Recently, human cerebral organoids (hCOs) emerged as powerful three-dimensional in vitro models recapitulating key aspects of early human cerebral cortex development. Improved protocols for hCO generation from induced pluripotent stem cells (hiPSC) paved the way for new disease modeling approaches and enable multimodal analyses of perturbed neurogenesis resulting from manipulation of signaling pathways or gene function. These prospects prompted us to adopt this technology as a model system to delineate the regulatory logic of local TH action during early human brain development and to model the consequences of impaired TH function for cortical neurogenesis. Here, we report results from our efforts to develop experimental strategies for targeted perturbation of TH signaling in hCOs and highlight critical quality measures to prevent experimental artifacts. Titration of graded amounts of exogenous TH to culture media is commonly regarded as the simplest means for manipulating TH signaling. Yet, we found that the variable amounts of TH present in culture media and commercial supplements (i.e. B27) can confound experimental outcomes and that rigorous analytical verification of TH levels is required to ensure reproducible endpoint measurements. Patient-derived hiPSC lines carrying genetic variants conferring reduced functionality of specific proteins gained great popularity for disease modeling. However, differences in the inherent propensity of individual hiPSC lines to generate cerebral cortex tissue in vitro can result in enormous variation and hamper robust phenotypic comparisons. We therefore developed CRISPR/Cas9-based strategies for generation of isogenic wildtype and mutant hiPSC lines to minimize this source of variation. In developing hCOs, progenitor and neuronal cell types position themselves in three-dimensional in vivo-like laminar structures along with lineage specification. We validated a panel of canonical cell type and layer markers to distinguish germinal zones and a cortical plate-like zone and define the timing of the sequential appearance of deep and upper layer neurons consistent with the in vivo inside-out manner of cortical layer emergence. Due to the complexity of the tissue, we apply single cell transcriptome techniques to comprehensively capture the temporal and cell type-specific expression of TH transporters, receptors and deiodinases in developing hCOs and use single molecule fluorescent in situ hybridization to register cell type-specific expression patterns in the spatial context of the laminar cortical organization. Collectively, we present a strategy combining various endpoint measurements and quality measures to faithfully exploit the enormous potential of hCO for studies on TH action in a human model system.
10 Sep 2022 - 13 Sep 2022