A temporal factorial dissection of kidney organoid differentiation identifies the impact of WNT, FGF, BMP and GDF signaling on ‘off-target’ patterning

Human pluripotent stem cell-derived organoids are multicellular models of developing tissues proposed to recapitulate developmental stages of lineage commitment across time. Assessing how accurately protocols recapitulate development is challenged by the lack of accurate reference data sets for early human development, the presence of ‘off target’ states and the potential to form cellular states not presentin vivo. This study addresses these challenges with respect to differentiation of human pluripotent stem cells to kidney organoids. Based on a factorial single-cell transcriptomic analysis across a 27-day differentiation protocol, including 150,957 cells collected at five time points, we present a comprehensive classification of predicted identity, representing anin vitrotemporal transcriptional atlas. A significant component of all timepoints were neuromesodermal ‘off target’ populations which resulted in a variety of neural/neural crest and muscle endpoints. Transitional mesodermal populations with no clearin vivoequivalent were also present and varied with duration of initial mesodermal commitment. The analysis of cell trajectory across the timecourse identified distinct asynchrony of cell responses duringin vitrodifferentiation, with this driving ‘off target’ patterning.