Stochasticity in response to rostrocaudal and mediolateral determinants influences inaccurate specification within kidney organoids

The differentiation of human pluripotent stem cells to kidney requires the accurate recapitulation of complex morphogenetic patterning events within an in vitro environment. Current protocols are assumed to accurately pattern through intermediate mesoderm to kidney with outcomes described based on formation of recognized renal structures. However, these protocols show significant technical variability and the presence of ‘off target’ cell types. We hypothesise that such ‘off-target’ cells represent heterogeneity in commitment along predictable rostrocaudal or mediolateral axes with respect to intermediate mesoderm. To test this hypothesis, we perform a temporal (4 stages across 27 days) factorial (21 growth factor conditions) screen evaluating the impact of perturbation to a commonly used kidney organoid protocol and evaluate the relative cellular composition at a single cell level. We test growth factor variation during early mesodermal patterning via variation in FGF, BMP and RA signalling. While the prevalence of ‘off-target’ endpoints shifts predictably along rostrocaudal and mediolateral axes, patterning to neural/glial, paraxial and intermediate mesoderm endpoints is evident in all conditions. Heterogeneity of specification is observed using different cell lines in the same protocol and in a distinct differentiation protocol designed to generate neuromesodermal progenitors. We suggest a stochasticity in early mesodermal patterning in vitro with heterogeneous trajectories arising early in the protocol and leading to indeterminate mesodermal states with considerable resulting in plasticity.