Hospitals: Wuhan: 85 Patients Shiyan: 50 Patients Shenzhen: 35 Patients Guangzhou: 3 Patients

Hospitals: Wuhan: 85 patients Shiyan: 50 patients Shenzhen: 35 patients Guangzhou: 3 patients

173 eligible patients

Allocated by ages

Gender and age Severity and Laboratory index and group analysis age group analysis age group analysis

Figure S1. Diagram of patient enrolment and data analysis.

The flowchart presents the patients’ recruitment and 173 patients were grouped into infants (0-

1 yrs.-old), younger children (1-5 yrs.-old), and older children (5-15 yrs.-old) by ages to analyse the gender distribution, disease severity (Table 1), and laboratory findings (Figure 1).

A B Lung progenitor cells(Day15) Lung progenitor cells(Day25) In vitro SOX2 SOX2 differentiation Undifferentiated human PSCs hPSCs(Day0) Day0

FOXA2 FOXA2 Endoderm cells Endoderm cells (Day3.5) Day3

Lung progenitor NKX2.1 NKX2.1 Lung progenitor cells cells (Day15-25) Day15-25

Lung cells NKX2.1/DAPI NKX2.1/DAPI Lung cells (Day50) Day50 scRNA-seq (10X)

C Positive control Negative control 4 ACE2/DAPI IgG/DAPI

0 s

n UMAP_2 −4 a

u Human lung

H −10 −5 0 5 10 UMAP_1

Figure S2. Directed differentiation of hESC into lung cells

(A). Scheme of directed differentiation of hESC into lung cells. Biomarkers and typical morphology at different differentiating stages were indicated.

(B) Representative and IF images in the hESC-derived cell cultures at day 15-25. The cells that are triple-positive for SOX2, FOXA2 and NKX2.1 at anterior foregut endoderm/lung progenitor stage or lung progenitor stage. Scale bars = 100 µm.

(C) The positive and negative controls of immunostaining. ACE2 stained in human testis was used as positive control, and IgG isotype stained in human lung was used as negative control.

Scale bar = 50 µm.

A B C Sample1 Sample2 PC1 (26.6% ) 6000 50k 5 2 Top 40 components 40k selected, capturing 0

2 95% of total variance 4000 30k 5 1 20k

2000 0 PC2 (8.5%) 1 10k PC2 (8.5%) Number of UMIs per cell 5 0 0 0 Number of genes detected per cell

Sample1 Sample2 Sample1 Sample2 Percentages of variance explained 0 10 20 30 40 50 60 Principal components (10238 cells)(11058 cells) (10238 cells) (11058 cells) PC1 (26.6% ) D E Higher in cluster 1 Higher in cluster 2 Variable genes SAA1 LRP1CD109 CD81 (2000) CD44 CD63 MUC1 PGC THY1 FOXQ1 30 ACTA2 IL1R1 BCAM DSP iance FGFR1CREB3L1 Non-variable genes PRRX1 ALCAM a r MAFB ZEB1 KLF5 PLA2G2A MEIS3 CDH1 (31538) Inf PDGFRAAPOE COL1A1 TWIST1 IFI16 ZEB2 CD24

ed V GYPC NFIC ELN LCN15 FBN1 HES1 PDGFRB ID3 FABP1 ATF3 20 SFTPB KRT17 PRRX2 SHOX2 MRC2 SNAI2 ARID3A SERPINA1 TCF4 ZNF521 EPCAM MMP1 MT2A CD248 ELF3 IGFL2 MME GAL CXCL13 MT1X TFF3 (FDR)

IL1R2 10 UBE2C SERPINE1 IL1RL1 300 PRSS2 DLK1 CXCL8 CD99 FCGBP MMP10 HMGA1 CTGF GDF15 TFF1 CD276 GNRH2 TOP2A -log TRPS1 SOX4 CD46 10 SAA2 MMP7 WFDC2 MMP3 CCL20 FOSL2 JUN NR2F6 CENPF STMN2 MGP MMP11 200 NCOR2MEIS2 LGALS4 ITGB1 ZFP36L2 CEACAM7 TSC22D3 ZFP36 CD9 PAWR KLF6 NFIL3 ZKSCAN1TAX1BP1

100 SOX11 CD164 ELF1TNFRSF12A Gene-wise Standardi z HMGB1 HIF3A FOSYY1XBP1ETS2 CD151 KLF3 CD47 ZNF91 0 0 PLIN2HIST1H1C 1e−3 1e−1 1e+1 −0.8 −0.6 −0.4 −0.2 0.0 0.2 0.4 0.6 Gene-wise Average Expression Margin of expressed ratios

Cluster1 Cluster2 F G ACTA2 ELN H (Stromal) (Epithelial) Representative GO terms in cluster 1 (Stromal) 20 4 20 4 3 3 10 10 4 ECM organisation 2 2 Skeletal system 0 1 0 1 0 0 Biological adhesion −10 −10 0 Collagen Fibril −20 −10 0 10 20 −20 −10 0 10 20 Epithelium development UMAP_2 PDGFRA Mesenchyme development COL1A1 −4 Mesenchymal cell differentiation 4 20 20 0 20 40 60 3 6 -log (FDR q) 10 2 10 10 4 −10 −5 0 5 10 1 Representative GO terms in cluster 2 (Epithelial) 0 0 2 I UMAP_1 0 0 + −10 −10 SFTPC cells subpopulations Biological adhesion −20 −10 0 10 20 −20 −10 0 10 20 SFTPC SOX9 HOPX ACE2 Secretion (11 cells) (3 cells,27.2%)(4 cells,36.3%)(0 cell, 0%) EPCAM MUC1

Regulation of transporters el 5 0.1 Epithelial cell differentiation 5 4 v 0.6 2.0 20 20 4 Epithelium development 4 3 1.5 3 Cell proliferation 10 10 2 3 0.4 0.05 Cell motility 2 1.0 0 1 0 1 2 0.2 0 0 0.5 0 5 1015 20 PC2 (8.5%) −10 −10

Expression L e 1 −20 −10 0 10 20 −20 −10 0 10 20 0.0 0.0 0.0 -log10(FDR q) PC1 (26.6% )

SFTPC+ SFTPC+ SFTPC+ SFTPC+ J K SOX9+ cells subpopulations L HOPX+ cells subpopulations ACE2 ACE2 SOX9 SFTPC HOPX ACE2 SOX2 HOPX SFTPC SOX9 ACE2 SOX2 (0 cell, 0%) (0 cell, 0%) (1275 cells) (3 cells,0.2%) (63 cells,4.9%)(49 cells,3.8%) (160 cells,12.5%) (625 cells) (4 cells,0.6%)(63 cells,10.1%)(7 cells,1.1%)(91 cells,14.6%)

el 5 el 2.0 2.5

v 3 2.5 v el 2.0 e 2.0 v e 3 4 1.5 2.0 2 e 3 2.0 1.5 1.5 2 3 1.5 1.5 0.05 0.05 1.0 2 2 1.0 2 1.0 1 1.0 1.0 1 1 0.5 0.5 1 1 0.5 0.5 0.5 Expression L Expression L 0.0 0.0 0 0.0 0.0 0 Expression L 0 0.0 0.0 0.0

HOPX+ SOX9+ SOX9+ SOX9+ SOX9+ SOX9+ SOX9+ HOPX+ HOPX+ HOPX+ HOPX+ HOPX+ SOX9+ SOX2+ (63 cells) (160 cells)

Figure S3. Single cell sequencing of hESC-derived cells

(A) Numbers of genes (left) and total UMIs (right) detected per biological replicate (n = 2).

(B) Principal component analyse shows that the two samples are representative to each other.

(C) Bar-chart showing the percentages of variance explained per each of the principal components, top 40 of which were selected for later dimension reduction approaches.

(D) Agnostic approach identified top 40 most informative genes (by dispersion) of the data.

(E) Volcano plot showing the most differentially expressed genes between cluster 1 and cluster

2, highlighting transcription factors (black) and surface markers (purple). Larger dot-size represents higher absolute value of log₂ (fold-change). “Margin of expressed ratios is the difference between the percentage of cells expressing one gene in one cluster minus that in another cluster. Black texts are transcription factors. Red text genes are reported markers of lung epithelial cells, and blue text genes are reported markers of lung stroma cells.

(F) Representative gene-ontology (GO) terms enriched for the DEGs higher in cluster 1 (cyan) or in cluster 2 (orange).

(G) Representative markers selected from the DEGs between cluster 1 and cluster 2, and were also reported by Travaglini et al. 2019.

(H) Scatterplot showing the first two components a UMAP reduction result, wherein the colors refer to the two major clusters identified by PCA.

(I) The violin plot shows the expression of SOX9, HOPX, and ACE2 in SFTPC+ subpopulation.

(J) The violin plot shows the expression of ACE2 in HOPX+SOX9+ and SOX9+SOX2+ cells.

(K) The violin plot shows the expression of SFTPC, HOPX, ACE2, and SOX2 in the SOX9+ subpopulation.

(L) The violin plot shows the expression of SFTPC, SOX9, ACE2, and SOX2 in the HOPX+ subpopulation.

Supplementary Methods and Materials

Key Resources Table

Antibodies Goat polyclonal anti-ACE2, R&D system, Cat# AF933, Rabbit monoclonal anti-KRT5, Abcam, Cat# ab52635, Mouse monoclonal anti-SCGB1A1, Santa Cruz Biotechnology, Cat# sc-365992 Rabbit polyclonal anti-SFTPC, Abcam, Cat# ab90716, Rabbit monoclonal anti-SOX9, Millipore, Cat# AB5535, Rabbit polyclonal anti-GFP, Thermo Fisher Scientific, Cat# A-11122, Mouse monoclonal anti-Luciferase, Thermo Fisher Scientific, Cat# 35-6700 Goat polyclonal anti-FOXA2, Santa Cruz Biotechnology, Cat# sc-6554 Rabbit polyclonal anti-SOX2, Abcam, Cat# ab97959, Rabbit polyclonal anti-NKX2.1, Seven Hills Bioreagents, Cat# WRAB-1231

Kits Trizol, Thermo Fisher Scientific, Cat# 15596026, PrimeScript RT Reagent Kit with gDNA Eraser, Takara, Cat# RR047B, Green Premix Ex Taq II (Tli RNase H Plus), Takara, Cat# RR820B, QIAGEN Plasmid Maxi Kit (25), Qiagen, Cat# 12163, H&E Staining Kit, Abcam, Cat# ab245880,

Cell Lines HEK293T, ATCC, Cat# CRL-3216,

Oligonucleotides SOX2: CCCATGCACCGCTACGACG CGGACTTGACCACCGAACCC SOX9: GAACGCCTTCATGGTGTGG GGGTGGTCCTTCTTGTGCTG SFTPB: CTTCCAGAACCAGACTGACTCA GCTCGGAGAGATCCTGTGTG SFTPC: GCAAAGAGGTCCTGATGGAG TGTTTCTGGCTCATGTGGAG ACE2: GGACCCAGGAAATGTTCAGA GGCTGCAGAAAGTGACATGA

Method Details

RNA extraction and cDNA synthesis Trizol reagent was used to extract total RNA of the lung samples according to the instructions. Briefly, 50-100 mg of lung tissues were homogenized in liquid nitrogen, then were transferred into 1.5 mL EP tubes filled with 1 mL trizol. The chloroform was added for vortex, which was followed with centrifuge (4°C, 12,000g, 15 mins). The supernatant was transferred into the new EP tubes, and the isopropanol was added to deposit the RNA. After the centrifuge and washing, total RNA was used for cDNA synthesis according to the instructions of reverse transcription kit of Takara. qPCR The qPCR was set and run according to the instruction of Takara Premix Ex Taq II. Briefly, 2 μL of cDNA was used as template to generate a 20 μL reaction system for each sample by mixing with the 0.4 μL primers (10 μM), 10 μL Takara SYSB Taq II mixture, and ddH2O. Step One & Step One Plus Real-Time PCR Systems (Thermo Fisher Scientific) was used for qPCR with the reaction condition: 95 °C 30 s, and 40 cycles of 95 °C 5 s, 60 °C 31 s. For data analysis, 2-ΔΔCT method was used.

H&E staining The lung section was stained following the instruction of H&E staining kit. After the standard process of deparaffinization and hydration, the section was incubated with hematoxylin for 5 mins at room temperature, and followed with the incubation of bluing reagent (10-15 s). Then, the slide was rinsed by distilled water, and stained with eosin Y solution for 2-3 mins. Finally, the section could be mounted for observation after the standard rehydration.