Abstract
Experimental models of the central nervous system (CNS) are imperative for developmental and pathophysiological studies of neurological diseases. Among these models, three-dimensional (3D) induced pluripotent stem cell (iPSC)-derived brain organoid models have been successful in mitigating some of the drawbacks of 2D models; however, they are plagued by high organoid-to-organoid variability, making it difficult to compare specific gene regulatory pathways across 3D organoids with those of the native brain. Single-cell RNA sequencing (scRNA-seq) transcriptome datasets have recently emerged as powerful tools to perform integrative analyses and compare variability across organoids. However, transcriptome studies focusing on late-stage neural functionality development have been underexplored. Here, we combine and analyze 8 brain organoid transcriptome databases to study the correlation between differentiation protocols and their resulting cellular functionality across various 3D organoid and exogenous brain models. We utilize dimensionality reduction methods including principal component analysis (PCA) and uniform manifold approximation projection (UMAP) to identify and visualize cellular diversity among 3D models and subsequently use gene set enrichment analysis (GSEA) and developmental trajectory inference to quantify neuronal behaviors such as axon guidance, synapse transmission and action potential. We showed high similarity in cellular composition, cellular differentiation pathways and expression of functional genes in human brain organoids during induction and differentiation phases, i.e., up to 3 months in culture. However, during the maturation phase, i.e., 6-month timepoint, we observed significant developmental deficits and depletion of neuronal and astrocytes functional genes as indicated by our GSEA results. Our results caution against use of organoids to model pathophysiology and drug response at this advanced time point and provide insights to tune in vitro iPSC differentiation protocols to achieve desired neuronal functionality and improve current protocols.
Highlights
The resulting shortcomings are twofold: (i) native cellular processes occur in the 3D microenvironment of the brain tissue and are altered when the cultures are performed in monolayer cultures, and (ii) due to a lack of fundamental understanding of the specific gene regulatory pathways governing cellular differentiation and behavior, most current in vitro differentiation protocols rely on trial and error to reprogram induced pluripotent stem cell (iPSC) into adopting specific cell fates and cellular functionalities
The enrichment of the gene signature was evaluated by gene set enrichment analysis (GSEA) [26] (v4.1.0) by choosing chip platform: Human_Gene_Symbol_with_Remapping_MSigDB.v7.4. chip, max size: 5000 and mix size: 1. The enrichment of pathway-specific gene signatures was evaluated with 1000 permutations
We identified and annotated major cell types of the human central nervous system including neural progenitor cells (NPCs; HES1 and SOX2), excitatory neurons (NEUROD1, NEUROD2 and SLA), interneurons (DLX1, GAD1 and GAD2), microglia (CD68 and PTPRC), astrocytes (GFAP, AQP4 and S100 calcium binding protein B (S100B)), oligodendrocyte precursor cells (OPCs; OLIG1 and OLIG2), radial glial cells (PAX6, VIM, NES and HES5), and glutamatergic (SLC17A7 and SLC32A1) and GABAergic neurons with cell-type-specific marker expression depicted in (Figures 1c and S1)
Summary
To address the integration challenges mentioned above, we used Harmony, a PCA-based method, which overall ranks higher in this integration benchmark with more robust batch integration and better conservation of biological variation This approach enables the generation of a unified and well-annotated map of the cellular diversity in the 3D brain organoids being investigated and an understanding of cell type-specific differentiation trajectories in organoids and fetal brain. We studied differential gene expression in gene sets related to axon guidance, axonogenesis, axon development, neuronal action potential, and neuronneuron synaptic transmission between in vitro organoids and in vivo fetal samples This comparative analysis enabled the accession of comprehensive correlations among the various differentiation methods and the resulting cellular functionality in matured organoids. It can provide researchers with a tool to tune differentiation protocols for achieving their desired functionality in 3D organoid cultures for tissue regeneration and disease modeling purposes
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
