Transplanted GFP-OECs in the center of the lesion core associate with numerous axons.

Sagittal sections show rostral and caudal glial scar borders of the injury site which are identified with glial fibrillary acidic protein (GFAP, blue). The GFAP-negative lesion core contains GFP-OECs (green) and is marked with asterisks. Arrowheads mark axons crossing into the lesion core. (a-f) Serotonergic axons (5-HT, red) are found in the rostral spinal cord stump and associate with OECs (green) in the lesion core. Single channels for GFAP (c), OECs (d), 5-HT (e), and a combination of 5-HT and OECs (f). (g-l) Nearby injury site section from the same rat (a). Numerous neurofilament-positive axons (white) are associated with the OECs (green) in the lesion core. Single channels for GFAP (i), OECs (j), neurofilament (k), and a combination of neurofilament and OECs (l). Scale bars: a, g = 500 µm; b, h = 100 µm. Reprinted from: Dixie, KL, 2019, UCLA. ProQuest ID: Dixie_ucla_0031D_18445.

scRNA-seq results show distinct clusters of OEC and leftover cell samples.

(a) Cells in tSNE plot colored by sample source, with cells from leftover samples in pink and cells from immunopurified OECs in cyan. (b) Clustering analysis revealed a total of 7 distinct cell clusters, each indicated with a different color. (c) A heatmap showing expression patterns of top marker genes of individual cell clusters. (d) Cell clusters showed distinct expression patterns for known cell type markers for fibroblasts, microglia, and OECs. Clusters 0, 1, and 5 had high expression of fibroblast markers and are labeled as fibroblast in the Y-axis; clusters 4, 6, and 7 showed high expression of microglia markers and are labeled as microglia; clusters 2, 3, and 7 showed high expression of OEC markers and are labeled as OEC. Known marker genes for different cell types are on the X-axis. Feature plots and violin plots for select marker genes are in Suppl. Figure 1. (e) Based on known cell type markers, cell clusters in tSNE plot were labeled with the corresponding cell types. (f) The expression of cell-type specific marker genes is depicted in a dot plot. (g) Genes that distinguish purified OECs vs OECs in leftover cultures are shown. The top 3 genes were higher in purified OECs, whereas the bottom 3 genes were higher in ‘leftover’ cultures.

Well-established OEC markers are revealed by scRNA-seq and immunofluorescence.

OEC cultures were replated from leftover cells prepared for scRNA-seq. Immunolabeled OECs are marked with arrows and all cell nuclei are stained with Hoechst (blue nuclei). tSNE maps of the gene expression in the 5 clusters are shown next to the protein expression in a-l. (a, b) Cultured OECs express Ngfrp75 protein and Ngfrp75 gene expression. (c, d) Blbp and Sox10 immunoreactive OECs with Fabp7 gene expression. (e, f) S100β-labeled OECs together with S100β expression. (g, h) L1cam and Sox 10 labeling next to L1cam expression. (i, j) Ncam1 protein and gene expression. (k, l) N-Cadherin and Sox10 markers with Cdh2 expression. (m-p) scRNA-seq data for Sox10, Cntf, Itga7, and Mpz (references in text). Scale bars: a, c, e, g, i, k = 50 µm.

OEC subclusters, marker genes, and enriched pathways.

(a) Clustering analysis of OECs revealed five separate clusters (0-4). (b) Heatmap depicts the expression patterns of known marker genes of glial cell types (x-axis) versus microglia, all OECs and OEC subclusters (y-axis). OEC subclusters express select markers of other glial cell types. (c) Heatmap depicts the top five marker genes (y-axis) for each OEC subcluster (x-axis). (d) Pathways associated with marker genes of different OEC clusters are shown. The dashed line indicates the false discovery rate (FDR) <0.05 in the pathway analysis. (e) Dot plot showing that cluster 3 has higher potential lysosomal function based on lysosome pathway genes than the other clusters. (f) Both cluster 3 and 4 express select genes involved in positive regulation of cell migration. (g) Trajectory analysis reveals two trajectories, one including subclusters 2, 0, and 4, and another involving subclusters 2, 0, 1, and 3. (h) NicheNet ligand-receptor network plot demonstrates intracellular communication between OEC subtypes. Hexagons represent subclusters 0 to 4 and circles indicate ligands secreted from subclusters. Edges (arrows) point to clusters where receptors of the ligands are expressed. Different colors represent different OEC subclusters.

Confirmation of Ccn2/Ctgf (Connective tissue growth factor) in cultured OECs and following OEC implantation after spinal cord injury.

(a, b) The scRNA-seq plots of the 9th (Ccn2/Ctgf) and 4th (Ccn3/Nov) highest-ranked marker genes are strongly expressed in subcluster 0. (c, c1, c2) The well-known matricellular protein Connective tissue growth factor (Ctgf) identifies OECs in cell cultures (red, c, c2), and Sox10 nuclear expression (white nuclei, c, c2) confirms they are neural crest-derived cells. (d, d1, d2) A sagittal section from a rat that received a complete spinal cord transection followed by Green Fluorescent Protein (GFP)-OEC implantation was fixed 2 weeks postinjury. Glial fibrotic acidic protein (d, Gfap, blue) marks the edges of the borders of the glial scar. GFP-OECs (green) that express Ctgf (red) in the injury site are outlined by the box in d. High expression of Ctgf is detected by GFP-OECs that bridge part of the injury site in d1. The single channel of Ctgf is shown in d2. Scale bars: c-c2 = 50 µm, d-d2 = 250 µm.

Subcluster 2 is characterized by cell cycle and proliferative markers.

(a-f) These scRNA-seq plots show high expression of cell proliferation markers in cluster 2, supporting their function as OEC progenitor cells. Only Stmn1 (e, Stathmin, a microtubule destabilizing protein) is broadly expressed across all five clusters. (g-g2, h-h2) Most cultured OECs are spindle-shaped and have high Ngfrp75 expression (red, white arrows). Of the OEC progenitors that express Ki67, 76% ± 8 of them display low levels of Ngfrp75 immunoreactivity and a “flat” morphology (g2, h2; green nuclei, arrowheads). The remainder of Ki67-expressing OECs express high levels of Ngfrp75 and are fusiform in shape (24% ± 8%, n=4 cultures, p= 0.023). Hoechst marks all nuclei (g1, h1, blue nuclei, arrowheads). Scale bars: g, h = 50 μm.

Spatial confirmation of the defined OECs subclusters within the olfactory nerve layer.

The protein expression of a number of top 20 genes from this scRNA-seq study of purified OEC cultures is verified in olfactory bulb sections. In all images, the olfactory nerve layer (ONL, layer I) is at the bottom of the image, the glomeruli (GL, layer II) next, and the remainder of the olfactory bulb toward the top. Suppl. figure 2 illustrates the corresponding tSNE plots for each gene in Figure 7. (a-c) The top gene in the largest cluster, Peripheral myelin protein 22, is highly expressed throughout the ONL. Gliomedin, the third-ranked gene in cluster 0, is detected as small discrete dot-like structures overlaying the ONL (b, box enlarged in c). (d-f) OECs in cluster 1 are immunolabeled by antibodies against the axonal growth factor Atf3 (d) and the intermediate filament Nestin (f). High levels of Gap43 in the ONL (e) are due to expression by OECs and axons of immature olfactory sensory neurons. (g-i) Strong immunoreactivity of Stathmin-1 in the ONL reflects labeled axons from immature olfactory sensory neurons and OECs. Ube2c, a G2/M cell cycle regulator, is expressed in a small number of cells in the ONL, whereas Mki67-labeled cells are widespread. (j-l) The immune function of this cluster is confirmed by antibodies against Apoe (j), Anxa3 (k), and Aif1/Iba1, markers expressed by microglia and macrophages. Scale bars: a, b, d-l = 50 µm; c, insets in j-l = 25 µm.

Reelin-signaling pathway marker genes in OEC clusters and Reelin immunoreactivity in embryonic olfactory system.

(a-f) These six tSNE plots show OEC gene expression levels associated with Reelin signaling. Reln is highly expressed in most OEC clusters (a), Disabled-1 (Dab1, b), and Apolipoprotein E receptor 2 (Apoer2, c) show little expression, and Very-low-density lipoprotein receptor has moderate expression (Vldlr, d). The serine protease tissue Plasminogen Activator (e, tPA) is highly expressed in all OEC subsets and cleaves secreted Reelin only at its C-terminus. ADAMTS-4 cleaves both the N- and C-terminals of Reelin, and is expressed at low levels by OECs (f). (g-g2) Sagittal section of the olfactory epithelium (OE) and olfactory bulb from an E16.5 Reln+/+mouse immunolabeled for Blbp (g, g1; red) and Reelin (g, g2; green). Axons of olfactory sensory neurons (arrows) are surrounded by peripheral OECs that express both Blbp (g1, arrowheads) and Reelin (g2, arrowheads). High Reln expression is obvious in olfactory bulb neurons (g, g1, green) but the olfactory nerve layer (ONL) only expresses Blbp (g, g2). (h, h1) No Reelin is detected in a sagittal section from a Reln-/- mouse, yet Blbp expression in the ONL appears normal. Scale bars: g-h = 50 µm; g1, g2 = 50 µm.

Reelin is expressed and secreted by olfactory ensheathing cells.

(a-c) Rat OEC cultures were treated with Brefeldin-A to inhibit protein transport and subsequent secretion. Reelin immunoreactivity (red) was detected in GFP-labeled OECs (a, arrows), but not in another cell type (arrowhead) in this primary culture. GFP-labeled OECs which were immunopurified with anti-Ngfrp75 also express Reelin (red; b, c). (d) Western blot confirms the expression of Reelin in rat olfactory nerve layer I and II (ONL; lane 1 of western blot). Reln+/+ and Reln-/- mouse olfactory bulbs were used as positive and negative controls, respectively (lanes: 2 and 3). Reelin synthesized by cultured OECs was found in whole cell lysates (WCL; lanes: 4, 6, and 8), whereas Reelin secreted by cultured OECs into tissue culture medium was measured in OEC “conditioned medium” (CM; lanes: 5 and 7). GAPDH was the loading control for tissue homogenates (lanes 1-4, 6, 8). (e) All three Reelin isoforms (400, 300, and 150 kDa) were visualized using a 4-15% gradient gel. Reln+/+ and Reln-/- mouse cortices were used as controls (lanes: 1 and 2). Reelin was detected in the rat ONL (lane 3) and in three rat OEC-CM samples (lanes: 4, 5, and 6). GAPDH was the loading control for tissue homogenates (lanes: 1-3). Scale bars: a-c = 40 μm.