Direct lineage conversion of postnatal mouse cortical astrocytes to oligodendrocyte lineage cells

Justine Bajohr
Erica Y Scott
Arman Olfat
Mehrshad Sadria
Kevin Lee
Maria Fahim
Hiba T Taha
Daniela Lozano Casasbuenas
Ann Derham
Scott A Yuzwa
Gary D Bader
Maryam Faiz author has email address

Department of Surgery, Temerty Faculty of Medicine; University of Toronto; Toronto, Ontario, M5S 1A8, Canada
Institute of Medical Science, Temerty Faculty of Medicine; University of Toronto; Toronto, Ontario, M5S 1A8, Canada
Institute of Biomedical Engineering, Faculty of Applied Science and Engineering; University of Toronto; Toronto, Ontario, M5S 3G9, Canada
Department of Chemistry, Faculty of Arts and Science; University of Toronto; Toronto, Ontario, M5S 3G3, Canada
Donnelly Centre for Cellular and Biomolecular Research, Temerty Faculty of Medicine; University of Toronto; Toronto, Ontario, M5S 3E, Canada
Department of Applied Mathematics, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
Department of Molecular Genetics, Temerty Faculty of Medicine; University of Toronto; Toronto, Ontario, M5S 1A8

https://doi.org/10.7554/eLife.98632.1

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Figures and data

Sox10, Olig2 and Nkx6.2 convert GFAP+ cells to oligodendrocyte lineage cells.
(A) Experimental design and timeline. (B) Quantification of PDGFRα⁺tdTomato⁺ iOPCs, O4⁺tdTomato⁺ iCOPs and MBP⁺tdTomato⁺ iOLs at 8 (n=5), 10 (n=4), 12 (n=5) and 14 (n=7) days post transduction (DPT). Data are presented as mean ± SEM, each data point represents one individual cell culture experiment; at each time point and for each cell type marker, a matched pairs one-way ANOVA or Kruskal-Wallis (d8, d10, d12) or one-way ANOVA (d14) was performed with Dunnet’s post testing (*= p<0.05, *** = p< 0.001). (C) Representative images of PDGFRα⁺tdTomato⁺,O4⁺tdTomato⁺ and MBP⁺ tdTomato⁺ cells at 12DPT. Single channel images are shown of the boxed cells (arrows indicate double positive cells). (D) UMAP clustering of Olig2-, Sox10-, Nkx6.2- and control (Cre) transduced cells at 14DPT (E) UMAP of (D) overlayed with treatment (Olig2-, Sox10-, Nkx6.2- and control (Cre)) (F) Proportion analysis of clusters found in Olig2-, Sox10-, Nkx6.2-, and control (Cre) transduced cells. (G) Heatmap of top upregulated genes from each cluster in (D).

Lineage tracing confirms true conversion of astrocytes to oligodendrocyte lineage cells.
(A) Experimental design, timeline and outcomes. (B) Quantification of tdTomato⁺zsGreen⁺PDGFRα⁺ iOPCs at 12DPT (n= 3). Data are presented as mean ± SEM, each data point represents one individual cell culture, a paired t-test was performed (** = p<0.01). (C) Quantification of tdTomato⁺zsGreen⁺O4⁺ iCOPs at 14DPT (n = 4). Data are presented as mean ± SEM, each data point represents one individual cell culture, a Wilcoxon test was performed (ns). (D) Quantification of tdTomato⁺zsGreen⁺MBP⁺ iOLs at 12DPT (n= 4 for Sox10, Nkx6.2, n= 3 for Cre). Data are presented as mean ± SEM, each data point represents one individual cell culture, a one-way ANOVA with Dunnet’s post testing was performed (* = p<0.05). (E) Representative image of PDGFRα⁺tdTomato⁺zsGreen⁺ cells 12DPT. Single channel images are shown of the boxed cells (arrows indicate triple positive cells, scale bar = 50um (merge) and 20um (single channel)). (F) Representative image of MBP⁺tdTomato⁺zsGreen⁺ cells 12DPT. Single channel images are shown of the boxed cells (arrows indicate triple positive cells, scale bar = 50um (merge) and 20um (single channel)). (G) Representative tdTomato⁺zsGreen⁺ cell with astrocyte-like morphology at onset (7DPT) and OLC expression at the end (12DPT) of live cell tracking (arrow indicates tracked cell, scale bar = 50um).

Characterization of DLR using scRNA-seq shows terminal oligodendrocyte cluster of cells at day 14 driven by Sox10.
(A) UMAP clustering of Sox10 and Cre control treated cells from prior to transduction, as well as 3, 8 and 14DPT (B) Canonical astrocyte gene expression (log-normalized, y-axis) separated by cluster (x-axis) and coloured by timepoint and treatment group. (C) Canonical gene expression (log-normalized, y-axis) of NG2 glia, VLMC, OLC, microglia and proliferation, separated by cluster (x-axis) and coloured by timepoint and treatment group. (D) UMAP clustering from (A) overlayed with timepoint and treatment group (E) Slingshot lineage analysis of Sox10 and Cre control treated clusters overlaid with UMAP embeddings. (F) Monocle3 lineage analysis of Sox10 and Cre control treated clusters overlaid on UMAP plot and coloured by pseudotime predictions. (G) The number of cells (y-axis) per cluster (x-axis) originating from each of the coloured timepoint+treatment groups. (H) CellOracle modeling of in silico Sox10 knock out (KO) overlaid onto UMAP plot. Arrows indicate trajectory prediction with Sox10 KO. (I) Cell Oracle modeling of in silico Sox10 knock in (KI) overlaid onto UMAP plot. Arrows indicate trajectory prediction with Sox10 KI (J) Clustered, top differentially expressed genes (dots, y-axis) between 14DPT Sox10 (D14_S, Beige, left) and Cre (D14_C, Blue, right) control treated cells from cluster 6. Size of dot is scaled to percent of cells in the cluster expressing that gene, colour of dot represents the average scaled expression of the gene across cells. (K) Predicted cell types following Fatecode perturbation on node 16 of the latent layer in the Sox10 and control treated dataset. Blue represents the number of cells prior to perturbation. Orange (overlayed) represents the number of cells following perturbation.

14DPT scRNA-seq processing for initial UMAP generation.
(A) Distribution of genes (nFeature), reads (nCount) and percent of mitochondrial reads (percent.mt) per sample. (B) UMAP clustering prior to removal of microglia cluster. (C) Top upregulated genes characterizing each cluster observed in (B).

Characterization of DLR cultures.
(A) Quantification of contaminating iOLCs in post-natal astrocyte cultures 3DPT. (B) Quantification of MBP expression in tdTomato⁺ transduced cells 22DPT (n=3, data are presented as mean ± SEM, each data point represents one individual cell culture, a matched pairs one way ANOVA was performed (ns)).

Comparison of DLR clusters 14DPT to established datasets of OLCs.
(A) Violin plots of cell types annotated by Marques et al. 2016 within each cluster (OPC = oligodendrocyte progenitor cells, COPs = committed oligodendrocyte progenitors, NFOL = newly formed oligodendrocyte, MFOL = myelin forming oligodendrocyte, mOL = mature oligodendrocyte).(B) Violin plots of cell types annotated by Dugas et al. 2006 within each cluster (OL = oligodendrocyte, OPC = oligodendrocyte progenitor cells).

(A) Quantification of (i) tdTomato⁺ and (ii) zsGreen⁺ PDGFRα⁺ iOPCs at 12DPT (n= 3). Data are presented as mean ± SEM, each data point represents one individual cell culture, a paired t-test for (i) and a Wilcoxon test for (ii) was performed (** = p<0.01). (B) Quantification of (i) tdTomato⁺ and (ii) zsGreen⁺ O4⁺ iCOPs at 14DPT (n = 4). Data are presented as mean ± SEM, each data point represents one individual cell culture, a paired t-test was used. (C) Quantification of (i) tdTomato⁺ (ii) zsGreen⁺ MBP⁺ OLs at 12DPT (n= 3). Data are presented as mean ± SEM, each data point represents one individual cell culture, a matched pairs one way ANOVA with Geisser –Greenhouse correction and Dunnet’s post testing was used (* = p<0.05). (D) Representative tdTomato^negzsGreen⁺ cell with astrocyte-like morphology at onset (7DPT) and OLC expression at the end (12DPT) of live cell tracking (arrow indicates tracked cell, scale bar = 50um).

DLR timecourse scRNA-seq processing for initial UMAP generation.
(A) Distribution of genes (nFeature), reads (nCount) and percent of mitochondrial reads (percent.mt) per sample. (B) UMAP clustering prior to removal of microglia and VLMC clusters. (C) Top upregulated genes characterizing each cluster observed in (B).

Differential gene expression of clusters in the Monocle3 trajectory.
(A) Genes enriched in leaf 14 of Monocle3 trajectory. Cells in UMAP colored according to log-normalized gene expression values. (B) Genes enriched in leaf 11 of Monocle3 trajectory. Cells in UMAP colored according to log-normalized gene expression values.

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