key: cord-0832752-ex99dwh2
authors: Ebisudani, Toshiki; Sugimoto, Shinya; Haga, Kei; Mitsuishi, Akifumi; Takai-Todaka, Reiko; Fujii, Masayuki; Toshimitsu, Kohta; Hamamoto, Junko; Sugihara, Kai; Hishida, Tomoyuki; Asamura, Hisao; Fukunaga, Koichi; Yasuda, Hiroyuki; Katayama, Kazuhiko; Sato, Toshiro
title: Direct Derivation of Human Alveolospheres for SARS-CoV-2 Infection Modeling and Drug Screening
date: 2021-05-19
journal: Cell Rep
DOI: 10.1016/j.celrep.2021.109218
sha: 82281f0373830e38afc58451e747e4a637f0ae55
doc_id: 832752
cord_uid: ex99dwh2

Although the main cellular target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is thought to be alveolar cells, the absence of their tractable culture system has precluded the development of a clinically-relevant SARS-CoV-2 infection model. Here, we established an efficient human alveolosphere culture method and sphere-based drug testing platform for SARS-CoV-2. Alveolospheres exhibited indolent growth in a Wnt and R-spondin dependent manner. Gene expression, immunofluorescence and electron microscopy analyses revealed the presence of alveolar cells in alveolospheres. Alveolospheres expressed ACE2 and allowed SARS-CoV-2 to propagate nearly 100,000- fold in three days of infection. While lopinavir and nelfinavir, protease inhibitors used for the treatment of HIV infection, had a modest anti-viral effect on SARS-CoV-2, remdesivir, a nucleotide prodrug, showed anti-viral effect at the concentration comparable to the circulating drug level. These results demonstrated the validity of alveolosphere culture system for the development of therapeutic agents to combat SARS-CoV-2.

The pandemic of coronavirus disease-2019 , caused by severe 55 acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently posing a 56 major public health issue. Due to the extremely rapid global spread of the 57 disease, the treatment of COVID-19 currently relies on supportive therapy, 58 such as the use of dexamethasone (Horby et al., 2020) , and the 59 compassionate use of antiviral drugs which has been developed and clinically 60 used for the treatment of other viral infections (Beigel et al., 2020b) . In vitro 61 validation of the anti-SARS-CoV-2 effect of the repurposed drugs has been 62 performed on infection-competent yet clinically irrelevant cell lines, such as 63

Vero cells (Zhou et al., 2020b) . It remains unknown to what extent such 64 experimental cell lines recapitulate the therapeutic effect of the drugs on 65 COVID-19, given that species-unique genetic variants in viral receptors, such 66 as ACE2, can potentially affect viral entry into target cells (Hoffmann et al., 67 2020) . For instance, genetic variants in mouse and hamster Ace2 account for 68 the species difference of disease severity during SARS-CoV-2 infection (Sia et 69 al., 2020) . Vero cells, which are derived from African green monkey kidney, 70 require artificial overexpression of TMPRSS2 for optimal SARS-CoV-2 71 infection (Takayama, 2020) . Therefore, there has been an urgent need for 72 human cell-based infection models that are amenable for the screening of 73 anti-SARS-CoV-2 drugs (Elbadawi and Efferth, 2020) . 74

suggesting the contribution of autocrine/paracrine secretion of Wnt ligands (Fig. 122 2A, 2B) . Indeed, RNA-seq analysis identified robust expression of WNT7B that 123 was previously shown to activate canonical Wnt signaling (Seino et al., 2018) 124 in alveolospheres (Fig. 2C ). R-spondin-dependent sphere growth was 125 abolished upon treatment with a porcupine inhibitor which abrogates the 126 production of active Wnt ligands. Supplementation of Wnt-3A rescued this 127 growth-suppressive effect, reinforcing the dependence of alveolospheres on 128 autocrine/paracrine Wnt signaling (Fig. 2D) . Additionally, the expression of 129 R-spondin receptor, LGR4, in alveolospheres was consistent with the 130 requirement of R-spondin for efficient Wnt activation (de Lau et al., 2011) (Fig. 131 2C) . In addition, alveolospheres exhibited better growth when supplied with 132 serum-free Afamin-Wnt-3A medium (Mihara et al., 2016) compared to the 133 standard serum-stabilized Wnt-3A conditioned medium. The difference 134 between the effects of these Wnt conditioned media implied growth inhibition 135 by serum components. In support of this, a treatment with serum blunted the 136 growth promoting effect of Afamin-stabilized Wnt-3A medium (Fig. 2E, 2F) . 137

Recombinant Wnt3A and Wnt7B failed to propagate alveolospheres, 138

presumably due to their lower Wnt-activating potency than that of 139 afamin-stabilized Wnt3A (Mihara et al., 2016) (Fig. S2A, S2B) . 140

Alveolospheres exhibited steady yet slow growth and enabled bi-or 141 tri-weekly passaging at a split ratio of 1:2. To improve scalability, we further 142 screened other candidate niche factors that were nominated based on 143 previous literatures (Cao et al., 2016; Katsura et al., 2019; Liang et al., 2016; 144 culture system. 156 157

We next sought to develop a SARS-CoV-2 infection model using 159 human alveolospheres. ACE2, a receptor for SARS-CoV-2, was robustly 160 expressed in alveolospheres at RNA and protein levels (Fig. 1F, S3A) . We 161 obtained viral particles from a patient diagnosed with COVID-19 in Japan. 162

Following removal of Matrigel with a non-enzymatic matrix depolymerizer, the 163 spheres were exposed to SARS-CoV-2 and re-embedded in Matrigel (Fig. 3A) . We next applied human alveolospheres to drug testing. A number of 189 clinical trials are running to repurpose or reposition several drugs for the 190 treatment of COVID-19. The budding of SARS-CoV-2 from ERGIC which was 191 known to be protease-dependent prompted us to select lopinavir and nelfinavir, 192 protease inhibitors used for the treatment of human immunodeficiency virus 193 (HIV) infection, from drug candidates (Cao et al., 2020; Chu et al., 2004; 194 Musarrat et al., 2020) . A recent study using Vero E6 cells demonstrated that 195 lopinavir has an anti-viral effect on SARS-CoV-2 with IC 50 and CC 50 values of 196 9.12 and >50 µM, respectively (Jeon et al., 2020) . Lopinavir induced a 197 cytotoxic response in alveolospheres at a concentration of 40 µM (data not 198 shown), which is lower than the CC 50 value in Vero E6 cells, and we thus 199 tested lopinavir within the range of 0-30 µM. Importantly, treatment with 10-30 200 µM lopinavir led to a significant decrease in SARS-CoV-2 viral RNA copy 201 numbers on 1 dpi (Fig. 4A ). The treatment did not induce apparent cytotoxicity 202 (Fig. 4B) , and alveolospheres tolerated a three-day treatment with 20 µM 203 lopinavir. Of note, a comparable level of drug concentration was observed in 204 the sera of lopinavir-treated patients (Cao et al., 2020) . Despite the therapeutic 205 effect on 1 dpi, the viral titer increased over time (Fig. 4A) . A similar trend was 206 observed for nelfinavir (Fig. 4C, D) , which is known to have anti-viral effect at a 207 lower concentration (Musarrat et al., 2020) , and therefore these protease 208 inhibitors may have a modest therapeutic potential. We next tested, remdesivir, 209 a viral RNA-dependent RNA polymerase inhibitor, given its efficacy in the 210 treatment of COVID-19 (Beigel et al., 2020b; Grein et al., 2020; Wang et al., 211 2020) . Strikingly, remdesivir dramatically inhibited the replication of 212 SARS-CoV-2 in alveolospheres over a 3-day period ( The pandemic of COVID-19 infection has been casting a devastating impact 218 on the global society, and the development of its preventative and therapeutic 219 measures is of utmost urgency for world-wide healthcare. To promptly respond 220 to this crisis, the scientific community has established a number of in vitro SARS-CoV-2 infection models that facilitate our understanding of the disease 222 pathophysiology and the development of therapeutic strategies. These models 223 mainly employ classic cell lines as well as human tissue-based systems, 224 including colon and airway organoids (Lamers et al., 2020) , and ex 225 vivo-cultured respiratory tract epithelium (Hui et al., 2020) . Human 226 tissue-based SARS-CoV-2 infection models have not been applied to drug 227 screening to date. A tractable culture system for adult alveolar epithelium, 228

which is considered to be the critical site of SARS-CoV-2 infection, has also 229 been lacking. Therefore, there has been an urgent demand for the 230 development of a tissue-relevant culture system for human alveolar cells that 231 enables robust propagation of SARS-CoV-2 and drug testing. 232

The culture of alveolar cells has classically adopted a 2D-culture 233 format, in which AT2 cells preferentially differentiate into AT1 cells and lose 234 self-renewing capacity within a few days (Wang et al., 2007) . Due to this 235 drawback, previous study has failed to efficiently propagate SARS-CoV-2 in 236 the 2D-cultured alveolar epithelium (Hou et al., 2020) . Recently, researchers 237 succeeded in the short-term propagation of alveolar epithelium using a 3D 238 culture platform (Barkauskas et al., 2013; Evans and Lee, 2020) . The growth 239 of 3D-cultured alveoli in this system was strictly dependent on co-cultured 240 fibroblasts, suggesting the essential role of fibroblast-derived niche factors in 241 the maintenance of alveolar epithelium (Nabhan et al., 2018; Zepp et al., 2017) . 242

In the current study, we defined niche factors that enable the long-term 243 self-renewal of the human alveolar epithelium. Similar niche factors, including 244 Wnt, FGF and EGF ligand families, are expressed in lung fibroblasts (Nabhan 245 et al., 2018; Zepp et al., 2017) , and such molecules may be key 246

fibroblast-secreted factors that support alveolar growth in the previous 247 co-culture system. In contrast to our culture condition, the co-culture format 248 nevertheless did not allow long-term expansion of alveolospheres. This 249 shortened life span may be attributed to undefined substances in the 250 co-culture medium including those included in the serum, which had a 251 detrimental effect on alveolospheres. The standard Wnt-3A conditioned 252 medium contains serum to stabilize the lipophilic Wnt protein, and its use has 253 also complicated the establishment of alveolospheres. In the course optimizing Wnt-3A conditioned medium with Afamin-stabilized serum-free Wnt-3A 256 conditioned medium (Mihara et al., 2016) , which enabled robust propagation of 257 alveolospheres. During the preparation of our manuscript, reports on the 258 propagation of the human alveolar epithelium have appeared (Katsura et al., 259 2020; Salahudeen et al., 2020; Youk et al., 2020) . These studies also 260 underscored the importance of activating Wnt signal without serum in 261 expanding the alveolar epithelium (Katsura et al., 2020; Youk et al., 2020) . 262

a single-cell RNA-seq analysis of alveolospheres revealed the co-expression 264 of AT1 and AT2 markers (Youk et al., 2020) . Although other studies showed 265 that human serum (Katsura et al., 2020) or culture on glass (Salahudeen et al., 266 2020) induced AT1 differentiation, further investigation will be required to 267 derive a robust condition for efficient maturation of AT1 and AT2 cells. In the 268 current study, we established alveolospheres using non-cancer lung tissues 269 from patients with lung cancers or a lung hamartoma. Future studies will reveal 270 to what extent non-tumor alveolar epithelium from cancer-bearing patients 271 harbor (epi)genetic lesions. as-yet-unanswered questions in the future. 293

We are currently facing a major challenge in developing effective 294

anti-viral drugs against SARS-CoV-2, and the establishment of efficient drug 295 screening systems has been warranted. However, recent studies on 296 alveolosphere culture did not perform drug screening (Katsura et al., 2020; 297 Salahudeen et al., 2020; Youk et al., 2020) . Using the alveolosphere-based 298 SARS-CoV-2 infection model, we determined the anti-viral effect of 299 FDA-approved drugs that have been used for the treatment of COVID-19. Viral 300 proteinase is an essential molecule that allows SARS-CoV-2 to be released 301 from ERGIC and is a possible anti-viral drug target. Although two protease 302 inhibitors, lopinavir and nelfinavir, significantly decreased viral titers at 1 dpi, 303 both drugs could not terminate viral replication. In contrast to this result but 304 consistent with human clinical data (Beigel et al., 2020b; Grein et al., 2020; 305 Wang et al., 2020) , remdesivir showed potent anti-viral effect at concentrations 306 within the range of serum drug levels in patients. Current screening of 307 anti-SARS-CoV-2 drugs mainly utilizes Vero cells as host cells. Immortalized 308 cell lines are, however, less relevant to native human tissues than primary cells, 309

and their routine use may lead to the underestimation of drug-induced 310 cytotoxicity and potential adverse effects. Vero cells indeed tolerate an 311 exposure to high-concentration lopinavir whereas alveolospheres do not. 312

and clinical practice using appropriate models for SARS-CoV-2 infection, 324

including those using human alveolospheres, will contribute to the accurate 325 evaluation of candidate drugs for the treatment of COVID-19. (Corning) and cultured with the medium for alveolospheres (Table S1 ). We 473 seeded 30,000 cells per well (48-well plate, Corning), and typically observed 474 around 10-30 organoid colonies per well. The screening of growth factors 475 including recombinant human neuregulin-1 (Heregulinβ-1) is described below. 476

Airway organoids were established as previously reported (Sachs et al., 2019) . 477

Spheres were passaged every 1-4 weeks using TrypLE express (Thermo 478

Fisher Scientific) with gentle pipetting. We added 10 µM Y-27632 (FUJIFILM 479 Wako Pure Chemical) for two days after passaging. Clinical characteristics of 480 the tissue donors are available in Table S2 . 481 482

SARS-CoV-2 (JPN/Kanagawa/KUH003) was isolated in April 2020 from of a 484 patient who had complained of sore throat. The patient's nasopharyngeal swab 485 sample tested positive for SARS-CoV-2 by PCR with a viral titer of 2.6 × 486 10 5 /mL, and the patient was referred to Kitasato University Hospital in Japan. 487

On admission, the patient did not complain symptoms except for a low-grade 488 fever and showed a normal blood oxygen partial pressure. The serum was 489 negative for SARS-CoV-2 S1 antibody. Viral particles were prepared by 490 culturing virus-infected Vero E6/TMPRSS2 cells (JCRB1819) (Matsuyama et 491 al., 2020) with DMEM with 2% FBS, 1 mg/mL G418 and 100 U/mL 492 penicillin/100 µg/mL streptomycin for 48 h at 37 °C in 5% CO 2 . The 493 supernatant was cleared of cell debris and was stored at -80 °C. The total 494 nucleotide sequence data of this viral strain is deposited in DNA Data Bank of

Alveolospheres 500

To screen growth factors that promote the growth of human alveolospheres, 501

we FACS-sorted and expanded HT2-280 + EpCAM + alveolospheres as 502 described above for three passages. Alveolospheres were dissociated into 503 single cells with TrypLE Express and filtered using a 20-µm pore cell strainer 504 where indicated. Images of each well were captured using a BZ-X800 digital 517 microscope (Keyence). Sphere area was automatically calculated using 518 BZ-X800 Analyzer (Keyence). Colonies with areas > 2,500 µm 2 were used for 519 area measurement. 520 521

GFP labelling of alveolospheres was performed by co-electroporating a 523 piggyBac GFP-Puro expression vector (PB513B-1, System Biosciences) and a 524

PBase vector (System Biosciences) into human normal alveolospheres as 525 previously described (Fujii et al., 2015) . Following selection with puromycin, 526 GFP-positive clones were manually isolated and expanded. 527

Human alveolospheres and airway organoids were cultured from single cells 530 for 7-14 days in the identical culture medium containing Afamin-Wnt-3A, 531 R-spondin-1, EGF, Noggin and A83-01. RNA was extracted from spheres 532 using the RNeasy Plus Mini Kit (QIAGEN) according to the manufacturer's 533 instructions. Total RNA from mock-infected or SARS-CoV-2 infected (2 dpi) 534 human alveolospheres cultured with optimized condition was extracted using 535 the Direct-zol RNA MicroPrep Kit (Zymo Research) according to the 536 manufacturer's instructions. 537 538

RNA quality was evaluated with an Agilent 2100 bioanalyzer (Agilent). 540

Sequence library was prepared with TruSeq RNA Library Prep Kit v2 (Illumina) 541 and sequenced with HiSeq X or NovaSeq 6000 (Illumina). Adaptors were 542 removed from raw fastq files with cutadapt (version 1.18) (Martin, 2011) and 543 the reads were aligned to human genome (hg38) using STAR (version 2.6.1b) 544 (Dobin et al., 2013) . The expression levels of the human genes in Ensembl 545 release 81 were estimated with RSEM (version 1.3.3) (Li and Dewey, 2011) . 546

Differentially expressed gene analysis was performed using nbinomLRT in the 547 R Bioconductor package DESeq2 (Love et al., 2014) . 548

Alveolospheres 551 RNA quality was evaluated with an Agilent 2100 bioanalyzer (Agilent). 552

Sequence library was prepared with TruSeq RNA Library Prep Kit v2 (Illumina) 553 and sequenced with HiSeq X Ten (Illumina). Adaptor sequences were removed 554 from raw fastq files using cutadapt (version 1.18). Then, the reads were 555 aligned to a combined reference genome comprising the hg38 and the 556 SARS-CoV2 genome (NC_007605.1) using STAR (version 2.6.1b). The immunofluorescence with the primary antibodies from the same spices, we 667 utilized microwaving between the first and second staining cycles. Nuclei were 668 counterstained with Hoechst 33342 (Thermo Fisher Scientific, 1:1000). Images 669 were captured using a confocal microscope (SP8, Leica). 670 671

Difference between means from separate groups was determined using 673

Welch's unpaired t-tests. The level of significance is indicated as the P value in 674 each experiment. Asterisks in figures indicate the following: *, P value < 0.05; 675 **, P value < 0.01; ***, P value < 0.001; n.s., P value > 0.05. The data are 676 demonstrated as mean ± SEM. Graphs with statistical analysis were made 677 with the R software. For further statistical details, refer to each figure legend. LGRs J o u r n a l P r e -p r o o f 

Type 2 alveolar cells are stem 682 cells in adult lung

Remdesivir for the 685 Treatment of Covid-19 -Final Report

Remdesivir for the 688 Treatment of Covid-19 -Preliminary Report

A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe 691

Targeting of the pulmonary capillary 694 vascular niche promotes lung alveolar repair and ameliorates fibrosis

Inflammatory Signals Induce AT2 Cell-Derived Damage-Associated Transient 698 Progenitors that Mediate Alveolar Regeneration

Role of lopinavir/ritonavir in the 701 treatment of SARS: initial virological and clinical findings

Lgr5 homologues 704 associate with Wnt receptors and mediate R-spondin signalling

STAR: ultrafast universal RNA-seq aligner

Organoids of human airways to study infectivity 709 and cytopathy of SARS-CoV-2

HTII-280, a biomarker specific to the apical plasma membrane of human lung alveolar 723 type II cells

Compassionate Use of 726

Remdesivir for Patients with Severe Covid-19

Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven 730

Hospitalized Patients with Covid-19 -Preliminary Report

Genetics Reveals a Variable Infection Gradient in the Respiratory Tract

Tropism, replication competence, 740 and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory 741 tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures

Tropism, replication competence, and innate 745 immune responses of influenza virus: an analysis of human airway organoids and 746 ex-vivo bronchus cultures

Identification of Antiviral Drug Candidates against SARS-CoV-2 from FDA-Approved 749

Contribute to the Inflammatory Niche to Enhance Alveolar Regeneration

RSEM: accurate transcript quantification from 765

RNA-Seq data with or without a reference genome

Hyaluronan and TLR4 promote 768 surfactant-protein-C-positive alveolar progenitor cell renewal and prevent severe 769 pulmonary fibrosis in mice

Moderated estimation of fold change 771 and dispersion for RNA-seq data with DESeq2

Cutadapt Removes Adapter Sequences From High-Throughput

Enhanced isolation of SARS-CoV-2 by 776 TMPRSS2-expressing cells

Active and water-soluble form of lipidated Wnt protein 779 is maintained by a serum glycoprotein afamin/α-albumin

The anti-HIV drug nelfinavir mesylate (Viracept) is a potent 782 inhibitor of cell fusion caused by the SARSCoV-2 spike (S) glycoprotein warranting 783 further evaluation as an antiviral against COVID-19 infections

Single-cell Wnt signaling niches maintain stemness of alveolar type 2 cells

Long-term expanding human airway organoids for disease modeling

Progenitor 792 identification and SARS-CoV-2 infection in human distal lung organoids

Human Pancreatic Tumor 795

Organoids Reveal Loss of Stem Cell Niche Factor Dependence during Disease 796 Progression

Pathogenesis and 799 transmission of SARS-CoV-2 in golden hamsters

The intracellular sites of early replication and 802 budding of SARS-coronavirus

In Vitro and Animal Models for SARS-CoV-2 research

Differentiated human alveolar 811 epithelial cells and reversibility of their phenotype in vitro

Remdesivir in adults with severe COVID-19: a randomised, 815 double-blind, placebo-controlled, multicentre trial

Pathological findings of COVID-19 associated with acute 818 respiratory distress syndrome

Three-Dimensional Human Alveolar Stem Cell Culture 821

Models Reveal Infection Response to SARS-CoV-2

Distinct Mesenchymal Lineages and Niches Promote

Epithelial Self-Renewal and Myofibrogenesis in the Lung

Infection of bat and human intestinal organoids by 827 SARS-CoV-2

A pneumonia outbreak associated with a new coronavirus 830 of probable bat origin

1 • A fibroblast-free sphere culture system for human adult alveolus 2• Activation of Wnt and ERK/AKT pathways enable the growth of 3 alveolospheres 4• Development of a robust antiviral drug testing model using human 5 alveolospheres 6• Remdesivir inhibits SARS-CoV-2 replication in human alveolospheres 7 8 9 eTOC 10Ebisudani et al. establish a clinically relevant culture platform for human 11alveolus. They apply this sphere-based system to SARS-CoV-2 infection. The 12reproduction of SARS-CoV-2 in alveolospheres enables evaluation of antiviral 13 drug inhibitory effects, such as with remdesivir. This model will contribute to 14 the accurate evaluation of candidate drugs against COVID-19.