DevKidCC allows for robust classification and direct comparisons of kidney organoid datasets

<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>While single-cell transcriptional profiling has greatly increased our capacity to interrogate biology, accurate cell classification within and between datasets is a key challenge. This is particularly so in pluripotent stem cell-derived organoids which represent a model of a developmental system. Here, clustering algorithms and selected marker genes can fail to accurately classify cellular identity while variation in analyses makes it difficult to meaningfully compare datasets. Kidney organoids provide a valuable resource to understand kidney development and disease. However, direct comparison of relative cellular composition between protocols has proved challenging. Hence, an unbiased approach for classifying cell identity is required.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>The R package, <jats:italic>scPred</jats:italic>, was trained on multiple single cell RNA-seq datasets of human fetal kidney. A hierarchical model classified cellular subtypes into nephron, stroma and ureteric epithelial elements. This model, provided in the R package <jats:italic>DevKidCC</jats:italic> (<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://github.com/KidneyRegeneration/DevKidCC">github.com/KidneyRegeneration/DevKidCC</jats:ext-link>), was then used to predict relative cell identity within published kidney organoid datasets generated using distinct cell lines and differentiation protocols, interrogating the impact of such variations. The package contains custom functions for the display of differential gene expression within cellular subtypes.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p><jats:italic>DevKidCC</jats:italic> was used to directly compare between distinct kidney organoid protocols, identifying differences in relative proportions of cell types at all hierarchical levels of the model and highlighting variations in stromal and unassigned cell types, nephron progenitor prevalence and relative maturation of individual epithelial segments. Of note, <jats:italic>DevKidCC</jats:italic> was able to distinguish distal nephron from ureteric epithelium, cell types with overlapping profiles that have previously confounded analyses. When applied to a variation in protocol via the addition of retinoic acid, <jats:italic>DevKidCC</jats:italic> identified a consequential depletion of nephron progenitors.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>The application of <jats:italic>DevKidCC</jats:italic> to kidney organoids reproducibly classifies component cellular identity within distinct single-cell datasets. The application of the tool is summarised in an interactive Shiny application, as are examples of the utility of in-built functions for data presentation. This tool will enable the consistent and rapid comparison of kidney organoid protocols, driving improvements in patterning to kidney endpoints and validating new approaches.</jats:p> </jats:sec>