DOI: 10.5061/dryad.1c59zw48q
title: Implementing a trilineage differentiation in the ReproTracker assay for improved teratogenicity assessment

description: Exposure to teratogenic compounds during pregnancy can lead to significant
 birth defects. Given the considerable variation in drug responses across
 species, along with the financial and ethical challenges associated with
 animal testing, the development of advanced human-based in vitro assays is
 imperative for effectively identifying potential human teratogens.
 Previously, we developed a human induced pluripotent stem cell
 (hiPSCs)-based biomarker assay, ReproTracker, that follows the
 differentiation of hiPSCs into hepatocytes and cardiomyocytes. The assay
 combines morphological profiling with the assessment of time-dependent
 expression patterns of cell-specific biomarkers to detect developmental
 toxicity responses. To further increase the predictability of the assay in
 identifying potential teratogens, we added differentiation of hiPSCs
 towards neural rosette-like cells. We evaluated the performance of the
 extended assay with a set of 51 well-known in vivo teratogens and
 non-teratogens, including the compounds listed in the ICH S5 reference
 list. The optimized assay correctly identified (neuro)developmental
 toxicants that were not detected in the hepatocyte and cardiomyocyte
 differentiation assays. These compounds selectively downregulated gene and
 protein expression of the neuroectodermal marker PAX6 and/or neural
 rosette marker NESTIN in a concentration-dependent manner and disrupted
 the differentiation of hiPSCs towards neural rosette-like cells. Overall,
 based on the current dataset, the addition of neural commitment improved
 the assay accuracy (from 72.55% to 86.27%) and sensitivity (from 67.50% to
 87.50%), when compared to the previously described assay. In summary,
 trilineage differentiation expanded the spectrum of teratogenic agents
 detectable by ReproTracker, making the assay an invaluable tool for early
 in vitro teratogenicity screening.
description: # Implementing a trilineage differentiation in the ReproTracker assay for
 improved teratogenicity assessment Dataset DOI:
 [10.5061/dryad.1c59zw48q](https://doi.org/10.5061/dryad.1c59zw48q) ##
 Description of the data and file structure **Readme for the data files**
 **Contents** The data described in the manuscript is composed of the
 following: 1. Dose-range finding data 2. qPCR data 3. Immunofluorescence
 data 4. LOAEL data Data are shared within this package. The contents of
 the package are as follows: **Dose-range finding data** – all data
 generated using the AlamarBlue cell viability assay. The data are combined
 into an Excel file (‘Dose-range finding data’), where the Overview sheet
 displays the data location per compound: | **Column name** |
 **Description** | | :-------------- |
 :--------------------------------------------------- | | Entry | Code
 provided to each compound, ranging between 1-51 | | Compound name | Tested
 compound | | Data location | Description of where the relevant data can be
 found | In the data sheets ‘Compounds 21-30’, ‘Compounds 31-40’, and
 ‘Compounds 41-51’, data are sorted per compound, each row corresponding to
 tested concentration, with the following columns: | **Column name** |
 **Description** | | :-------------- |
 :------------------------------------------------------------------- | |
 Entry | Code provided to each compound | | Compound name | Tested compound
 | | DF data | Concentration tested, with value 1 displaying the lowest
 tested dose | | Conc (..) | Concentration (unit) | | Viability (%) | Cell
 viability for replicate 1 | | Raw values | Obtained results for replicate
 1 | | Viability (%) | Cell viability for replicate 2 | | Raw values |
 Obtained results for replicate 2 | | Viability (%) | Cell viability for
 replicate 3 | | Raw values | Obtained results for replicate 3 | | AVERAGE
 | Average cell viability | | SD | Standard deviation of cell viability | |
 N | Number of replicates | **qPCR data** – all raw data generated using
 Quantitative real-time PCR. The excel file ‘Decoding file_qPCR’ contains
 an overview of all data files of the 51 tested compounds. Data are listed
 per compound, each row corresponding to a compound, with the following
 columns: | **Column name** | **Description** | |
 :--------------------------------------------- |
 :------------------------------------------------------------------------------------------------ | | Entry | Code provided to each compound, ranging between 1-51 | | Compound name | Tested compound | | Cardiomyocyte data\_Exposed samples CSV file# | Reference to the coded data file of exposed samples from the cardiomyocyte differentiation assay | | Hepatocyte data\_Exposed samples CSV file# | Reference to the coded data file of exposed samples from the hepatocyte differentiation assay | | Neural data\_Exposed samples CSV file# | Reference to the coded data file of exposed samples from the neural differentiation assay | | Cardiomyocyte data\_Solvent controls CSV file# | Reference to the coded data file of solvent controls from the cardiomyocyte differentiation assay | | Hepatocyte data\_Solvent controls CSV file# | Reference to the coded data file of solvent controls from the hepatocyte differentiation assay | | Neural data\_Solvent controls CSV file# | Reference to the coded data file of solvent controls from the neural differentiation assay | Coded data files are sorted per lineage, in folders: ‘Raw qPCR data files_CM’, ‘Raw qPCR data files_LVR’, ‘Raw qPCR data files_NRL’. Each folder contains the following: 1. A folder named ‘CSV files-exposed’ with the coded data of exposed samples. 2. A folder named ‘CSV files-solvents’ with the coded data of solvent controls 3. An excel file named ‘RNA codes_Exposed samples_lineage’ with the relevant RNA sample numbers listed per experiment 4. An Excel file named ‘RNA codes_Solvent controls_lineage’ with the relevant RNA sample numbers listed per experiment Both the folders ‘CSV files-exposed’ and ‘CSV files-solvents’ contain .CSV files, in which data are sorted per technical replicate value, where columns capture the following: | **Column name** | **Description** | | :-------------------- | :---------------------------------------------------------------------------- | | Well | Well in the 384-WP used for qPCR | | Omit | Allows for selection (FALSE) or deselection (TRUE) of data | | Sample | The sample name assigned as ‘RNA code\_technical replicate’ (e.g. R064235\_1) | | Target | Target gene | | Task | Allows for extra analysis within the qPCR analysis software | | Dyes | Probe | | Cq | Quantification cycle value | | Cq Conf | Confidence of the algorithm in calling the Cq value | | Amp score | Amplification score | | Amp status | Amplification status | | Annotated | User annotation | | Curve quality | Qualitative call on the amplification curve | | Result quality issues | Flags any problems the software detected | The files ‘RNA codes_Exposed samples’ for each lineage contain an overview of the relevant RNA codes per differentiation experiment, where columns capture the following: | **Column name** | **Description** | | :------------------------------ | :-------------------------------------------------------------- | | RNA number | RNA sample code | | Experimental code | Code assigned to the regarding differentiation assay experiment | | Replicate # (1 or 2) | Replicate number | | Experiment type | Differentiation assay (lineage) | | Toxys compound code or Biogroup | Compound or solvent to which the sample was exposed | | Day of differentiation | Day during differentiation at which the sample was collected | | Concentration | Tested concentration coded from 1 (low) to 5 (high) | The files ‘RNA codes_Solvent controls’ for each lineage contain an overview of the relevant RNA codes per differentiation experiment, where columns capture the following: | **Column name** | **Description** | | :------------------------------ | :-------------------------------------------------------------- | | RNA number | RNA sample code | | Experimental code | Code assigned to the regarding differentiation assay experiment | | Replicate # (1 or 2) | Replicate number | | Experiment type | Differentiation assay (lineage) | | Toxys compound code or Biogroup | Compound or solvent to which the sample was exposed | | Day of differentiation | Day during differentiation at which the sample was collected | The following data can be found in folder ‘Raw Data All Figures’: Additional qPCR data used for Figure 1D and 1G are collected in ‘Raw Data_Figure_1’. Data are sorted per target gene and per timepoint (D0, D7, D13). Columns capture the following: | **Column name** | **Description** | | :--------------- | :---------------------------------------------------------------------------- | | Gene of interest | Ct value obtained for the indicated target gene | | HKG (GAPDH) | Ct value obtained for the housekeeping gene GAPDH | | ΔCt | Difference in Ct value obtained from the housekeeping gene vs the target gene | | ΔΔCt | ΔCt values normalized to those on D0 | | LogΔΔCt | POWER(2, ΔΔCt)  | | Average | Average LogΔΔCt of all replicates | | STDEV | Standard deviation of the LogΔΔCt of all replicates | Additional qPCR data used for Figure 2 are collected in ‘Raw Data_Figure_2’, both containing an overview of the raw data as well as the performed analysis. Columns capture the following depending on the provided sheets (raw data vs analysis): Sheet 1. | **Column name** | **Description** | | :-------------- | :------------------------------------------------------------------ | | Experiment | Number of experimental replicate | | Compound | Tested compound | | Data-type | Indicates the type of data (ΔCt vs log ΔCt) | | Target | Indicates the gene of interest | | Day | Day during differentiation at which the sample was collected | | Conce.0 | Ct value obtained for the indicated target gene at concentration 0. | | Conce. 1 | Ct value obtained for the indicated target gene at concentration 1. | | Conce.2 | Ct value obtained for the indicated target gene at concentration 2. | | Conce. 3 | Ct value obtained for the indicated target gene at concentration 3. | | Conce.4 | Ct value obtained for the indicated target gene at concentration 4. | | Conce. 5 | Ct value obtained for the indicated target gene at concentration 5. | Sheet 2. | **Column name** | **Description** | | :---------------------------- | :------------------------------------------------------------------------------- | | Replicate | Replicate number | | Compound | Compound and concentration (1-5) | | Day | Day during differentiation at which the sample was collected | | GAPDH | Ct value obtained for the indicated target gene | | PAX6 | Ct value obtained for the indicated target gene | | NESTIN | Ct value obtained for the indicated target gene | | ΔCt | Difference in Ct value obtained from the vs target gene vs de housekeeping gene | | Average D0 | Average of the ΔCt values of all D0 samples for an specific gene | | ΔΔCt | Difference in Ct value obtained from reducing the Average D0 to the sample’s ΔCt | | Fold Change (Gene expression) | 2\^-ΔΔCt | Additional qPCR data used for Supplementary Figure 3 are collected in ‘Raw data_Suppl-qPCR data for biomarker kinetics’ and sorted by Compound, Concentration, Timepoint, Replicate and Target gene (NGN2, OTX2 and SOX1). The information is divided in two sheets. Sheet 1. Raw data. | **Column name** | **Description** | | :-------------- | :---------------------------------------------------------------------------- | | Well | Well in the 384-WP used for qPCR | | Omit | Allows for selection (FALSE) or deselection (TRUE) of data | | Sample | The sample name assigned as ‘RNA code\_technical replicate’ (e.g. R064235\_1) | | Target | Target gene | | Task | Allows for extra analysis within the qPCR analysis software | | Dyes | Probe | | Cq | Quantification cycle value | Sheet 2. Analysis | **Column name** | **Description** | | :-------------- | :---------------------------------------------------------------------------- | | Sample | The sample name assigned as ‘RNA code\_technical replicate’ (e.g. R064235\_1) | | GAPDH | Ct value obtained for the indicated target gene | | NGN2 | Ct value obtained for the indicated target gene | | OTX2 | Ct value obtained for the indicated target gene | | SOX1 | Ct value obtained for the indicated target gene | | Conc. | Concentration of the compound | | Compound | Name of the compound of exposure | | Timepoint | Day during differentiation at which the sample was collected | | Replicate | Indicates the replicate number | **Immunofluorescence data** – all data generated using immunofluorescent staining and analysis using the Revvity Harmony Software. Data used for Figure 2 are collected in folder ‘IF and analysis-Figure 2’, in which file ‘Data Analysis #1 2’ contains the data for Saccharin, Retinoic Acid and Tacrolimus, where columns capture the following: | **Column name** | **Description** | | :--------------------------------------------------------------------- | :--------------------------------------------------------------------------- | | Row | Plate coordinates of the well | | Column | Plate coordinates of the well | | Nuclei Selected – Number of Objects | Total nuclei initially detected in the well | | Nuclei – Number of Objects | Total nuclei objects before filtering (raw count) | | PAX6 positive – Number of objects | Number of selected nuclei that meet the PAX6 threshold | | PAX6 Positive - PAX6 Nuclear Intensity Mean - Mean per Well | Average nuclear fluorescence intensity of PAX6 | | NESTIN Positive - Number of Objects | Number of cells classified as Nestin-positive | | NESTIN Positive - PAX6 Nuclear Intensity Mean - Mean per Well | Among NESTIN-positive cells, average nuclear intensity of PAX6 staining | | NESTIN Positive - NESTIN Intensity - CELL Mean - Mean per Well | Among NESTIN-positive cells, average cellular intensity of NESTIN | | NESTIN Highly-Positive - Number of Objects | Number of cells classified as highly NESTIN-positive (above upper threshold) | | NESTIN Highly-Positive - PAX6 Nuclear Intensity Mean - Mean per Well | Average nuclear PAX6 intensity of highly NESTIN-positive cells | | NESTIN Highly-Positive - NESTIN Intensity - CELL Mean - Mean per Well | Average cellular NESTIN intensity of highly NESTIN-positive cells | | Nuclei Accepted - Number of Objects | Nuclei that passed all quality filters and were used in final analysis | | Nuclei Accepted - PAX6 Nuclear Intensity Mean - Mean per Well | Average nuclear PAX6 intensity of accepted nuclei | | Nuclei Accepted - NESTIN Intensity - CELL Mean - Mean per Well | Average cellular NESTIN intensity of accepted cells | | Nuclei Accepted - N-Cadherin Intensity - MEMBRANE Mean - Mean per Well | Average membrane N-cadherin intensity of accepted cells | | PAX6 % Positive | Percentage of accepted nuclei classified as PAX6-positive | | NESTIN % Positive | Percentage of accepted cells classified as NESTIN-positive | | NESTIN-High % Positive | Percentage of accepted cells classified as highly NESTIN-positive | Additional data used for Supplementary Figure 1 are collected in file ‘Raw data_Suppl-PAX6 Expression levels % from IF.’ | **Column name** | **Description** | | :------------------------------------------------------------------- | :--------------------------------------------------------------------------- | | Row | Plate coordinates of the well | | Column | Plate coordinates of the well | | Nuclei Selected – Number of Objects | Total nuclei initially detected in the well | | Nuclei – Number of Objects | Total nuclei objects before filtering (raw count) | | NESTIN positive – Number of objects | Number of selected nuclei that meet the PAX6 threshold | | NESTIN Positive - PAX6 Nuclear Intensity Mean - Mean per Well | Average nuclear fluorescence intensity of PAX6, per well | | NESTIN Positive – NESTIN intensity-CELL Mean-Mean per well | Average cellular NESTIN intensity of NESTIN-positive cells, per well | | NESTIN Highly-positive- Number of objects | Number of cells classified as highly NESTIN-positive (above upper threshold) | | NESTIN Highly-positive-PAX6 Nuclear Intensity Mean-Mean per well | Average nuclear PAX6 intensity of highly NESTIN-positive cells, per well | | NESTIN Highly positive NESTIN intensity CELL mean-Mean per well | Average cellular NESTIN intensity of accepted nuclei, per well | | Nuclei Accepted – N-Cadherin intensity- Membrane mean- Mean per well | Average membrane N-Cadherin intensity of accepted nuclei/cells, per well | | PAX6% Positive | Percentage of accepted nuclei classified as PAX6-Positive | **LOAEL data** – all LOAELs (Lowest Observed Adverse Effect Levels) were obtained from the ReproTracker assay results applying the assay criteria. In file ‘Raw Data_Figure_4’, all obtained values are summarized and sorted per compound, including the lower (L) and higher (H) limits of the Cmax (from literature) and the ReproTracker LOAELs. | **Column name** | **Description** | | :----------------- | :---------------------------------------- | | # | Compound number | | Compounds | Compound name | | Cmax L | Lowest Cmax Value | | Cmax H | Highest Cmax Value | | LOAEL L | Lowest LOAEL value | | LOAEL H | Highest LOAEL Value | | Log Cmax L | Log transformation of Lowest Cmax Value | | Log Cmax H | Log transformation of Highest Cmax Value | | Log LOAEL L | Log transformation of Lowest LOAEL value | | Log LOAEL H | Log transformation of Highest LOAEL value | | Sorted Log LOAEL L | Sorted Log LOAEL L (decreasing) | | Sorted Log LOAEL H | Sorted Log LOAEL H (decreasing) | ### Files #### File: Dose-range_finding_data.xlsx **Description:** Information describing the dose-range finding experiments and results #### File: Decoding_file_qPCR.xlsx **Description:** Information on how to decide qPCR files  #### File: Raw_qPCR_data_files_CM.zip **Description:** Raw qPCR files for cardiomyocyte differentiations #### File: Raw_qPCR_data_files_NRL.zip **Description:** Raw qPCR files for neural differentiations #### File: Raw_qPCR_data_files_LVR.zip **Description:** Raw qPCR files for hepatocyte differentiations #### File: Raw_Data_All_Figures.zip **Description:** All raw data and analyses performed in all main and supplementary figures ## Human subjects data Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.