ABSTRACT
The division potential of individual stem cells and the molecular consequences of successive rounds of proliferation remain largely unknown. Here, we developed an inducible cell division counter (iCOUNT) that reports cell division events in human and mouse tissues in vitro and in vivo. Analyzing cell division histories of neural stem/progenitor cells (NSPCs) in the developing and adult brain, we show that iCOUNT can provide novel insights into stem cell behavior. Further, we use single-cell RNA sequencing (scRNA-seq) of iCOUNT-labeled NSPCs and their progenies from the developing mouse cortex and forebrain-regionalized human organoids to identify functionally relevant molecular pathways that are commonly regulated between mouse and human cells, depending on individual cell division histories. Thus, we developed a tool to characterize the molecular consequences of repeated cell divisions of stem cells that allows an analysis of the cellular principles underlying tissue formation, homeostasis, and repair.
Subject(s)
Neural Stem Cells , Animals , Brain , Cell Division , Cell Proliferation , Mice , Organoids , Sequence Analysis, RNAABSTRACT
Neural stem cells (NSCs) generate neurons throughout life in the mammalian hippocampus. However, the potential for long-term self-renewal of individual NSCs within the adult brain remains unclear. We used two-photon microscopy and followed NSCs that were genetically labeled through conditional recombination driven by the regulatory elements of the stem cell-expressed genes GLI family zinc finger 1 (Gli1) or achaete-scute homolog 1 (Ascl1). Through intravital imaging of NSCs and their progeny, we identify a population of Gli1-targeted NSCs showing long-term self-renewal in the adult hippocampus. In contrast, once activated, Ascl1-targeted NSCs undergo limited proliferative activity before they become exhausted. Using single-cell RNA sequencing, we show that Gli1- and Ascl1-targeted cells have highly similar yet distinct transcriptional profiles, supporting the existence of heterogeneous NSC populations with diverse behavioral properties. Thus, we here identify long-term self-renewing NSCs that contribute to the generation of new neurons in the adult hippocampus.
Subject(s)
Hippocampus/growth & development , Neural Stem Cells/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors/biosynthesis , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Lineage , Female , Gene Expression Profiling , Hippocampus/cytology , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/genetics , Intravital Microscopy , Male , Metallothionein 3 , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Fluorescence, Multiphoton , Nerve Regeneration , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Single-Cell Analysis , Zinc Finger Protein GLI1/biosynthesis , Zinc Finger Protein GLI1/geneticsABSTRACT
This protocol presents a plate-based workflow to perform RNA sequencing analysis of single cells/nuclei using Smart-seq2. We describe (1) the dissociation procedures for cell/nucleus isolation from the mouse brain and human organoids, (2) the flow sorting of single cells/nuclei into 384-well plates, and (3) the preparation of libraries following miniaturization of the Smart-seq2 protocol using a liquid-handling robot. This pipeline allows for the reliable, high-throughput, and cost-effective preparation of mouse and human samples for full-length deep single-cell/nucleus RNA sequencing. For complete details on the use and execution of this protocol, please refer to Bowers et al. (2020).
Subject(s)
Sequence Analysis, RNA/instrumentation , Single-Cell Analysis/instrumentation , Single-Cell Analysis/methods , Animals , Base Sequence/genetics , Brain/cytology , Brain/metabolism , Cell Nucleus/metabolism , Cell Separation/methods , Gene Expression/genetics , Gene Expression Profiling/methods , Gene Library , High-Throughput Nucleotide Sequencing/methods , Humans , Mice , Miniaturization , RNA/genetics , Sequence Analysis, RNA/methods , Transcriptome/genetics , Exome Sequencing/methods , WorkflowABSTRACT
Altered neural stem/progenitor cell (NSPC) activity and neurodevelopmental defects are linked to intellectual disability. However, it remains unclear whether altered metabolism, a key regulator of NSPC activity, disrupts human neurogenesis and potentially contributes to cognitive defects. We investigated links between lipid metabolism and cognitive function in mice and human embryonic stem cells (hESCs) expressing mutant fatty acid synthase (FASN; R1819W), a metabolic regulator of rodent NSPC activity recently identified in humans with intellectual disability. Mice homozygous for the FASN R1812W variant have impaired adult hippocampal NSPC activity and cognitive defects because of lipid accumulation in NSPCs and subsequent lipogenic ER stress. Homozygous FASN R1819W hESC-derived NSPCs show reduced rates of proliferation in embryonic 2D cultures and 3D forebrain regionalized organoids, consistent with a developmental phenotype. These data from adult mouse models and in vitro models of human brain development suggest that altered lipid metabolism contributes to intellectual disability.
