Single-cell transcriptome analyses reveal signals to activate dormant neural stem cells

Abstract

The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133(+)/GFAP(-) ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133(+)/GFAP(-) quiescent cells were enriched for immune-responsive genes, as well as genes encoding receptors for angiogenic factors. Administration of vascular endothelial growth factor (VEGF) activated CD133(+) ependymal neural stem cells (NSCs), lining not only the lateral but also the fourth ventricles and, together with basic fibroblast growth factor (bFGF), elicited subsequent neural lineage differentiation and migration. This study revealed the existence of dormant ependymal NSCs throughout the ventricular surface of the CNS, as well as signals abundant after injury for their activation.

Figure 1. Quality Controls of Single-Cell RNA-Seq Analysis

(A) Quadruplicate independent sequencing of the same cDNA sample demonstrating good reproducibility as shown by the Pearson correlation coefficient being 0.996–0.997 among all four sequencing results. P, pooled sample. (B) The same single-cell cDNA sample (S10) was subjected to two independent sequencings in two batches, with the exact sequencing time being 6 months apart. High Pearson correlation coefficient (0.998) indicates minimum sequencing batch effect and, therefore, reliable sequencing technology. TR, technical repetition. (C) Estimation of technological and biological variations of single-cell sequencing analysis. Two-cell-stage mouse embryos were sequenced either as a whole or separately as individual cells. Pearson correlation coefficients among the four datasets are between 0.901 and 0.95. Assuming these cells are identical, our technical variation is ranging between 0.05 and 0.1. E, embryo. (D) Pearson correlation coefficient of the three pairs of half-cell RNA-seq. (E) Saturation curves of RNA-seq data with FPKM ≥ 0.1. (F) Number of genes detected in pooled samples and single-cell samples (FPKM ≥ 0.1). See also Figures S1, S2, S3, and S4 and Table S1.

Figure 2. WGCNA Revealed Gene-Network Modules Enriched in CD133⁺ E Cells, GFAP⁺/CD133⁺ B Cells, DLX2⁺ A Cells, and SOX10⁺/OLIG1⁺ O Cells

(A) Unsupervised hierarchical clustering of 28 single-cell samples and four pooled-cell datasets with FPKM cutoff of 0.1. (B) WGCNA dendrogram indicating expression of different gene modules in all 28 single-cell samples. (C) Eigengene expression of four selected modules across all 28 single-cell samples. Color code of the modules is preserved. Expressions of a few known markers for E, B, O, and A cells were also shown in 28 samples. Based on these two sets of information, it is obvious that the blue module has enriched expression in 8 CD133 single-positive cells, the brown module is expressed in 3 O cells, the red module is highly expressed in 11 DLX2⁺ A cells, and the royal blue module is expressed in 2 GFAP⁺ and CD133⁺ B cells. (D) Expanded view of expression of all genes in each of the four modules across all 28 single-cell samples. (E) Hub-gene network of the red module (the A module). Size of the dots represents hubness. Red highlights the genes known for neurogenesis. (F) Hub-gene network of the brown module (the O module). Size of the dots represents hubness. Brown highlights the genes known for oligodendrocyte differentiation. See also Figure S5.

Figure 3. Mitotic Activation of Ependymal CD133⁺ Cells by VEGF

(A) Hub-gene network of the blue module (the CD133-specific E module). The size of the dots represents hubness. Blue highlights the genes being discussed in the text. (B) A representative sagittal section from the adult SVZ region stained with CD133 (red) and Flt1 (green). (C–F) Fluorescent photomicrographs of representative sagittal sections from the adult SVZ region stained with CD133 (red) and the proliferation marker Ki67 (green). The animals were injected with saline (C), bFGF (D), VEGF (E), or bFGF + VEGF (F). Arrows and the insets in (E) and (F) indicate CD133⁺/Ki67⁺ ependymal cells from the same panels. (G) A representative sagittal section from the SVZ region of adult mouse that was injected with bFGF + VEGF followed by 7-day oral BrdU administration. BrdU⁺ nuclei (red) are present within the SVZ. The arrows and the lower inset show BrdU⁺ nuclei within the ependymal layer from the same panel. The upper inset is taken from a different section and shows additional BrdU⁺ cells in the ependymal layer adjacent to the lateral ventricle. (H) The percentage of Ki67⁺ cells in the subependymal region under saline, bFGF+VEGF−, bFGF−VEGF+, and bFGF+VEGF+ conditions. (I) The percentage of Ki67⁺/CD133⁺ ependymal cells under saline, bFGF+VEGF−, bFGF−VEGF+, and bFGF+VEGF+ conditions. (J) Quantification of BrdU⁺ cells in the ependymal layer under saline, bFGF+VEGF−, bFGF-VEGF+, and bFGF+VEGF+ conditions. Scale bars, 100 µm in (B)-(G). Error bars indicate the SD from three different experiments. *p < 0.05; **p < 0.01. CC, corpus callosum; cp, choroid plexus; LV, lateral ventricle.

Figure 4. Labeling of the Progeny of CD133⁺ Cells Surrounding the Lateral Ventricles

(A) A diagram illustrating the injection of mP2 plasmid into the lateral ventricle of ROSA26-tdTomato animals followed by electroporation. (B–E) Sagittal sections of P14 ROSA26-tdTomato reporter mice showing tdTomato⁺ cells (red) around the lateral ventricle; the animals were injected with mP2 followed by electroporation at P7. Arrows in (E) point out migrating tdTomato⁺ cells in the RMS. (F–I) Fluorescent images of ROSA26-tdTomato forebrain sections stained with CD133 (F, green), GFAP (Gand H, green), and doublecortin (I, green). The arrows in (F), (H), and (I) denote tdTomato⁺ cells that are immunoreactive for CD133, GFAP, and doublecortin, respectively. Scale bars, 250 µm in (B) and (C); 50 µm in (D)–(H); and 40 µm in (I). CC, corpus callosum, LV, lateral ventricle, cp, choroid plexus.

Figure 5. Activation of Dormant CD133⁺ NSCs in the Fourth Ventricle by Treatment of FGF and VEGF

(A) A sagittal section of adult fourth ventricular region stained with CD133 (red). (B–D) Immunostained sections of the postnatal fourth ventricular region from control (B, saline) and experimental (C, bFGF, and D, bFGF + VEGF administration) groups showing numerous proliferating Ki67⁺ cells (green, arrows). (E) A fluorescent image showing BrdU⁺ cells (green) double-labeled with CD133 (red) lining the fourth ventricle following the local administration of bFGF and VEGF. (F) Diagram illustrating the experimental paradigm of CD133⁺ ependymal cell lineage-tracing studies. The prominin 1 minimum promoter-driven Cre construct used for electroporation is shown. (G–L) Sagittal sections of P14 ROSA26-tdTomato reporter mice showing tdTomato⁺ cells (red) around the fourth ventricle; the animals were injected with mP2 + saline (G), mP2 + bFGF (H), or mP2 + FGF + VEGF (I–L) followed by electroporation at P7. The inset in (I) shows the higher magnification of tdTomato⁺ cells. (K and L) Fluorescent images of ROSA26-tdTomato mice stained with CD133 showing tdTomato⁺ (red) and CD133⁺ (green) cells lining the fourth ventricle following the injection of FGF + VEGF and electroporation of mP2. (M–P) tdTomato⁺ cells (red) in the parenchyma of ROSA26-tdTomato reporter mice at P21; the animals were injected with mP2 + FGF + VEGF followed by electroporation at P7. The bracket in (M) indicates a group of cells at the ependymal layer expressing tdTomato, while arrows point to some of the apparently migrating tdTomato⁺ cells. The arrows in (N) denote tdTomato⁺ cells with bushy processes resembling astrocytes. (O) and (P) show tdTomato⁺ cells expressing MAP2 or GFAP, respectively. Scale bars, 100 µm in (A) and (G)–(J); 40 µm in (B)–(E); and 75 µm in (M)–(P). 4V, fourth ventricle; cb, cerebellum; cp, choroid plexus.