basal cells of bronchial organoids, but only TMPRSS2 is expressed in ciliated cells (pre-print).33

Single-cell RNA sequencing of SEAM eye organoids revealed ACE2 may be predominantly expressed in the limbus, conjunctiva and a subset of ocular surface ectoderm cells (pre-print).36 The TMPRSS2 receptor was expressed in corneal cells in SEAM eye organoids (pre-print).36 Single-cell profiling of kidney organoids revealed the proximal tubule and podocyte II cell clusters express ACE2.39

Organoids have allowed researchers to identify specific cell populations that express SARS-CoV-2 receptors in the lung,31-33 eye36 and kidney.39 Understanding the localisation and enrichment of host cell receptors that are utilised by SARS-CoV-2 during infection not only indicates the cell types that are more permissive to infection in specific organs, but also can potentially inform development of targeted therapeutics to these vulnerable cell populations.

Although organoids have facilitated the detection of target cell receptors in different organ types, this alone is not sufficient to determine the organ's permissiveness to SARS-CoV-2 infection, nor the organs' ability to support viral replication. Yang et al. reported that despite the expression of ACE2 in human pluripotent stem-cell-derived macrophages, cortical neurons and endothelial cells, they had little to no permissiveness to SARS-CoV-2 or SARS-CoV-2 pseudo-entry virus, suggesting other factors are involved in determining viral tropism.42 Therefore, supplementing viral receptor studies with those specifically investigating the tropism of different organoids are necessary to ascertain their effectiveness as models for SARS-CoV-2 infection.

2.2 Permissiveness of organoids to SARS-CoV-2 infection

In a study using autopsy tissue samples from patients who had died from Covid-19, SARS-CoV-2 was detected in the lungs, pharynx, brain, heart, liver and kidneys, suggesting viral tropism in these organs.48 Additionally, monocultures have been utilised to model and establish SARS-CoV-2 tropism in cell lines representative of different organ types including Caco2 (intestinal epithelial carcinoma), Calu3 (lung epithelial adenocarcinoma) and U251 (glioblastoma).49

Studies investigating the permissiveness of different types of organoids to SARS-CoV-2 infection are detailed in Table 1.31-39, 41-43, 50-52

TABLE 1. Permissiveness to SARS-CoV-2 infection in various organoid models Organoid type Source SARS-CoV-2 strain Permissiveness Lung Human pluripotent stem cells (hPSC) Permissive and supported robust viral replication.32 Distal lung epithelial cells and MRC5 human lung fibroblast cells Intact alveolar organoids were refractory to viral infection. Gentle physical and enzymatic disruption to organoids made them permissive to infection and viral replication (pre-print).50 Distal airway cells from patient lung tissue Permissive to infection. Organoids required everting so cells would be relocated and the ACE2 receptor would face outward (pre-print).31 Normal human bronchial epithelial cells Permissive to infection and viral replication (pre-print).33 Brain spheres and cerebral organoids Induced pluripotent stem cells (iPSC) Permissive to infection. Small fraction of neural cells contained viral particles. Increased viral RNA indicative of replication.43 iPSC Choroid plexus organoids are permissive to productive infection.34 iPSC Brain organoids are permissive to infection but do not support active viral replication.35 SEAM eye organoid Human embryonic stem cells (hESC) Permissive to infection (pre-print).36 Liver iPSC Liver hepatocyte organoids. Permissive in ALB + hepatocytes and supported robust replication.42 Liver bile duct-derived progenitor cells Liver ductal organoids. Permissive and supported robust replication. Infected cholangiocytes formed syncytia.41 hPSC Cholangiocyte organoids. Permissive in CK19+ cholangiocytes and supported robust replication.42 Enteroids/Intestinal organoids Intestinal samples from patients Enteroids are permissive to infection. Most infected cells were Villin+, indicating enterocytes are the predominant target cells for infection.37 Patient tissue Duodenal organoids are permissive to infection.38 Patient tissue Ileum-derived organoids are permissive to infection and support robust viral replication.38 Patient tissue Colon-derived organoids are permissive and support viral replication.38 Patient tissue Small intestinal organoids are permissive and support productive infection. Viral particles detected in the lumen of the organoid.51 Stem cells isolated from human tissue Permissive to infection, supports viral replication, and de novo infectious virus production.52 Colonoids/Colonic organoids Colonic sample from patient Permissive to infection and robust viral replication.30 hPSC Permissive.32 Capillary iPSC Permissive to active viral replication.39 Kidney hESC Permissive to viral replication.39 Note: Table summarising the tropism of different strains of SARS-CoV-2 in various organoid types as well as their ability to support viral replication when specified. Abbreviations: ACE2, angiotensin I converting enzyme 2; hESC, human embryonic stem cells; hPSC, human pluripotent stem cells; iPSC, induced pluripotent stem cells; SARS-CoV-2, severe acute respiratory syndrome related coronavirus-2; SEAM, self-formed ectodermal autonomous multi-zone. 2.3 SARS-CoV-2 and the immune response

Increased production of cytokines has been implicated in the immunopathology of disease in patients with Covid-19, resulting in what is commonly referred to a ‘cytokine storm'.53, 54 Various types of organoids have been utilised to model this immunological phenomenon. A Th1 cytokine response was elicited following SARS-CoV-2 infection in alveolar (pre-print),50 choroid plexus34 and intestinal,37, 51 organoids (Table 2). In a study comparing post-mortem lung samples from Covid-19 patients to biopsies from healthy lung tissue from uninfected individuals, transcriptional analysis revealed high chemokine signatures in Covid-19 patients including CCL2 (MCP-1), CCL8 (MCP-2) and CCL11.53 Similarly, induction of chemokines or chemokine transcripts were detected in SARS-CoV-2 infected lung,32 hepatocyte,42 cholangiocyte,42 intestinal37, 51 and colonic32 organoids (Table 2).

TABLE 2. Expression profiles of cytokines and chemokines in organoid models following SARS-CoV-2 infection Organoid type Expression of cytokines and chemokines following SARS-CoV-2 infection Downregulation of cytokines and chemokines following SARS-CoV-2 infection Alveolar (pre-print) IFNB150 .. Lunga CXCL2, CXCL3, CXCL5, CCL2, and CCL2032 .. Choroid plexus CCL7, IL-32, CCL2 (MCP1), IL-18, and IL-834 .. Intestinal/intestinal enteroids CCR1, CCR8, IL16, IL337 CXCL10 (IP10)37,51 CCR2, CCR5 and IL537 Hepatocyte CXCL1, CXCL3, CXCL5, CXCL6 (GCP-2), and CCL20 (MIP3α)42 .. Cholangiocyte CXCL1, CXCL2 (MIP-2α), CXCL3, and CCL2 (MCP-1)42 .. Colonica CXCL6, CXCL8, CXCL11, IL-1α, IL-1β32 .. Note: Table summarising the expression profiles of different cytokines and chemokines in a variety of SARS-CoV-2 infected organoid models. These data are indicative of the induction of cytokine and chemokines during infection in a majority of organoid types. Abbreviation: SARS-CoV-2, severe acute respiratory syndrome related coronavirus-2. a Data extrapolated from volcano plots of gene expression profiles from mock and SARS-CoV2-infected organoids in Han et al.32 study.

Among the consequences of viral infection induced cytokine storms in the lung are cellular apoptosis, vascular leakage, insufficient T-cell response and the development of acute respiratory distress syndrome (ARDS).55, 56 A recent cohort study reported up to 85% of Covid-19 patients admitted to the ICU had developed ARDS,57 and this was associated with increased mortality rates.58, 59 The SARS-CoV-2-mediated increase in cytokine and chemokine signatures observed in both organoid models and clinical settings may reflect the immunopathological mechanism for the development of further Covid-19-related sequelae.

Interferons (IFNs) are innate immune response proteins secreted by host cells and are responsible for inducing and regulating antiviral mechanisms following viral infection.60, 61 The effects of SARS-CoV-2 infection on IFN signalling have been summarised in Table 3. Transcriptomics analysis of infected alveolar organoids 2 days post-infection (dpi) revealed IFN signalling was the most upregulated canonical pathway (pre-print).50 Similarly, SARS-CoV-2 infection of bronchial organoids induced a moderate increase in type I IFN and IFN-stimulated genes (ISGs) at 5 dpi (pre-print).33 Upregulation of type III (IFN- λ)52 as well as modest expression of ISGs, such as ISG1551 were also observed in infected colon and intestinal organoids at 24 and 60 hours post-infection (hpi), respectively. Similarly, SARS-CoV-2 infection of intestinal enteroids induced expression of IFNL2 and IFNL3 at 48 hpi.37

TABLE 3. IFN response in different organoid types following SARS-CoV-2 infection Organoid type Induction of the IFN response following SARS-CoV-2 infection Minimal induction or downregulation of the IFN response following SARS-CoV-2 infection Hours/days post infection Alveolar (pre-print) Upregulated50 .. 2 dpi50 Bronchial (pre-print) Moderate increase in type I IFN and IFN-stimulated genes (ISGs)33 .. 5 dpi33 Intestinal/intestinal enteroids Modest expression of ISGs51 .. 60 hpi51 Induction of IFNL2 and IFNL337 IFN-α, IFN-β and IFN-γ were “barely” induced

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