Isogenic spheroid cultures identify asynchronous development of cortical excitatory neurons during brain development in Down syndrome

The intellectual disability (ID) in Down syndrome (DS) is thought to result from a variety of developmental cellular deficits such as altered neural progenitor division, abnormal neurogenesis and gliogenesis, aberrant cortical architecture, and reduced cortical volume. However, the molecular processes underlying these neurodevelopmental changes are still elusive, preventing an understanding of the mechanistic basis of ID in DS. In this study, we used a pair of isogenic (trisomic and euploid) induced pluripotent stem cell (iPSC) lines to generate cortical spheroids (CS) that model the impact of trisomy 21 on brain cells. CS contain neurons, astrocytes, and oligodendrocytes and they are widely used to approximate early neurodevelopment . Our single cell RNA sequencing (scRNA-seq) analysis from these CS uncovers cell-specific transcriptomic changes primarily affecting excitatory neurons in trisomic CS. Using advanced bioinformatic tools, we identify a specific population of excitatory neurons (transcriptomically corresponding to layer 4 cortical neurons) that display profound divergence in developmental trajectory between trisomic and euploid controls. Our results identify candidate genes potentially driving this developmental asynchrony. A direct comparison of our isogenic CS-derived scRNA-seq data to other published datasets highlights a shared transcriptomic signature across DS samples as well as specific differences that can be attributed to individual genetic background (sex, allelic composition), cell type, and differentiation stage, as well as to different experimental approaches. Altogether, our study highlights the importance of deeply analyzing cell-specific features within an individual sample in addition to broader examination of mixed samples in order to comprehensively evaluate cellular phenotypes in individuals with DS.