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

James A Gregory,Miguel Cuevas,Sally Temple,Emily Hoelzli,Kristen J Brennand,Catherine Braine,Madeline Halpern,Esther Cheng,Nadine Schrode,Steven Lotz,Kathryn Bowles,Natalie Barretto,Hemali Phatnani,Kristy Kang,Alison Goate,Celeste M Karch,Rawan Abdelaal,Susan Goderie,Güney Akbalik

doi:10.3390/cells9061406

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.

Highlights

The many cell types of the brain interact to influence each other’s function in health and disease [1].The growing list of rare and common risk loci associated with brain disease implicates a variety of cell types across disorders, with genetic risk sometimes enriched in cell types previously not thought to be related to disease pathology [2]
We demonstrate that RPL22-HA and RPL22-V5 IP efficiently enriched mRNA from mixed species co-cultures of immortalized cell lines
Our report demonstrates that restricting the expression of uniquely-tagged RPL22 (RiboTags) to specific cell types within a mixed co-culture allows for enrichment of cell type-specific translatomes

Summary

Introduction

The many cell types of the brain interact to influence each other’s function in health and disease [1].The growing list of rare and common risk loci associated with brain disease implicates a variety of cell types across disorders, with genetic risk sometimes enriched in cell types previously not thought to be related to disease pathology [2]. The many cell types of the brain interact to influence each other’s function in health and disease [1]. Non-neuronal and non-cell autonomous effects on neurons are increasingly recognized to play a critical role across psychiatric and neurodegenerative disease [3]. Human-induced pluripotent stem cell (hiPSC)-based models represent a powerful approach to study the complex etiology of brain disease [4]. Cells 2020, 9, 1406 complex in vitro models that better capture neuronal circuitry [5], astrocyte support [6], myelination [7], microglia [8], vasculature [9], and blood-brain-barrier functions [10]. Interrogating cell type-specific processes within increasingly complex networks, of cell types represented at low abundance, is difficult using conventional approaches

Methods

Results

Conclusion