Points to consider regarding the use and implementation of virtual controls in nonclinical general toxicology studies.

The replacement of a proportion of concurrent controls by virtual controls in nonclinical safety studies has gained traction over the last few years. This is supported by foundational work, encouraged by regulators, and aligned with societal expectations regarding the use of animals in research. This paper provides an overview of the points to consider for any institution on the verge of implementing this concept, with emphasis given on database creation, risks, and discipline-specific perspectives.

Cross study analyses of SEND data: toxicity profile classification.

A SEND toxicology data transformation, harmonization, and analysis platform were created to improve the identification of unique findings related to the intended target, species, and duration of dosing using data from multiple studies. The lack of a standardized digital format for data analysis had impeded large-scale analysis of in vivo toxicology studies. The CDISC SEND standard enables the analysis of data from multiple studies performed by different laboratories. This work describes methods to analyze data and automate cross-study analysis of toxicology studies. Cross-study analysis can be used to understand a single compound's toxicity profile across all studies performed and/or to evaluate on-target versus off-target toxicity for multiple compounds intended for the same pharmacological target. This work involved development of data harmonization/transformation strategies to enable cross-study analysis of both numerical and categorical SEND data. Four de-identified SEND datasets from the BioCelerate database were used for the analyses. Toxicity profiles for key organ systems were developed for liver, kidney, male reproductive tract, endocrine system, and hematopoietic system using SEND domains. A cross-study analysis dashboard with a built-in user-defined scoring system was created for custom analyses, including visualizations to evaluate data at the organ system level and drill down into individual animal data. This data analysis provides the tools for scientists to compare toxicity profiles across multiple studies using SEND. A cross-study analysis of 2 different compounds intended for the same pharmacological target is described and the analyses indicate potential on-target effects to liver, kidney, and hematopoietic systems.

GeneMarkeR: A Database and User Interface for scRNA-seq Marker Genes.

Single-cell sequencing (scRNA-seq) has enabled researchers to study cellular heterogeneity. Accurate cell type identification is crucial for scRNA-seq analysis to be valid and robust. Marker genes, genes specific for one or a few cell types, can improve cell type classification; however, their specificity varies across species, samples, and cell subtypes. Current marker gene databases lack standardization, cell hierarchy consideration, sample diversity, and/or the flexibility for updates as new data become available. Most of these databases are derived from a single statistical analysis despite many such analyses scattered in the literature to identify marker genes from scRNA-seq data and pure cell populations. An R Shiny web tool called GeneMarkeR was developed for researchers to retrieve marker genes demonstrating cell type specificity across species, methodology and sample types based on a novel algorithm. The web tool facilitates online submission and interfaces with MySQL to ensure updatability. Furthermore, the tool incorporates reactive programming to enable researchers to retrieve standardized public data supporting the marker genes. GeneMarkeR currently hosts over 261,000 rows of standardized marker gene results from 25 studies across 21,012 unique genomic entities and 99 unique cell types mapped to hierarchical ontologies.

The Integrated Stress Response Regulates Cell Health of Cardiac Progenitors.

The discovery of mammalian cardiac progenitor cells has suggested that the heart consists of not only terminally differentiated beating cardiomyocytes, but also a population of self-renewing stem cells with the potential to generate new cardiomyocytes (Anderson, D., Self, T., Mellor, I. R., Goh, G., Hill, S. J., and Denning, C. 2007. Transgenic enrichment of cardiomyocytes from human embryonic stem cells. Mol. Ther. 15, 2027-2036; Bearzi, C., Rota, M., Hosoda, T., Tillmanns, J., Nascimbene, A., De Angelis, A., Yasuzawa-Amano, S., Trofimova, I., Siggins, R. W., Lecapitaine, N., Cascapera, S., Beltrami, A. P., D'Alessandro, D. A., Zias, E., Quaini, F., Urbanek, K., Michler, R. E., Bolli, R., Kajstura, J., Leri, A., et al. 2007. Human cardiac stem cells. Proc. Natl. Acad. Sci. U.S.A. 104, 14068-14073; Wu, S. M., Chien, K. R., and Mummery, C. 2008. Origins and fates of cardiovascular progenitor cells. Cell 132, 537-543). A consequence of longevity is continual exposure to environmental and xenobiotic stresses, and recent literature suggests that hematopoietic stem cell pools tightly control cell health through upregulation of the integrated stress response and consequent cellular mechanisms such as apoptosis. However, whether or not this biological response is conserved in progenitor cells for later lineages of tissue-specific stem cells is not well understood. Using human-induced pluripotent stem cells (iPSC) of both cardiac progenitor and mature cardiomyocyte lineages, we found that the integrated stress response was upregulated in the iPSC cardiac progenitors leading to an increased sensitivity for apoptosis relative to the mature cardiomyocytes. Of interest, C/EBP homologous protein (CHOP) signaling plays a mechanistic role in the cell death phenotype observed in iPSC progenitors, by which depletion of CHOP prevents cell death following cellular stress by thapsigargin exposure. Our studies suggest that the integrated stress response plays a unique role in maintaining iPSC cardiac progenitor cellular integrity by removing unhealthy cells via apoptosis following environmental and xenobiotic stresses, thus preventing differentiation and self-renewal of damaged cells.