Endocrine Abstracts

Perturbation of thyroid hormone (TH) function during prenatal brain development can cause a spectrum of neurological disorders as evident in pediatric patients with untreated congenital hypothyroidism or patients with mutations in THRA and SLC16A2 genes. However, scarcity and inaccessibility of human brain tissue from early developmental stages has been a major roadblock for experimental approaches to decipher local TH action in early development. Advanced in vitro models including human cerebral organoids (hCOs) hold great promises of accelerating our understanding of early human brain development and the hormonal signals that govern the complex processes underlying cortical neurogenesis. In this study, we differentiated hCOs from human induced pluripotent stem cells (hiPSC) in media containing either 1.5 nM T3 (BASAL group) or 20 nM T3 (CHRONIC group) for 9 weeks. Starting at day 13 of differentiation, hCOs were harvested at various developmental stages and analyzed by single cell RNA-seq for T3 treatment effects on the transcriptome. In addition, a subset of hCOs from the BASAL group was acutely treated with 50 nM T3 for 48 h (PULSE) at various stages and similarly analyzed by single cell RNA-seq. To avoid hiPSC line-inherent bias, all treatments were replicated for three different hiPSC lines resulting in 27 multiplexed single cell libraries. Global analysis of all cells collected during this study identified 31 neural cell clusters. Following cluster annotation, we could assign more than 95% of all cells to a major lineage trajectory from neural stem cell state towards excitatory neurons. Our single cell atlas comprised major cortical cell types including various radial glia cell (RGC) populations, three intermediate progenitor (IP) subtypes, as well as nine excitatory neuron subtypes. We defined developmental and T3-responsive expression profiles for genes related to TH action with cell type-specific resolution. For a subset of genes, smFISH was used to correlate spatial mRNA expression patterns with the laminar cell type distribution. We identified partially overlapping gene signatures in response to acute and chronic T3 treatment. Cell type- and developmental stage-specific gene set enrichments highlighted T3 effects on metabolic pathways in RGC, cell cycle regulation in IP and synapse function in neurons. T3-induced gene signatures in hCOs showed limited overlap with published mouse data sets. Chronic T3 treatment did not cause differentiation of aberrant cell types but altered the relative proportion of neuronal cell types in late stage hCOs. This comprehensive atlas of the T3-responsive transcriptome will serve as a unique resource to propel the use of hCOs in delineating the regulatory logic of local TH action in human brain development.