Novel Tools and Investigative Approaches for the Study of Oligodendrocyte Precursor Cells (NG2-Glia) in CNS Development and Disease

Abstract

Oligodendrocyte progenitor cells (OPCs), also referred to as NG2-glia, are the most proliferative cell type in the adult central nervous system. While the primary role of OPCs is to serve as progenitors for oligodendrocytes, in recent years, it has become increasingly clear that OPCs fulfil a number of other functions. Indeed, independent of their role as stem cells, it is evident that OPCs can regulate the metabolic environment, directly interact with and modulate neuronal function, maintain the blood brain barrier (BBB) and regulate inflammation. In this review article, we discuss the state-of-the-art tools and investigative approaches being used to characterize the biology and function of OPCs. From functional genetic investigation to single cell sequencing and from lineage tracing to functional imaging, we discuss the important discoveries uncovered by these techniques, such as functional and spatial OPC heterogeneity, novel OPC marker genes, the interaction of OPCs with other cells types, and how OPCs integrate and respond to signals from neighboring cells. Finally, we review the use of in vitro assay to assess OPC functions. These methodologies promise to lead to ever greater understanding of this enigmatic cell type, which in turn will shed light on the pathogenesis and potential treatment strategies for a number of diseases, such as multiple sclerosis (MS) and gliomas.

Keywords: NG2-glia; genetic alteration; heterogeneity; imaging; oligodendrocyte precursor cells; sequencing.

Figure 1

Cre-LoxP based systems to study Oligodendrocyte progenitor cell (OPC) functions. (A) Fluorescent proteins can be specifically expressed in OPCs in a Cre-LoxP system where Cre-recombinase or Cre-ERT2 (Cre fused to a mutant estrogen ligand-binding domain) is expressed downstream of an OPC-specific marker, such as NG2 or PDGFRα. Upon Tamoxifen administration (for Cre-ERT2), Cre-recombinase excises a floxed stop signal, which then permits expression of fluorescent protein under the ubiquitous Rosa26 promoter. Fluorescently-labeled OPCs will transmit the excised allele to their progeny. Lineage tracing of labeled-NG2-glia showed that OPCs give rise primarily to MBP^+ve, MAG^+ve, MOG^+ve, PLP^+ve oligodendrocytes, but could also generate GFAP^+ve astrocytes or NeuN^+ve neurons. (B) Diphtheria toxin receptor (DTR) can be specifically expressed in OPCs by the Cre-LoxP system described above. Cre-recombinase excises a floxed stop signal, which then permits expression of DTR under the ubiquitous Rosa26 promoter. Injection of diphtheria toxin (DT) then elicits apoptotic cell death in DTR-expressing cells. In a non-inducible system, such as the one depicted, DTR will be expressed in NG2⁺ cells and their progeny which (in the case of OPCs) will result in cell death of OPCs but also any derived progeny, including oligodendrocytes. Such models must be validated for specificity of expression of DTR in the desired cell type, such as via immunostaining for DTR and OPC-specific markers, or by quantifying loss of OPCs and other cell types following DT-administration.

Figure 2

Methodologies for charaterizing OPC transcriptomics and heterogeneity. OPCs can be isolated from whole brain or spinal cord tissue. Manual dissection of different regions of the brain or spinal cord allows for anatomical comparisons. OPCs can be enriched and isolated from tissues via FACS (Fluorescence-activated cell sorting) or magnetic activated cell sorted (MACS, magnetic-activated cell sorting), based on expression of OPC-specific cell-surface markers, such as NG2 or PDGFRα. These sorted OPCs can then be processed and sequenced in bulk (bulk RNA-seq), or can be sequenced as single cells (scRNA-seq), yielding information on OPC heterogeneity and diversity. Alternatively, sequencing may be performed in situ on tissue sections [sc spatial RNA-seq or in situ sequencing (ISS)], producing single-cell transcriptomic data in a spatial context, and potentially mapping OPC heterogeneity to distinct anatomical regions.