An iTSC-derived placental model of SARS-CoV-2 infection reveals ACE2-dependent susceptibility in syncytiotrophoblasts

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   ScreenIT

   Nov 19, 2021

   SciScore for 10.1101/2021.10.27.465224: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IRB: Placental tissue preparation and Immunohistochemistry: Ethics approval for the use of first trimester human placental tissues for research was obtained from the Human Ethics Committee at Monash Health (RES-19-0000-399A, Melbourne, Australia), all patients provided informed written consent. Consent: Placental tissue preparation and Immunohistochemistry: Ethics approval for the use of first trimester human placental tissues for research was obtained from the Human Ethics Committee at Monash Health (RES-19-0000-399A, Melbourne, Australia), all patients provided informed written consent.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not …

   SciScore for 10.1101/2021.10.27.465224: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IRB: Placental tissue preparation and Immunohistochemistry: Ethics approval for the use of first trimester human placental tissues for research was obtained from the Human Ethics Committee at Monash Health (RES-19-0000-399A, Melbourne, Australia), all patients provided informed written consent. Consent: Placental tissue preparation and Immunohistochemistry: Ethics approval for the use of first trimester human placental tissues for research was obtained from the Human Ethics Committee at Monash Health (RES-19-0000-399A, Melbourne, Australia), all patients provided informed written consent.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Sections were then incubated with primary antibodies (ACE2, ab15438, Abcam,	ACE2 suggested: None
   Human IgG1 antibodies were purified using Protein-A affinity chromatography.	Human IgG1 suggested: None
   Assessment of human antibody binding specificity by ELISA: 96-well flat-bottomed MaxiSorp plates were coated with 50 μl of 125 nM recombinant human or mouse ACE2 protein in PBS at room temperature for one hour.	mouse ACE2 suggested: None
   Goat anti-Human IgG secondary antibody (1:5000).	anti-Human IgG suggested: None
   For affinity measurements against human ACE2, antibodies were loaded by submerging sensor tips for 200 s and then washed in kinetics buffer for 60 s.	human ACE2 suggested: None
   Primary antibodies used in the study were: anti-HCG (ab9582, abcam, 1:200), anti-dsRNA (MABE1134, Merck, 1:200), anti-ACE2 (ab15348, abcam, 1:200), anti-GATA3 (MA1-028, Invitrogen, 1:100), and anti-HLA G (ab7759, abcam, 1:50).	anti-HCG suggested: (Abcam Cat# ab9582, RRID:AB_296507) anti-dsRNA ( MABE1134 suggested: None anti-GATA3 ( MA1-028 suggested: None anti-HLA G suggested: (Abcam Cat# ab7759, RRID:AB_306053)
   Secondary antibodies used in the study (all 1:400) were Alexa Fluor 488 goat anti-mouse IgG1 (A21121, Invitrogen, Thermo Fisher Scientific)	anti-mouse IgG1 suggested: (Thermo Fisher Scientific Cat# A-21121, RRID:AB_2535764) A21121 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Median TCID50 in supernatants were determined by 10-fold serial dilution in Vero cells and calculated using the Reed and Muench method.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   Fabs from positive phage were reformatted into IgG1 expression plasmids and used for transient expression in Expi293 cells.	Expi293 suggested: RRID:CVCL_D615)
   Software and Algorithms
   Sentences	Resources
   Image Analysis/Cell Quantification: Cell quantification was performed using the particle analysis option of the ImageJ software (http://rsb.info.nih.gov/ij/).	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Gene expression analyses: Pre-processing RNA-seq: Raw next generation RNA sequencing (RNA-seq) reads were obtained in FASTQ format, and prior to demultiplexing the forward read FASTQ was trimmed with trimmomatic to 18 nucleotides (nt) (the targeted read length as described above) with the following parameters: SE -phred33 CROP:18 MINLEN:18 (Bolger et al., 2014)	trimmomatic suggested: (Trimmomatic, RRID:SCR_011848)
   Sequencing reads were then mapped to a customised genome, composed of both GENCODE’s GRCh38.p13 and human SARS-CoV2 (RefSeq - NC_045512.2; see “Custom genome for mapping” below for further details), with STAR v2.5.2b (Dobin et al., 2013) and the parameters: --outSAMattributes All --alignIntronMax 1000000 --alignEndsType	RefSeq suggested: (RefSeq, RRID:SCR_003496) STAR suggested: (STAR, RRID:SCR_004463)
   Prior to library size normalisation, normalisation factors were calculated with EdgeR’s (v3.32.1) calcNormFactors function (Robinson et al., 2010).	EdgeR’s suggested: None
   Hierarchical clustering was performed utilizing base R’s package stats (functions: dist and hclust), with the distance measure canberra and linkage method Ward.D.	hclust suggested: (HCLUST, RRID:SCR_009154)
   Dendrogram visualization was performed with dendexted v1.15.1 (parameter: k = 3) (Galili, 2015); 3D visualizations were performed with plotly v4.9.4.1 (Sievert, 2020); heatmap visualizations were performed with ComplexHeatmap v2.6.2 (Gu et al., 2016); all other visualizations were performed with ggplot2 v3.3.5 (Villanueva and Chen, 2019) and where required ggrepel v0.9.1 (Slowikowski et al., 2018)	ComplexHeatmap suggested: (ComplexHeatmap, RRID:SCR_017270) ggplot2 suggested: (ggplot2, RRID:SCR_014601)
   Gene ontology and pathway analyses were performed with Metascape (http://metascape.org) (Zhou et al., 2019).	Metascape suggested: (Metascape, RRID:SCR_016620)

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   Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Although, as demonstrated by our results, this infection model can be of great utility, there are limitations. For example, the decidua contains 10-20% of macrophages but we cannot model their effects in this system (Kreis et al., 2020, Manaster and Mandelboim, 2010). In the future, complex models that include immune cells could be used to enhance current models, as has been done with brain slice cultures and microglia from iPSC derived models (Grubman et al., 2021). Recent studies have reported that host cell factors such as ACE2, TMPRSS2 or cathepsins are vital for SARS-CoV-2 entry and could be utilized as potential therapeutic targets against infection (Dong et al., 2020, Hoffmann et al., 2020). We also reported that blocking viral infection through ACE2 blockade restores the functional phenotype in STs, similar to the rescue of function in lung and cardiac cells through the inhibition of ACE2 or TMPRSS2 activity (Huang et al., 2020, Hoffmann et al., 2021, Pei et al., 2020, Bojkova et al., 2020, Perez-Bermejo et al., 2021). More importantly, this in vitro derived placental model allowed us to generate a quick and effective system. We envision that our model will facilitate a deeper understanding of COVID-19 pathogenesis and will provide a platform for drug discovery and potential treatments.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 35, 19, 36 and 7. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

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   Results from scite Reference Check: We found no unreliable references.

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   Version published to 10.1101/2021.10.27.465224v1 on bioRxiv

   Oct 29, 2021