Studying SARS-CoV-2 infectivity and therapeutic responses with complex organoids

Abstract

Clinical management of patients with severe complications of COVID-19 has been hindered by a lack of effective drugs and a failure to capture the extensive heterogeneity of the disease with conventional methods. Here we review the emerging roles of complex organoids in the study of SARS-CoV-2 infection, modelling of COVID-19 disease pathology and in drug, antibody and vaccine development. We discuss opportunities for COVID-19 research and remaining challenges in the application of organoids.

Fig. 1:. SARS-CoV-2 (CoV-2) infection cycle, immunological response, molecular targets, and intervention strategies.

(a) The infection cycle includes spike glycoprotein (S-gp) binding to the human angiotensin-converting enzyme 2 (ACE2) receptor, pre-cleavage by the host cellular protease furin to dissociate the S1 subunit from the S2 subunit of S-gp^,, and S2 activation mediated by serine protease TMPRSS2 co-receptor. Notably, cleavage by furin is required for the entry of CoV-2 into human lung cells. S2 activation triggers viral and host cell membrane fusion. Within the host cell cytoplasm, the positive-sense CoV-2 genomic RNA is transcribed to yield full-length negative-sense RNAs (for genome replication) and subgenomic negative-sense RNAs (-sgRNA, for producing subgenomic mRNAs). Subgenomic mRNAs, converted from -sgRNAs, are translated into viral structural proteins, including S-gp, envelope (E), membrane (M), and nucleocapsid (N) proteins^–. Finally, viral genome encapsulation and reassembly enable virus maturation and export out of cells for the next infection cycle. (b and c) CoV-2 induces immunological responses through viral antigen presentation in macrophages, naive T cell activation, and release of cytokines. (d) A possible dual role of B-cell-mediated humoral immune response: B cells generate the neutralising antibodies to protect the lung from CoV-2 infection and contribute to cytokine-induced damage through FcγR-mediated and antibody-dependent enhancement of CoV-2 infection. (e) CoV-2-induced organ damage via an unbalanced presence of pro-inflammatory cytokines or absence of antiviral factors. (f) Representative intervention strategies, such as the development of drugs, vaccines, antibodies, recombinant proteins and repurposing of approved drugs against CoV-2 infection, based on molecular targets in Figure 1a. Abbreviations: ACE2, Angiotensin-converting enzyme 2; ADE, antibody-dependent enhancement; APC, antigen-presenting cells; CXCL10, C-X-C motif chemokine ligand 10; ER, endoplasmic reticulum; FcγR, Fc-gamma receptor; IFN, interferon; IL-6, interleukin 6; IL-6R, Interleukin 6 receptor; JAK, Janus kinase; JAKi, Janus kinase inhibitor; mAbs, monoclonal antibodies; NA, data not available; NF-κB, nuclear factor kappa B; Rc, replicase and transcriptase complex; NSPs, non-structural proteins; rc-ACE2-Ig, recombinant ACE2-Ig; STAT, signal transducer and activator of transcription; TMPRSS2, transmembrane protease serine 2; TNF-α, tumour necrosis factor alpha.

Fig. 2:. COVID-19-related assays.

(a) Assays are categorised as in vitro cell-free molecular and biochemical, pseudotyped virus, and live virus assays. Pseudotyped virus experiments are exemplified by pseudotyped VSV harbouring VSV-G and a SARS-CoV-S-gp chimeras. At 16-hour post inoculation, the pseudotyped viral entry is analysed by determining luciferase activity in cell lysates. No envelope glycoprotein pseudo-viral control is used for normalization. (b) Assays can be animal models, 2D-monolayer cell culture, 2D air-liquid interface (ALI) transwell culture, and 3D organoids. The combination of platforms empowers the utility of these assays for Covid-19 drug and vaccine development. Abbreviations: ASCs, adult stem cells; CoV2, SARS-CoV-2; ECM, extracellular matrix; hPSCs, human pluripotent stem cells; rc-proteins, recombinant proteins; S-gp, Spike glycoprotein; VSV-G, wild-type vesicular stomatitis virus; VSVΔG, vesicular stomatitis virus with deletion of the envelope glycoprotein (G).

Fig. 3:. Lung cell types and organoids.

(a) Human lung anatomy. (b) Schematic of the major cell types in different compartments of the human lung, partially adapted from references^,–. (c) A representative protocol for the generation of lung organoids containing cell types of interest. (d) Schematic of lung organoids that model different cellular compartments of the lung. (e) Cell types in panels b and d, with gene and protein markers listed alphabetically^,–,,. (f) Representative single-cell RNA sequencing analysis of CoV-2 receptor gene expression and co-expression (co-exp) in major cell types of the respiratory airways and alveoli. Nasal secretory cells are used as control for comparison. The size of the dots is proportional to the percentage of cells that express indicated genes (adapted from the published data in reference). Abbreviations: ABCA3, ATP-binding cassette subfamily A member 3; ACE2, angiotensin-converting enzyme 2; AQP5, aquaporin 5; ASCL3, Achaete-Scute family BHLH transcription factor 3; ATRA, all-trans retinoic acid; BMP4, bone morphogenetic protein 4; BV, blood vessels; CFTR, CF transmembrane conductance regulator; CYP4B1, cytochrome P450 family 4 subfamily B member 1; d, day(s); FBS, foetal bovine serum; FGF, fibroblast growth factor; FOXI1, forkhead box I1; FOXJ1, forkhead box J1; FOXN4, forkhead box N4; GSKβ, glycogen synthase kinase 3 beta; hiPSC, human induced pluripotent stem cells; inh, inhibitor; KRT5/14, keratin 5/14; LAMP3, lysosomal associated membrane protein 3; LGR5, leucine-rich repeat-containing G-protein coupled receptor 5; PDGFRA/B, platelet derived growth factor receptor alpha/beta; PDPN, podoplanin; SCGB1A1, secretoglobin family 1A member 1; SFTPB/C, surfactant protein B/C; SPDEF, SAM pointed domain containing ETS transcription factor; TMPRSS2, transmembrane serine protease 2; TUBB4, tubulin beta 4B class IVb.

Fig. 4:. Stem-cell-based organoids to assess SARS-CoV-2 (CoV-2) susceptibility.

(a) CoV-2 receptor gene expression and coexpression (co-exp) in human cells (adapted from reference). Isogenic organoids can be generated from adult stem cells (ASCs) and human induced pluripotent stem cells (hiPSCs). The size of the dots in the left panel is proportional to the percentage of cells that express the indicated genes. (b) Development of multi-dimensional organoids to model the complexity of immunological and hyper-inflammatory complications in COVID-19 patients. Abbreviations: mTEC (III), medullary thymic epithelial cells of the foetal thymus; PC-atrial and PC-vent, pericytes in the atrium and ventricle of the heart; r. respiratory; secretory (u.r.a), secretory cells from the upper respiratory airway.