Modeling Down syndrome neurodevelopment with isogenic cerebral organoids

Abstract

As a model of early fetal brain development in Down syndrome, this study examines cortical organoids generated from isogenic trisomic and disomic iPSC lines. Initially pools of organoids from a trisomic versus disomic line found broad transcriptomic differences and modest differences in cell-type representation, suggesting a potential neurodevelopmental phenotype due to Trisomy 21. To better control for multiple sources of variation, we undertook a very robust study of ~1,200 organoids, using an expanded panel of six isogenic subclones (three disomic and three trisomic). The power of the experimental design was indicated by exceptionally strong detection of the ~1.5-fold difference in most chr21 genes. Despite some variability in secreted Aβ-40 levels between “identical” cell lines, this Alzheimer-related phenotype was detected as clearly correlated with Trisomy 21. However, the many statistically significant non-chr21 DEGs found in the small experiment fell away in the expanded study design, such that just three non-chr21 DEGs correlated to T21 status. Similarly, differences in cell-type representation of organoids varied somewhat between the six isogenic lines, but did not correlate with T21 status. Overall, our results indicate that even when organoid and batch variability are better controlled, common, subtle differences between isogenic cell lines (even subclones) may obscure, or be confused with, differences due to Trisomy 21. Interestingly, the neurodegenerative increase in Aβ due to T21 was strong enough to be evident in “fetal” organoids. In contrast, any neurodevelopmental phenotype that may be present in the ~2nd trimester of DS brain development may be more subtle, and within the range of variability in neurodifferentiation potential (unrelated to Trisomy 21) of our isogenic iPSC lines. The potential significance of two non-Chr21 DEGs that results suggest correlate with T21 is discussed.

Large-scale organoid study suggests effects of trisomy 21 on early fetal neurodevelopment are more subtle than variability between isogenic lines and experiments

“…While this overall finding is clear, whether any individual gene is identified as a DEG can be modestly impacted by even small differences in estimated cell-type proportions and specific cut-offs. Thus, changes in the deconvolution methodology, filtering of lowly expressed genes and other computational details can affect whether a few off-chr21 genes are identified as significant, as in our earlier analysis of this data (Czerminski, 2019;Czerminski et al, 2022). Hence, our central conclusion here is that this expanded organoid study shows powerful detection of chr21 DEGs, but that we cannot affirm that chromosome 21 dosage broadly impacts the transcriptome in this model of fetal brain development.…”

“…We thank members of the Lawrence lab for their collegiality, assistance as needed, and input on this study. Much of this work was originally part of a dissertation by JC (Czerminski, 2019) (http://hdl.handle.net/20.500.14038/31257), and a version with some revisions and extensions has been posted as a preprint to bioRxiv (Czerminski et al, 2022). Many of the analyses have been updated and expanded in the present manuscript, in part in response to helpful suggestions by the reviewers.…”