Cell Type-Specific In Vitro Gene Expression Profiling of Stem Cell-Derived Neural Models

Abstract

Genetic and genomic studies of brain disease increasingly demonstrate disease-associated interactions between the cell types of the brain. Increasingly complex and more physiologically relevant human-induced pluripotent stem cell (hiPSC)-based models better explore the molecular mechanisms underlying disease but also challenge our ability to resolve cell type-specific perturbations. Here, we report an extension of the RiboTag system, first developed to achieve cell type-restricted expression of epitope-tagged ribosomal protein (RPL22) in mouse tissue, to a variety of in vitro applications, including immortalized cell lines, primary mouse astrocytes, and hiPSC-derived neurons. RiboTag expression enables depletion of up to 87 percent of off-target RNA in mixed species co-cultures. Nonetheless, depletion efficiency varies across independent experimental replicates, particularly for hiPSC-derived motor neurons. The challenges and potential of implementing RiboTags in complex in vitro cultures are discussed.

Enhanced oxidative phosphorylation, re-organized intracellular signaling, and epigenetic de-silencing as revealed by oligodendrocyte translatome analysis after contusive spinal cord injury

“…Such a contamination may originate from non-specific interactions between highly abundant non-OL mRNAs and IgG-coupled magnetic beads that occurs during sample preparation. Similar contamination has been recently reported in co-cultures of human and mouse neural cells 25 . The two-fold filtration process that took into account differential or constantly high OL enrichment reduced that contamination and identified high confidence components of the OL translatome that are regulated by SCI.…”

“…3 b). Such findings suggest that some mRNAs that are present in OL translatome represent a contamination from non-OL cells, as recently shown in heterologous culture systems 25 . Such a non-specific co-purification with cell type-tagged ribosomes may be particularly relevant acutely after CNS injury when tissue cellularity changes and some mRNAs such as those expressed by the inflammatory cells become extremely abundant 10 .…”

Enhanced oxidative phosphorylation, re-organized intracellular signaling, and epigenetic de-silencing as revealed by oligodendrocyte translatome analysis after contusive spinal cord injury

“…Such a contamination may originate from non-speci c interactions between highly abundant non-OL mRNAs and IgG-coupled magnetic beads that occurs during sample preparation. Similar contamination has been recently reported in co-cultures of human and mouse neural cells 23 . The two-fold ltration process that took into account differential or constantly high OL enrichment reduced that contamination and identi ed high con dence components of the OL translatome that are regulated by SCI.…”

“…3b). Such ndings suggest that some mRNAs that are present in OL tranlsatome represent a contamination from non-OL cells, as recently shown in heterologous culture systems 23 . Such a non-speci c co-puri cation with cell type-tagged ribosomes may be particularly relevant acutely after CNS injury when tissue cellularity changes and some mRNAs such as those expressed by the in ammatory cells become extremely abundant 10 .…”

Human tau mutations in cerebral organoids induce a progressive dyshomeostasis of cholesterol

“…iPSCs used in this study were established from the Tau Consortium iPSC line collection ( Karch et al., 2019 ), grown at NSCI core facility NeuraCell, and are available upon request ( www.neuralsci.org/tau ). hCOs were generated using described protocols ( Yoon et al., 2019 ; Gregory et al., 2020 ). For scRNA-seq, three to four hCOs were pooled for dissociation, and drop-seq was performed as described ( Macosko et al., 2015 ).…”