Human induced pluripotent stem cells as a tool for disease modeling and drug screening for COVID-19

Abstract

The emergence of the new corona virus (SARS-CoV-2) and the resulting COVID-19 pandemic requires fast development of novel prevention and therapeutic strategies. These rely on understanding the biology of the virus and its interaction with the host, and on agnostic phenotypic screening for compounds that prevent viral infection. In vitro screenings of compounds are usually performed in human or animal-derived tumor or immortalized cell lines due to their ease of culturing. However, these platforms may not represent the tissues affected by the disease in vivo, and therefore better models are needed to validate and expedite drug development, especially in face of the COVID-19 pandemic. In this scenario, human induced pluripotent stem cells (hiPSCs) are a powerful research tool due to their ability to generate normal differentiated cell types relevant for the disease. Here we discuss the different ways hiPSCs can contribute to COVID-19 related research, including modeling the disease in vitro and serving as a platform for drug screening.

Keywords:
Stem cell differentiation; COVID-19; drug screening; Brazil; human pluripotent stem cells

Introduction

The COVID-19 (Coronavirus Disease 2019) pandemic, caused by SARS-CoV-2, is a global emergency that affects countries worldwide and continues to spread rapidly ( Livingston et al. 2020; World Health Organization 2020). On July 24th, 2020, Brazil had the second-highest number of COVID-19 cases and number of deaths, behind only the USA (Dong et al. 2020). Being so severely affected, it is essential for the country to outline an action plan aiming to understand the biology of the virus and its interaction with the host which will eventually lead to the development of novel vaccines and therapies against the disease. In this review we discuss the role of human induced pluripotent stem cells (hiPSCs) for basic research and screening for drugs against SARS-CoV-2.

Drug Screening and COVID-19

Antiviral chemotherapy is a much sought-after strategy for control of COVID-19. However, this is a new disease for which there is scarce knowledge, and in vitro and in vivo models are not readily available. In the absence of an effective vaccine, drug discovery for the development of novel and COVID-19-specific antivirals is ideal. However, de novo drug discovery is a lengthy process, and thus, a strategy that is being pursued in parallel and may expedite the development of COVID-19 chemotherapy is drug repositioning or repurposing. This approach has been used with success in the past for several diseases, including viral infections. In fact, the first treatment approved by the FDA for HIV, Zidovudine (AZT, azidothymidine), was originally used in cancer treatment (Richman 1988; Maeda et al. 2019).

Usually drug discovery begins with screening, and modern programs in both Academia and Industry rely on two different approaches for interrogating compound activity, often used in complement to each other: target-based and cell-based or phenotypic screening (Moffat et al. 2017). The former refers to screening assays that normally measure compound activity (inhibition, activation) or binding in a biochemical assay with a single isolated molecular target, ideally previously shown to be important in the resolution of the disease and validated genetically or by chemical methods. The latter refers to assays that use cells, tissues, or even whole organisms, to measure compound capacity to modulate a phenotype that correlates with efficacious chemotherapy of the disease of interest, without previous knowledge of the target. While both strategies have their own advantages and limitations, phenotypic screening has been shown to outperform target-based screening in the discovery of novel “first in class” drugs, as they allow for the discovery of molecules on unknown targets, with novel molecular mechanisms of action - sometimes even with a multitarget or pleiotropic mechanism of action (Hopkins 2008). Phenotypic screening assays are also considered more physiologically relevant, as molecules are interrogated in a cellular milieu, and the phenotype measured in assay endpoint is usually closer to the efficacy endpoint measured later in the clinic (Swinney and Anthony 2011). One disadvantage of phenotypic screenings is that the molecular target of active compounds is often unknown and requires further work for their discovery - a process usually referred to as target deconvolution (Schirle and Jenkins 2016). In the case of COVID-19, an emerging disease, phenotypic screening can offer the possibility of discovering new drugs and repurposing existing ones, targeting either SARS-CoV-2 or host proteins and pathways. Viral proteins, as well as cellular receptors and pathways used by the virus might be amenable to modulation/inhibition by existing chemotherapies, thus expediting the development of new drugs. In fact, several FDA-approved drugs have already been proposed for repositioning for COVID-19 based on phenotypic screening campaigns carried out since the beginning of the pandemic (Riva et al. 2020; Heiser et al. 2020; Jeon et al. 2020; Mirabelli et al. 2020). However, these candidates still require demonstration of clinical efficacy to be repurposed for COVID-19 chemotherapy and currently there are several clinical trials evaluating candidates for COVID-19 chemotherapy.

Another aspect is the physiological relevance of the screening assay and the cell-based model adopted, as the cellular assay should try to replicate key aspects of the disease. In vitro screenings of compounds are usually performed in models that are easier to manipulate, such as tumor or immortalized cell lines like HeLa (Human cervix epitheloid carcinoma), CHO (Chinese Hamster Ovary cells) and HCT116 (human colorectal carcinoma) (Zhang et al. 2012). The choice of a cell line for phenotypic screening impacts on the results and drugs found to have the desired activity profile, the hits. So much so that compounds can have a remarkable difference in activity depending on the cell model used: a recent study showed that chloroquine may or may not inhibit SARS-CoV-2 infection in vitro depending on the cell model used (Hoffmann et al. 2020b).

However, even when working with relevant-tissue type cells, these lineages often do not represent or mimic the tissues affected by the disease in vivo. Thus, for the discovery/repositioning process of compounds to be more efficient, in vitro models that translate, in the most reliable way possible, the conditions observed in vivo are extremely important.

Human pluripotent stem cells as a study model for COVID-19

Pluripotent stem cells (PSC) are defined by their capability of differentiating into cell types derived from the three embryonic germ layers. Initially established in culture as embryonic stem cells (ESCs) derived from the inner cell mass of mouse blastocysts (Evans and Kaufman 1981; Martin 1981), these cells can be maintained practically indefinitely in culture in an undifferentiated state. Upon induction of differentiation in vitro, they can specialize in potentially any tissue of the adult (Shi et al. 2017). Although embryo-derived human ESCs (Thomson et al. 1998) are equivalently pluripotent, the development of methods to reprogram adult somatic cells to pluripotency has revolutionized the stem cell field (Takahashi and Yamanaka 2006; Takahashi et al. 2007). Human induced pluripotent stem cells (hiPSCs) can be derived from any individual with a phenotype/genotype of interest, providing a potent tool for in vitro disease modeling. Patient-specific hiPSC models make it possible to obtain differentiated cell types relevant for a particular disease, such as neurons or cardiomyocytes, that can be used to understand molecular mechanisms of pathogenesis and as a platform for drug screening.

More recently, hiPSC-derived cells have been shown to be valuable also as a model for infectious diseases (Shi et al. 2017). Our group has shown the infection capacity of the protozoan Trypanosoma cruzi in hiPSC-derived cardiomyocytes (iCMs), demonstrating the potential of these cells as a human model for studying cardiomyopathy in Chagas disease and also for the development of new therapies against the parasite (da Silva Lara et al. 2018). Similarly, hiPSC-derived hepatocytes infected with hepatitis B virus (Xia et al. 2017), hPSC-Neural progenitors infected with Zika virus (Zhou et al. 2017) and Herpes Simplex Virus-1 (D’Aiuto et al. 2017) have been reported. Also, infection susceptibility of numerous hiPSC-derived cell types with SARS-CoV-2 have already been demonstrated (Monteil et al. 2020; Yang L et al. 2020; Sharma et al. 2020). Table 1 shows the advantages and disadvantages of this model in comparison to others used during the drug development process.

Table 1 -
Advantages and disadvantages of the different models used for drug development.

Model	Advantages	Disadvantages
hiPSC-derived cells	Human model, patient (genotype)-specific, suitable for gene editing	Labor/time consuming and higher cost culture
	Can generate multiple relevant cell-types with same genetic background	Some differentiated cell types with fetal phenotype
	Increased complexity can be achieved with co-culture and organoids	Possible variations among batches of differentiations
Human cancer/ immortalized lines	Easy to keep in culture and maintain	Fail to represent or mimic the tissues affected by the disease in vivo
	Passage number is not a concern	Carry tumor-associated mutations, such as P53 mutations; some cell lines have mutations in genes controlling the innate immune response
Transgenic mice	Complete organism able to show systemic disease	Distinct from human biology and unlikely to capture key aspects of some diseases
	Can be engineered to express molecules of interest	Use increasingly discouraged for ethical reasons

Relevant cell types in the COVID-19 study

SARS-CoV-2 infection is mainly caused by the binding of the receptor-binding domain (RBD) of the spike (S) viral surface protein to the human angiotensin I converting enzyme 2 (ACE2) receptor, an interaction that occurs with high affinity (Hoffmann et al. 2020a; Shang et al. 2020a; Shang et al. 2020b; Wan et al. 2020; Zhang H et al. 2020; Walls et al. 2020). The host cell TMPRSS2 transmembrane protease action is also crucial, since it cleaves residues of the viral spike protein and allows the fusion of viral and cellular membranes (Matsuyama et al. 2020; Hoffmann et al. 2020a). Low expression of this protein does not protect against infection, since a furin preactivation can also facilitate viral entry in some types of cells regardless of TMPRSS2 expression levels (Shang et al. 2020a). Additionally, in HEK293 cells stably expressing ACE2, it was demonstrated that viral entry happens mainly through endocytosis, and that other proteases, such as cathepsin L, are critical (Ou et al. 2020). These data suggest that the range of possible infection mechanisms are broader than initially thought and can vary among different cell types.

ACE2 is expressed in the lungs (mainly in type 2 alveolar epithelial cells - AT2s) and also in the heart, intestine and kidneys, supporting mechanisms for dysfunction in multiple organs, which has also been observed in COVID-19 patients (Zhang H et al. 2020). The TMPRSS2 protease has high expression in the respiratory tract epithelium, a fact that justifies the tropism of SARS-CoV-2 to lungs; in addition, it is expressed in prostate, colon, stomach and salivary gland (Vaarala et al. 2001; Matsuyama et al. 2020; Hoffmann et al. 2020a). Despite representing the majority of infected cells by SARS-CoV-2 (Zheng et al. 2020), single cell RNA sequencing of pulmonary cells has shown that the expression of ACE2 is restricted to only a small subset (around 1.4%) of AT2s (Ziegler et al. 2020; Ackermann et al. 2020; Zhao et al. 2020), while expression of TMPRSS2 is found in approximately 30% of the same cell type (Ziegler et al. 2020; Lukassen et al. 2020). Co-expression of both ACE2 and TMPRSS2 was found mainly in AT2 and ciliated cells among all the cell types analyzed from fibrotic lung tissue, but at very low rates of 0.8% and 5.3%, respectively (Ziegler et al. 2020). These data show that, although the functional role of both genes in COVID-19 seems established, there is still much to learn about the influence of expression rates in susceptibility of infection and also in the complexity of disease outcome. For that, together with clinical data, both in vitro and in vivo models will be crucial.

In the lungs, AT2s perform central functions, the main one being the production and secretion of surfactants that prevent the collapse of the pulmonary tissue by regulating the surface tension of the alveoli (reviewed by Fehrenbach, 2001). They also proliferate and can differentiate into type 1 alveolar epithelial cells (AT1s) after lung injury and play a role in immune defense upon infection (Fehrenbach 2001; Jacob et al. 2019). Despite the fact that AT2s are highly proliferative in a renewing tissue, primary AT2s proliferate poorly in vitro and last only a few passages in the absence of mesenchymal support. In contrast, hiPSC-derived AT2s (iAT2s) can form alveolar organoids with proliferative potential, morphology, and molecular phenotype comparable to lung alveoli (Jacob et al. 2017; Jacob et al. 2019). Correction of a mutation in the surfactant protein-B encoding gene (SFTPB) in a patient-specific cell line restored the surfactant production and processing of the respective iAT2s, demonstrating the suitability of these cells for pulmonary disease modeling (Jacob et al. 2017). Thus, generation of AT2s from hiPSCs provides a powerful tool to model COVID-19 pulmonary phenotypes in vitro and to investigate the cellular and molecular changes in these cells after infection by SARS-CoV-2, as showed recently in a model of at air-liquid interface witch mimetic the lung environment (Abo et al. 2020). The screening of compounds against the virus in iAT2s 3D cultures is also a possibility, although phenotypic evaluation of 3D organoids is harder to manage in high content screening platforms in comparison to monolayer cell cultures (Li et al. 2016). Therefore, this highly relevant physiological model is more likely to be introduced as low throughput, downstream, secondary assays for validating and profiling antiviral activity and mechanism of action of compounds.

Infection of the endothelium by SARS-CoV-2 has also been proposed as a mechanism of viral migration to different organs (Monteil et al. 2020). Post-mortem histology revealed lymphocytic endotheliitis in lung (Ackermann et al. 2020), heart (Tavazzi et al. 2020), kidney, liver and in the submucosal vessels of the small intestine (Varga et al. 2020) of COVID-19 patients. Specifically, in endothelial cells, direct infection was reported by one study (Ackermann et al. 2020), despite the fact that this cell type usually has low levels of ACE2 expression (Nicin et al. 2020; Ackermann et al. 2020). Recently, Yang L et al. (2020) demonstrated that, although ACE2 was detected in endothelial cells differentiated from hiPSCs (iECs), infection by SARS-CoV-2 pseudo-entry virus was very low in comparison to other cells types, such as cardiomyocytes and dopaminergic neurons. In a pilot experiment, our group also observed no infection of iECs by SARS-CoV-2, even though (low) ACE2 expression was detected (unpublished data). These data strengthen the hypothesis that other factors, apart from expression of this receptor, may play a role for viral entry to occur. Furthermore, it is important to consider that, in vivo, the systemic response against infection could trigger similar molecular changes in endothelial cells as observed for nasal/pulmonary epithelial cells, for which it was shown that ACE2 is an interferon stimulated gene (ISG) (Ziegler et al. 2020). Interferons (IFNs) and ISGs are normally expressed in host cells as an antiviral and tissue-protective mechanism (Acharya et al. 2020; Ziegler et al. 2020), and it is hypothesized that upregulation of ACE2 by these responses could favor further SARS-CoV-2 infection in severe COVID-19 cases. However, although expression of ISG was shown to be increased in the respiratory tract of COVID-19 patients (Zhou et al. 2020) and IFN signaling was shown to be upregulated in primary human lung epithelial cells infected with SARS-CoV-2 (Mulay et al. 2020), the kinetics of IFN activation in the disease remains controversial (Acharya et al. 2020), as the same responses were not observed in infected primary cultures of dendritic cells and macrophages, for example (Yang D et al. 2020). In particular, the need of previous activation of endothelial cells by IFNs and/or proinflammatory cytokines to support direct SARS-CoV-2 infection remains uncertain and could also be addressed using iECs.

Another relevant fact is that, increasingly, cases of pulmonary embolisms and also microthrombosis in other organs have been reported in COVID-19 patients (Zuckier et al. 2020; Danzi et al. 2020; Dolhnikoff et al. 2020; Griffin et al. 2020). In line with that, anticoagulant treatment, mainly with heparin, has been shown as potentially beneficial in a subset of severe cases (Thachil 2020; Negri et al. 2020; Tang et al. 2020). Although a hypercoagulable state due to cytokine storm and indirect endothelial dysfunction is commonly observed in patients with infections, the same severe scenario was not observed in SARS patients (Madjid et al. 2020; Dolhnikoff et al. 2020) or in cases of acute respiratory distress syndrome (ARDS) secondary to influenza A (H1N1), where the presence of alveolar capillary microthrombi was 9 times less prevalent than in COVID-19 patients (Ackermann et al. 2020). The possible role of microcirculation abnormalities in hypoxia lead to the latter proposal of COVID-19 as a vascular-thrombotic disease instead of solely a respiratory disease. If confirmed, this would represent a paradigm shift that could possibly change the way new therapies are sought and developed. For that, iECs would also be very useful as an in vitro model system.

In addition to vascular dysfunction, the occurrence of cardiac manifestations in patients affected by COVID-19 (Zheng et al. 2020; Clerkin et al. 2020) also raises the hypothesis of direct cardiac myocyte infection by SARS-CoV-2 (Gallagher et al. 2008; Gomes et al. 2012). ACE2 is highly expressed in adult human hearts, especially by cardiomyocytes (Nicin et al. 2020), which indicates an intrinsic susceptibility of this organ to SARS-CoV-2. It has been shown that patients with heart failure disease exhibited increased expression of this gene and might have a higher possibility of developing a heart attack and progressing to severe condition upon SARS-CoV-2 infection (Chen et al. 2020). Also, high doses of highly sensitive troponin-I (hs-cTnI) have been found to predict a worse COVID-19 outcome (Madjid et al. 2020; Ruan et al. 2020; Wang D et al. 2020; Huang et al. 2020; Zhou et al. 2020), and in a group of patients treated in China, around 10% of those who died suffered heart damage, even when no previous cardiovascular disease was present (reviewed by Zheng et al., 2020). Myocardial injury, arrhythmias and viral myocarditis were also highly prevalent in groups of patients admitted to intensive care units (ICU) (Madjid et al. 2020; Zheng et al. 2020). In a case report, Tavazzi et al. (2020) show evidence of myocardial localization of viral particles in one patient with cardiogenic shock, although no direct cardiomyocyte or endothelial infection was observed. Therefore, it is not known whether all these cardiac alterations described are directly caused by viral infection of heart cells or were a secondary effect of impaired pulmonary function and/or systemic inflammatory state. Another question that remains open is whether patients with comorbidities have naturally increased ACE2 expression and/or whether the use of ACE inhibitors or angiotensin II receptor blockers (ARBs) would lead to this hypothetical increase (Fang et al. 2020; Zheng et al. 2020; Clerkin et al. 2020; Pinto et al. 2020).

Recently, our group (unpublished data) and others (Yang L et al. 2020; Sharma et al. 2020) have shown that hiPSC-derived cardiomyocytes (iCMs) are susceptible to SARS-CoV-2 infection. Also, Sharma and colleagues observed that infection caused apoptosis and contractility alterations, validating these cells as a potential model to study SARS-CoV-2-related myocarditis and for phenotypic drug screening.

Host genetics

The host response to a virus is generally not uniform, and infections can inflict different degrees of morbidity and mortality in different patients. For COVID-19, up to now, it can be noted among critically ill patients, the importance of a few preexisting comorbidities, which have been considered risk factors for a while. According to the USA CDC, the most critical comorbidities, and more commonly observed in fatal cases, were hypertension (48%), diabetes mellitus (31%), cardiovascular disease (13%) and age over 70 years. The average age of patients with the greatest disease severity was 52 years (40.0-65.0), the greatest severity being linked to an increase in patients' age (Wu and McGoogan 2020).

Understanding of virus-host interactions increases the chances of developing effective strategies to resolve infection. In this context, several studies have inquired the importance of variations in both ACE2 and TMPRSS2 expression and in individual responses to explain the wide range of symptoms and complications in COVID-19 (Lukassen et al. 2020; Asselta et al. 2020; Grifoni et al. 2020; Hou et al. 2020; Pinto et al. 2020). Small variations in gene expression are often related to the presence of non-pathogenic genomic variants which diverge widely between human populations (Cao et al. 2020). Cao and collaborators (2020) analyzed data from several large-scale genomic banks looking for variants in the ACE2 gene associated with differences in susceptibility to SARS-CoV-2 infection in different populations. Although no direct evidence was established, the authors observed increased frequency of allelic variants of expression quantitative trait loci (eQTL) associated with increased expression of ACE2 in East Asian populations. ACE2 expression has also been reported to increase with age and is generally higher in men than in women (Lukassen et al. 2020).

In addition, increasing evidence suggests that men are more vulnerable to COVID-19 than women. Epidemiological data have shown that the majority of hospitalized patients are men in different countries (Huang et al. 2020; Grasselli et al. 2020; Guan et al. 2020; Richardson et al. 2020) and mortality also seems higher for this particular group (Jin et al. 2020; Onder et al. 2020). Many different factors have been proposed to explain the mechanisms of these discrepancies, and because behavioral differences also may play a role, the answer might not be so simple. Despite that, a "not so complex" mechanism about androgens and their part in the modulation of TMPRSS2 expression has gathered strength. As mentioned, TMPRSS2 is a protease that cleaves the viral S protein, enabling viral and host membrane fusion. Interestingly, this protein is also known to be over expressed in more than 50% of prostate cancer patients, and it has been shown that its expression is subjected to androgen receptors activation in this organ (Vaarala et al. 2001). Androgen-deprivation therapies (ADTs) are a common class of treatment against prostate cancer, and recent studies showed that patients under ADT were less likely to contract COVID-19 and to be hospitalized in comparison to men that were not on ADT (Wadman 2020; Montopoli et al. 2020). Meanwhile, as mentioned, the degree of importance of TMPRSS2 for viral infection is not completely established, since many cell types do not express high levels of this gene/protein. Furthermore, it is still unclear whether this androgen-driven expression is also observed in the respiratory tract or in other COVID-19 relevant cell types. To investigate the molecular mechanisms of this complex sex bias, again, hiPSC models could be very helpful.

In addition to their capability of generating several different human cell types for the study of infectious diseases, hiPSCs can also be established to broadly encompass human genetic diversity (Turner et al. 2013), something important to consider in the drug development process. Recognizing the diversity and genetic admixture of the Brazilian population, our group established a collection of hiPSC lineages representative of the genetic variability of our population from participants in the ELSA-Brazil project (Estudo Longitudinal da Saúde de Adultos - Longitudinal Study of Adult Health) (Tofoli et al. 2016). Along with its genetic admixture, each hiPSC line is connected to a large database of relevant clinical data of the corresponding participant, such as the presence of common chronic diseases like hypertension and diabetes. In the current COVID-19 pandemic scenario, in addition to the importance of clinical factors such as the occurrence of comorbidities already associated with differences in the prognosis of the infection (Guan et al. 2020; Zhang J et al. 2020; Huang et al. 2020; Wang T et al. 2020; Zhou et al. 2020), the genomic analysis of hiPSCs lineages’ collection can also be used in the search for polymorphisms potentially associated with the greater or lesser severity of disease (Cao et al. 2020; Asselta et al. 2020), which would provide the possibility of establishing correlations between genotype and phenotype (Figure 1).

Figure 1 -
The potential uses of human induced pluripotent stem cells (hiPSCs) in COVID-19 research. Human iPS-derived cell types relevant for COVID-19 - alveolar type 2 epithelial cells (iAT2s), cardiomyocytes (iCMs) and endothelial cells (iECs), can be used for drug screening/repurposing and disease modeling. A hiPSC-bank of the Brazilian genetic admixture allows for the functional investigation of the role of host-genomics in SARS-CoV-2 infection and in COVID-19 severity.

Conclusion

Human iPSCs are a powerful tool for the development of novel therapies for COVID-19. With their capacity to generate differentiated cells relevant for the disease, hiPSCs can be used to validate anti-viral drugs identified in large scale screens, and as an in vitro model system to understand the biology of host-virus interaction. In the last 15 years, the Brazilian government has made substantial investments in the development of stem cell research that can now be leveraged to contribute to research in COVID-19. Finally, the unique genetic diversity of the Brazilian population represents both a challenge and an opportunity. While findings in genomic research of virus-host interaction made in populations of European ancestry may have limited value for Brazilians, those same explorations performed in our admixed population may unravel novel genetic variants, and therefore novel molecular pathways, involved in the diverse aspects of SARS-CoV-2 infection and of COVID-19.

References

• Abo KM, Ma L, Matte T, Huang J, Alysandratos KD, Werder RB, Mithal A, Beermann M Lou, Lindstrom-Vautrin J, Mostoslavsky G et al (2020) Human iPSC-derived alveolar and airway epithelial cells can be cultured at air-liquid interface and express SARS-CoV-2 host factors. bioRxiv 2020.06.03.132639.
• Acharya D, Liu GQ and Gack MU (2020) Dysregulation of type I interferon responses in COVID-19. Nat Rev Immunol 20:397-398.
• Ackermann M, Verleden SE, Kuehnel M, Haverich A, Welte T, Laenger F, Vanstapel A, Werlein C, Stark H, Tzankov A et al (2020) Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19. N Engl J Med 383:120-128.
• Asselta R, Paraboschi EM, Mantovani A and Duga S (2020) ACE2 and TMPRSS2 variants and expression as candidates to sex and country differences in COVID-19 severity in Italy. Aging (Albany NY) 12:10087-10098.
• Cao Y, Li L, Feng Z, Wan S, Huang P, Sun X, Wen F, Huang X, Ning G and Wang W (2020) Comparative genetic analysis of the novel coronavirus (2019-nCoV/SARS-CoV-2) receptor ACE2 in different populations. Cell Discov 6:4-7.
• Chen L, Li X, Chen M, Feng Y and Xiong C (2020) The ACE2 expression in human heart indicates new potential mechanism of heart injury among patients infected with SARS-CoV-2. Cardiovasc Res 116:1097-1100.
• Clerkin KJ, Fried JA, Raikhelkar J, Sayer G, Griffin JM, Masoumi A, Jain SS, Burkhoff D, Kumaraiah D, Rabbani L et al (2020) COVID-19 and Cardiovascular Disease. Circulation 141:1648-1655.
• D’Aiuto L, Williamson K, Dimitrion P, McNulty J, Brown CE, Dokuburra CB, Nielsen AJ, Lin WJ, Piazza P, Schurdak ME et al (2017) Comparison of three cell-based drug screening platforms for HSV-1 infection. Antiviral Res 142:136-140.
• da Silva Lara L, Andrade-Lima L, Magalhães Calvet C, Borsoi J, Lopes Alberto Duque T, Henriques-Pons A, Souza Pereira MC and Veiga Pereira L (2018) Trypanosoma cruzi infection of human induced pluripotent stem cell-derived cardiomyocytes: an in vitro model for drug screening for Chagas disease. Microbes Infect 20:312-316.
• Danzi GB, Loffi M, Galeazzi G and Gherbesi E (2020) Acute pulmonary embolism and COVID-19 pneumonia: a random association? Eur Heart J 41:1858-1858.
• Dolhnikoff M, Duarte‐Neto AN, Almeida Monteiro RA, Silva LFF, Oliveira EP, Saldiva PHN, Mauad T and Negri EM (2020) Pathological evidence of pulmonary thrombotic phenomena in severe COVID‐19. J Thromb Haemost 18:1517-1519.
• Dong E, Du H and Gardner L (2020) An interactive web-based dashboard to track COVID-19 in real time. Lancet Infect Dis 20:533-534.
• Evans MJ and Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154-156.
• Fang L, Karakiulakis G and Roth M (2020) Are patients with hypertension and diabetes mellitus at increased risk for COVID-19 infection? Lancet Respir 8:e21.
• Fehrenbach H (2001) Alveolar epithelial type II cell: Defender of the alveolus revisited. Respir Res 2:33-46.
• Gallagher PE, Ferrario CM and Tallant EA (2008) Regulation of ACE2 in cardiac myocytes and fibroblasts. Am J Physiol Circ Physiol 295:H2373-H2379.
• Gomes ERM, Santos RAS and Guatimosim S (2012) Angiotensin-(1-7)-mediated signaling in cardiomyocytes. Int J Hypertens 2012:1-8.
• Grasselli G, Zangrillo A, Zanella A, Antonelli M, Cabrini L, Castelli A, Cereda D, Coluccello A, Foti G, Fumagalli R et al (2020) Baseline Characteristics and Outcomes of 1591 Patients Infected with SARS-CoV-2 Admitted to ICUs of the Lombardy Region, Italy. JAMA - J Am Med Assoc 323:1574-1581.
• Griffin DO, Jensen A, Khan M, Chin J, Chin K, Saad J, Parnell R, Awwad C and Patel D (2020) Pulmonary embolism and increased levels of D-dimer in patients with coronavirus disease. Emerg Infect Dis 26:1941-1943.
• Grifoni A, Weiskopf D, Ramirez SI, Mateus J, Dan JM, Moderbacher CR, Rawlings SA, Sutherland A, Premkumar L, Jadi RS et al (2020) Targets of T cell responses to SARS-CoV-2 Coronavirus in humans with COVID-19 disease and unexposed individuals. Cell 181:1489-1501.
• Guan W, Ni Z, Hu Y, Liang W, Ou C, He J, Liu L, Shan H, Lei C, Hui DSC et al (2020) Clinical characteristics of Coronavirus Disease 2019 in China. N Engl J Med 382:1708-1720.
• Heiser K, Mclean PF, Davis CT, Fogelson B, Gordon HB, Jacobson P, Hurst B, Miller B, Alfa RW, Earnshaw BA et al (2020) Identification of potential treatments for COVID-19 through artificial intelligence-enabled phenomic analysis of human cells infected with SARS-CoV-2. bioRxiv 2020.04.21.054387.
• Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu N-H, Nitsche A et al (2020a) SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181:271-280.
• Hoffmann M, Mösbauer K, Hofmann-Winkler H, Kaul A, Kleine-Weber H, Krüger N, Gassen NC, Müller MA, Drosten C and Pöhlmann S (2020b) Chloroquine does not inhibit infection of human lung cells with SARS-CoV-2. Nature 1-5.
• Hopkins AL (2008) Network pharmacology: the next paradigm in drug discovery. Nat Chem Biol 4:682-690.
• Hou YJ, Okuda K, Edwards CE, Martinez DR, Asakura T, Dinnon KH, Kato T, Lee RE, Yount BL, Mascenik TM et al (2020) SARS-CoV-2 reverse genetics reveals a variable infection gradient in the respiratory tract. Cell 182:429-446.
• Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, Zhang L, Fan G, Xu J, Gu X et al (2020) Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395:497-506.
• Jacob A, Morley M, Hawkins F, McCauley KB, Jean JC, Heins H, Na CL, Weaver TE, Vedaie M, Hurley K et al (2017) Differentiation of human pluripotent stem cells into functional alveolar epithelial cells. Cell Stem Cell 21:472-488.
• Jacob A, Vedaie M, Roberts DA, Thomas DC, Villacorta-Martin C, Alysandratos KD, Hawkins F and Kotton DN (2019) Derivation of self-renewing lung alveolar epithelial type II cells from human pluripotent stem cells. Nat Protoc 14:3303-3332.
• Jeon S, Ko M, Lee J, Choi I, Byun SY, Park S, Shum D and Kim S (2020) Identification of antiviral drug candidates against SARS-CoV-2 from FDA-approved drugs. Antimicrob Agents Chemother 64:e00819-20.
• Jin J-M, Bai P, He W, Wu F, Liu X-F, Han D-M, Liu S and Yang J-K (2020) Gender differences in patients with COVID-19: focus on severity and mortality. Front Public Heal 8:152.
• Li L, Zhou Q, Voss TC, Quick KL and LaBarbera DV. (2016) High-throughput imaging: Focusing in on drug discovery in 3D. Methods 96:97-102.
• Livingston E, Bucher K and Rekito A (2020) Coronavirus disease 2019 and influenza 2019-2020. JAMA 323:1122.
• Lukassen S, Chua RL, Trefzer T, Kahn NC, Schneider MA, Muley T, Winter H, Meister M, Veith C, Boots AW et al (2020) SARS‐CoV‐2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells. EMBO J 39:e105114.
• Madjid M, Safavi-Naeini P, Solomon SD and Vardeny O (2020) Potential effects of Coronaviruses on the cardiovascular system: A review. JAMA Cardiol 10:1-10.
• Maeda K, Das D, Kobayakawa T, Tamamura H and Takeuchi H (2019) Discovery and development of anti-HIV therapeutic agents: Progress towards improved HIV medication. Curr Top Med Chem 19:1621-1649.
• Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 78:7634-7638.
• Matsuyama S, Nao N, Shirato K, Kawase M, Saito S, Takayama I, Nagata N, Sekizuka T, Katoh H, Kato F et al (2020) Enhanced isolation of SARS-CoV-2 by TMPRSS2- expressing cells. Proc Natl Acad Sci USA 117:7001-7003.
• Mirabelli C, Wotring JW, Zhang CJ, McCarty SM, Fursmidt R, Frum T, Kadambi NS, Amin AT, O’Meara TR, Pretto-Kernahan CD et al (2020) Morphological cell profiling of SARS-CoV-2 Infection infection identifies drug repurposing for COVID-19. bioRxiv 2020.05.27.117184.
• Moffat JG, Vincent F, Lee JA, Eder J and Prunotto M (2017) Opportunities and challenges in phenotypic drug discovery: an industry perspective. Nat Rev Drug Discov 16:531-543.
• Monteil V, Kwon H, Prado P, Hagelkrüys A, Wimmer RA, Stahl M, Leopoldi A, Garreta E, Hurtado del Pozo C, Prosper F et al (2020) Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2. Cell 181:905-913.
• Montopoli M, Zumerle S, Vettor R, Rugge M, Zorzi M, Catapano CV, Carbone GM, Cavalli A, Pagano F, Ragazzi E et al (2020) Androgen-deprivation therapies for prostate cancer and risk of infection by SARS-CoV-2: a population-based study (N = 4532). Ann Oncol 31:1040-1045.
• Mulay A, Konda B, Garcia Jr G, Yao C, Beil S, Sen C, Purkayastha A, Kolls JK, Pociask DA, Sainz de Aja J et al (2020) SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery for equal contributions Introduction. bioRxiv 2020.06.29.174623.
• Negri EM, Piloto B, Morinaga LK, Jardim CVP, Lamy SAE-D, Ferreira MA, D’Amico EA and Deheinzelin D (2020) Heparin therapy improving hypoxia in COVID-19 patients - a case series. medRxiv 2020.04.15.20067017.
• Nicin L, Abplanalp WT, Mellentin H, Kattih B, Tombor L, John D, Schmitto JD, Heineke J, Emrich F, Arsalan M et al (2020) Cell type-specific expression of the putative SARS-CoV-2 receptor ACE2 in human hearts. Eur Heart J 41:1804-1806.
• Onder G, Rezza G and Brusaferro S (2020) Case-fatality rate and characteristics of patients dying in relation to COVID-19 in Italy. JAMA - J Am Med Assoc 323:1775-1776.
• Ou X, Liu Y, Lei X, Li P, Mi D, Ren L, Guo L, Guo R, Chen T, Hu J et al (2020) Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat Commun 11: 1620.
• Pinto BGG, Oliveira AER, Singh Y, Jimenez L, Gonçalves ANA, Ogava RLT, Creighton R, Schatzmann Peron JP and Nakaya HI (2020) ACE2 Expression is increased in lungs of patients with comorbities associated with severe COVID-19. J Infect Dis 222:556-563.
• Richardson S, Hirsch JS, Narasimhan M, Crawford JM, McGinn T, Davidson KW, Barnaby DP, Becker LB, Chelico JD, Cohen SL et al (2020) Presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with COVID-19 in the New York City area. JAMA 323:2052.
• Richman DD (1988) The treatment of HIV infection. Azidothymidine (AZT) and other new antiviral drugs. Infect Dis Clin North Am 2:397-407.
• Riva L, Yuan S, Yin X, Martin-Sancho L, Matsunaga N, Burgstaller S, Pache L, Jesus P De, Hull MV, Chang M et al (2020) A large-scale drug repositioning survey for SARS-CoV-2 antivirals. bioRxiv 2020.04.16.044016.
• Ruan Q, Yang K, Wang W, Jiang L and Song J (2020) Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan, China. Intensive Care Med 46:846-848.
• Schirle M and Jenkins JL (2016) Identifying compound efficacy targets in phenotypic drug discovery. Drug Discov Today 21:82-89.
• Shang J, Wan Y, Luo C, Ye G, Geng Q, Auerbach A and Li F (2020a) Cell entry mechanisms of SARS-CoV-2. Proc Natl Acad Sci US A 2020:202003138.
• Shang J, Ye G, Shi K, Wan Y, Luo C, Aihara H, Geng Q, Auerbach A and Li F (2020b) Structural basis of receptor recognition by SARS-CoV-2. Nature 581:221-224 .
• Sharma A, Garcia G, Wang Y, Plummer JT, Morizono K, Arumugaswami V and Svendsen CN (2020) Human iPSC-derived cardiomyocytes are susceptible to SARS-CoV-2 infection. Cell Reports Med 1:100052.
• Shi Y, Inoue H, Wu JC and Yamanaka S (2017) Induced pluripotent stem cell technology: a decade of progress. Nat Rev Drug Discov 16:115-130.
• Swinney DC and Anthony J (2011) How were new medicines discovered? Nat Rev Drug Discov 10:507-519.
• Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T and Tomoda K (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861-872.
• Takahashi K and Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663-676.
• Tang N, Bai H, Chen X, Gong J, Li D and Sun Z (2020) Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy. J Thromb Haemost 18:1094-1099.
• Tavazzi G, Pellegrini C, Maurelli M, Belliato M, Sciutti F, Bottazzi A, Sepe PA, Resasco T, Camporotondo R, Bruno R et al (2020) Myocardial localization of coronavirus in COVID-19 cardiogenic shock. Eur J Heart Fail 22: 911-915.
• Thachil J (2020) The versatile heparin in COVID-19. J Thromb Haemost 18:1020-1022.
• Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS and Jones JM (1998) Embryonic stem cell lines derived from human fibroblasts blastocysts. Science 282:1145-1147.
• Tofoli FA, Dasso M, Morato-Marques M, Nunes K, Pereira LA, Silva GS, Fonseca SAS, Costas RM, Santos HC, Costa Pereira A et al (2016) Increasing the genetic admixture of available lines of human pluripotent stem cells. Sci Rep 6:34699.
• Turner M, Leslie S, Martin NG, Peschanski M, Rao M, Taylor CJ, Trounson A, Turner D, Yamanaka S and Wilmut I (2013) Toward the development of a global induced pluripotent stem cell library. Cell Stem Cell 13:382-384.
• Vaarala MH, Porvari K, Kyllönen A, Lukkarinen O and Vihko P (2001) The TMPRSS2 gene encoding transmembrane serine protease is overexpressed in a majority of prostate cancer patients: Detection of mutated TMPRSS2 form in a case of aggressive disease. Int J Cancer 94:705-710.
• Varga Z, Flammer AJ, Steiger P, Haberecker M, Andermatt R, Zinkernagel AS, Mehra MR, Schuepbach RA, Ruschitzka F and Moch H (2020) Endothelial cell infection and endotheliitis in COVID-19. Lancet 395:1417-1418.
• Wadman M (2020) Sex hormones signal why virus hits men harder. Science 368:1038-1039.
• Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT and Veesler D (2020) Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell 181:281-292.
• Wan Y, Shang J, Graham R, Baric RS and Li F (2020) Receptor Recognition by the Novel Coronavirus from Wuhan: an analysis based on decade-long structural studies of SARS Coronavirus. J Virol 94:1-9.
• Wang D, Hu B, Hu C, Zhu F, Liu X, Zhang J, Wang B, Xiang H, Cheng Z, Xiong Y et al (2020) Clinical characteristics of 138 hospitalized patients with 2019 novel Coronavirus-infected pneumonia in Wuhan, China. JAMA - J Am Med Assoc 323:1061-1069.
• Wang T, Du Z, Zhu F, Cao Z, An Y, Gao Y and Jiang B (2020) Comorbidities and multi-organ injuries in the treatment of COVID-19. Lancet 395:e52.
• Wu Z and McGoogan JM (2020) Characteristics of and Important Lessons from the Coronavirus Disease 2019 (COVID-19) outbreak in China: Summary of a report of 72314 cases from the Chinese Center for Disease Control and Prevention. JAMA - J Am Med Assoc 323:1239-1242.
• Xia Y, Carpentier A, Cheng X, Block PD, Zhao Y, Zhang Z, Protzer U and Liang TJ (2017) Human stem cell-derived hepatocytes as a model for hepatitis B virus infection, spreading and virus-host interactions. J Hepatol 66:494-503.
• Yang D, Chu H, Hou Y, Chai Y, Shuai H, Lee AC-Y, Zhang X, Wang Y, Hu B, Huang X et al (2020) Attenuated interferon and pro-inflammatory response in SARS-CoV-2-infected human dendritic cells is associated with viral antagonism of STAT1 phosphorylation. J Infect Dis 222:734-745.
• Yang L, Han Y, Nilsson-Payant BE, Gupta V, Wang P, Duan X, Tang X, Zhu J, Zhao Z, Jaffré F et al (2020) A human pluripotent stem cell-based platform to study SARS-CoV-2 tropism and model virus infection in human cells and organoids. Cell Stem Cell 27:125-136.
• Zhang H, Penninger JM, Li Y, Zhong N and Slutsky AS (2020) Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target. Intensive Care Med 46:586-590.
• Zhang J, Dong X, Cao Y, Yuan Y, Yang Y, Yan Y, Akdis CA and Gao Y (2020) Clinical characteristics of 140 patients infected with SARS‐CoV‐2 in Wuhan, China. Allergy 75:1730-1741.
• Zhang L, Wang X, Feng J, Jia Y, Xu F and Xu W (2012) Discovery of novel Vascular Endothelial Growth Factor Receptor 2 inhibitors: A virtual screening approach. Chem Biol Drug Des 80:893-901.
• Zhao Y, Zhao Z, Wang Y, Zhou Y, Ma Y and Zuo W (2020) Single-cell RNA expression profiling of ACE2, the receptor of SARS-CoV-2. Am J Respir Crit Care Med 202:756-759.
• Zheng YY, Ma YT, Zhang JY and Xie X (2020) COVID-19 and the cardiovascular system. Nat Rev Cardiol 17:259-260.
• Zhou F, Yu T, Du R, Fan G, Liu Y, Liu Z, Xiang J, Wang Y, Song B, Gu X et al (2020) Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet 395:1054-1062.
• Zhou T, Tan L, Cederquist GY, Fan Y, Hartley BJ, Mukherjee S, Tomishima M, Brennand KJ, Zhang Q, Schwartz RE et al (2017) High-content screening in hPSC- neural progenitors identifies drug candidates that inhibit Zika virus infection in fetal-like organoids and adult brain. Cell Stem Cell 21:274-283.
• Ziegler CGK, Allon SJ, Nyquist SK, Mbano IM, Miao VN, Tzouanas CN, Cao Y, Yousif AS, Bals J, Hauser BM et al (2020) SARS-CoV-2 Receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues. Cell 181:1016-1035.e19.
• Zuckier LS, Moadel RM, Haramati LB and Freeman LM (2020) Diagnostic evaluation of pulmonary embolism during the COVID-19 pandemic. J Nucl Med 61:630-631.

Internet Resources

• World Health Organization (WHO) (2020) Coronavirus disease 2019, World Health Organization (WHO) (2020) Coronavirus disease 2019, https://www.who.int/emergencies/diseases/novel-coronavirus-2019 (accessed 9 April 2020).
  » https://www.who.int/emergencies/diseases/novel-coronavirus-2019

Publication Dates

• Publication in this collection
  16 Nov 2020
• Date of issue
  2021

History

• Received
  16 June 2020
• Accepted
  25 Sept 2020