Enhanced metanephric specification to functional proximal tubule enables toxicity screening and infectious disease modelling in kidney organoids

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1. 

   Version published to 10.1101/2021.10.14.464320v2 on bioRxiv

   May 27, 2022
2. 

   ScreenIT

   Oct 22, 2021

   SciScore for 10.1101/2021.10.14.464320: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   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
   To determine the contribution of SIX2-mCherry + cells to EPCAM+ populations in organoids derived from the SIX2Cre lineage tracing iPSC line, dissociated and strained cells were stained using directly conjugated anti-EPCAM Alexa Fluor-647 antibody (see Table 1) diluted 1:100 in 100 µL of FACS wash (1% fetal calf serum [FCS] in PBS) for every 5 x105 cells.	anti-EPCAM suggested: None
   Cells were stained for 20 minutes on ice with 1µg of BioLegend TotalSeq-A anti-human hashtag oligo antibody …

   SciScore for 10.1101/2021.10.14.464320: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   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
   To determine the contribution of SIX2-mCherry + cells to EPCAM+ populations in organoids derived from the SIX2Cre lineage tracing iPSC line, dissociated and strained cells were stained using directly conjugated anti-EPCAM Alexa Fluor-647 antibody (see Table 1) diluted 1:100 in 100 µL of FACS wash (1% fetal calf serum [FCS] in PBS) for every 5 x105 cells.	anti-EPCAM suggested: None
   Cells were stained for 20 minutes on ice with 1µg of BioLegend TotalSeq-A anti-human hashtag oligo antibody (BioLegend TotalSeq-A0251 to A0258).	anti-human hashtag oligo suggested: None
   Fluorescent substrate uptake assays: For albumin uptake assays, D13+14 PT-enhanced organoids (triplicate wells per condition) were incubated in TRITC albumin (1:1000, Sigma Aldrich) and anti-MEGALIN/LRP2 (1:500, pre-incubated with an alpaca Nano-secondary Alexa Fluor 647 secondary antibody diluted in TeSR-E6 culture media via the basolateral compartment of Transwell tissue culture plates and incubated overnight (37°C, 5% CO2 and 5% O2).	anti-MEGALIN/LRP2 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Infectious virus titration (TCID50): Viral titrations were performed on confluent monolayers of Vero cells in 96-well plates.	Vero suggested: None
   Software and Algorithms
   Sentences	Resources
   Imaging was performed on the ZEISS LSM 780 confocal microscope (Carl Zeiss, Oberkochen, Germany) with acquisition and processing performed using ZEISS ZEN Black software (Zeiss Microscopy, Thornwood, NY) and Fiji ImageJ (Schindelin, et al., 2012).	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Images were acquired on a Zeiss Axio Imager A2 with Zeiss Zen software (Zeiss Microscopy, Thornwood, NY).	Zeiss Zen suggested: None
   Data were graphed and analysed in Prism 8 (GraphPad).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   The day before bead addition, 100µL of Affi-Gel Blue Gel 100 – 200 mesh crosslinked agarose beads (Bio-Rad Laboratories, Hercules, CA), were washed 3 times in PBS via centrifugation.	Bio-Rad Laboratories suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
   Cisplatin toxicity assay: D13+14 PT-enhanced organoids were exposed through the basolateral compartment of the Transwell tissue culture plate (Corning Incorporated, Corning, NY) to 1mL per well of 20 µM Cisplatin (Accord Healthcare, Durham, NC), or an equivalent volume of PBS, in TeSR-E6 for 24 hours (37°C, 5% CO2 and 5% O2).	Accord Healthcare suggested: None

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 57 and 53. 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.
   • No funding statement was detected.
   • No protocol registration statement was detected.

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

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3. 

   Version published to 10.1101/2021.10.14.464320v1 on bioRxiv

   Oct 15, 2021