Large-scale study suggests effects of trisomy 21 on fetal neurodevelopment may be more subtle than variability between organoids and isogenic lines

This study examines cortical organoids generated from a panel of isogenic trisomic and disomic iPSC lines (subclones) as a model of early fetal brain development in Down syndrome. An initial experiment comparing organoids from one trisomic and one disomic line showed many genome-wide transcriptomic differences and modest differences in cell-type proportions, suggesting there may be a neurodevelopmental phenotype that is due to trisomy of chr 21. To better control for multiple sources of variation, we undertook a highly robust study of ∼1,200 organoids using an expanded panel of six all-isogenic lines, three disomic, and three trisomic. The power of this experimental design was indicated by strong detection of the ∼1.5- fold difference in chr21 genes. However, the numerous expression differences in non-chr21 genes seen in the smaller experiment fell away, and the differences in cell-type representation between lines did not correlate with trisomy 21. Results suggest that the initial smaller experiment picked up differences between small organoid samples and individual isogenic lines, which “averaged out” in the larger panel of isogenic lines. Our results indicate that even when organoid and batch variability are better controlled for, variation between isogenic cell lines (even subclones) may obscure, or be conflated with, subtle neurodevelopmental phenotypes that may be present in ∼2nd trimester DS brain development. Interestingly, despite this variability between organoid batches and lines, and the “fetal stage” of these organoids, an increase in secreted Aβ40 peptide levels—an Alzheimer-related cellular phenotype—was more strongly associated with trisomy 21 status than were neurodevelopmental shifts in cell-type composition. Comparison of RNA-seq gene expression data from trisomic (T21) versus disomic cerebral organoids. In the pilot study, each RNA-seq library was from an individual organoid, with 5 samples each from one trisomic and one isogenic disomic cell-line. In the expanded study, each RNA-seq library was from a pool of ~6 organoids, with 8 samples (4 separate experiments x 2 replicate pools) each from three trisomic and three disomic cell-lines, all isogenic.