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Culture Protocol and Transcriptomic Analysis of Murine SVZ NPCs and OPCs

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Abstract

The mammalian adult brain contains two neural stem and precursor (NPC) niches: the subventricular zone [SVZ] lining the lateral ventricles and the subgranular zone [SGZ] in the hippocampus. From these, SVZ NPCs represent the largest NPC pool. While SGZ NPCs typically only produce neurons and astrocytes, SVZ NPCs produce neurons, astrocytes and oligodendrocytes throughout life. Of particular importance is the generation and replacement of oligodendrocytes, the only myelinating cells of the central nervous system (CNS). SVZ NPCs contribute to myelination by regenerating the parenchymal oligodendrocyte precursor cell (OPC) pool and by differentiating into oligodendrocytes in the developing and demyelinated brain. The neurosphere assay has been widely adopted by the scientific community to facilitate the study of NPCs in vitro. Here, we present a streamlined protocol for culturing postnatal and adult SVZ NPCs and OPCs from primary neurosphere cells. We characterize the purity and differentiation potential as well as provide RNA-sequencing profiles of postnatal SVZ NPCs, postnatal SVZ OPCs and adult SVZ NPCs. We show that primary neurospheres cells generated from postnatal and adult SVZ differentiate into neurons, astrocytes and oligodendrocytes concurrently and at comparable levels. SVZ OPCs are generated by subjecting primary neurosphere cells to OPC growth factors fibroblast growth factor (FGF) and platelet-derived growth factor-AA (PDGF-AA). We further show SVZ OPCs can differentiate into oligodendrocytes in the absence and presence of thyroid hormone T3. Transcriptomic analysis confirmed the identities of each cell population and revealed novel immune and signalling pathways expressed in an age and cell type specific manner.

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Data availability (Data Transparency)

The datasets generated and analysed during the current study are available in the GEO repository (GSE217507).

Code availability (Software Application or Custom Code)

Open-source and publicly available software tools were used for the analysis of data herein and no custom code or tools were generated. All software tools are listed in Material & Methods. Analysis scripts are available from the corresponding author upon reasonable requests.

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Acknowledgements

This work was supported by the Canada Research Chairs, Natural Sciences and Engineering Research Council of Canada (NSERC) grants (232418, RGPIN-2018-04669) awarded to AV. NLD was supported by Brad Mates E Drive Studentship in Parkinson's Disease and Movement Disorders and NSERC PGS-D; PT by NSERC USRA, UAlberta Neuroscience and Mental Health Institute as well as URI studentships; and AESW by Multiple Sclerosis Society of Canada Master’s [3821], WCHRI Graduate and CIHR CGSD Scholarships. The authors wish to thank Kin Chan for the RNA-Seq service performed at the Network Biology Collaborative Centre (nbcc.lunenfeld.ca), a facility supported by grants from Canada Foundation for Innovation, the Ontarian Government, and Genome Canada and Ontario Genomics (OGI-139). We wish to thank Beatrix Wang for technical assistance and Dr. Monique de Almeida for reading the manuscript.

Funding

This work was supported by the Canada Research Chairs and Natural Sciences and Engineering Research Council of Canada (NSERC) grants (232418, RGPIN-2018–04669).

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Authors and Affiliations

  1. Department of Medical Genetics, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, T6G 2H7, Canada

    Nicole L. Dittmann, Pouria Torabi, Adrianne E. S. Watson & Anastassia Voronova

  2. Neuroscience and Mental Health Institute, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, T6G 2E1, Canada

    Nicole L. Dittmann & Anastassia Voronova

  3. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, M5S 1A8, Canada

    Scott A. Yuzwa

  4. Women and Children’s Health Research Institute5-083 Edmonton Clinic Health Academy, University of Alberta, 11405 87 Avenue NW, Edmonton, Alberta, T6G 1C9, Canada

    Anastassia Voronova

  5. Department of Cell Biology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, T6G 2H7, Canada

    Anastassia Voronova

  6. Multiple Sclerosis Centre, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, T6G 2H7, Canada

    Anastassia Voronova

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Contributions

Conceptualization, NLD and AV.; Methodology, NLD, PT, AESW, SY, AV.; Formal analysis, NLD, PT, SY; Investigation, NLD, PT, AESW, AV; Resources, AV; Writing – original draft, NLD and AV; Writing – review & editing, NLD and AV; Supervision, AV; Funding acquisition, AV.

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Dittmann, N.L., Torabi, P., Watson, A.E.S. et al. Culture Protocol and Transcriptomic Analysis of Murine SVZ NPCs and OPCs. Stem Cell Rev and Rep 19, 983–1000 (2023). https://doi.org/10.1007/s12015-022-10492-z

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