Cultured Mesenchymal Cells from Nasal Turbinate as a Cellular Model of the Neurodevelopmental Component of Schizophrenia Etiology

Abstract

The study of neurodevelopmental molecular mechanisms in schizophrenia requires the development of adequate biological models such as patient-derived cells and their derivatives. We previously utilized cell lines with neural progenitor properties (CNON) derived from the superior or middle turbinates of patients with schizophrenia and control groups to study schizophrenia-specific gene expression. In this study, we analyzed single-cell RNA seq data from two CNON cell lines (one derived from an individual with schizophrenia (SCZ) and the other from a control group) and two biopsy samples from the middle turbinate (MT) (also from an individual with SCZ and a control). We compared our data with previously published data regarding the olfactory neuroepithelium and demonstrated that CNON originated from a single cell type present both in middle turbinate and the olfactory neuroepithelium and expressed in multiple markers of mesenchymal cells. To define the relatedness of CNON to the developing human brain, we also compared CNON datasets with scRNA-seq data derived from an embryonic brain and found that the expression profile of the CNON closely matched the expression profile one of the cell types in the embryonic brain. Finally, we evaluated the differences between SCZ and control samples to assess the utility and potential benefits of using CNON single-cell RNA seq to study the etiology of schizophrenia.

Keywords: mesenchymal cells; middle turbinate; neurodevelopment; scRNA-seq; schizophrenia.

Figure 1

Microscopic images of CNON cell (A) outgrowing from biopsy piece in Matrigel, (B) growing in 2D culture, and (C) growing in Matrigel after 2D culturing for several passages. All pictures are presented at the same magnification (objective 10×).

Figure 2

Single-cell reference mapping of CNON datasets (CNON-CTRL and CNON-SCZ) to human middle turbinate datasets (MT-CTRL and MT-SCZ) with cell cycle regression. (A) UMAP dimensionality reduction plot of 21,565 MT-CTRL cells displaying 13 distinct cell types (central panel). All cells from CTRL (left panel) and SCZ (right panel) are mapped to the MC cluster in MT-CTRL. All three datasets are shown in the same UMAP coordinates as MT-CTRL. (B) UMAP dimensionality reduction plot of 28,140 MT-CTRL cells with the same 13 annotated cell types as in MT-CTRL (central panel). CNON-CTRL cells are mapped exclusively to MC cluster (left panel); 3268 CNON-SCZ cells are mapped to MC cluster, while 35 cells are mapped to Basal cluster. (C) Heatmap representation of marker genes across different MT cell types.

Figure 3

(A) Average gene expression of selected cell markers in CNON. A gene is considered a MC marker gene if it is expressed in the MC cluster higher than in all other clusters with (a) statistical significance, (b) logFoldChange > 2, and (c) expressed in at least 50% of cells of MC. MC markers TAGLN, COL1A2, COL1A1, CALD1, TPM2, COL3A1, TPM1, and LGALS1; housekeeping markers GAPDH and ACTB; and the neural stem cell marker ITGB1 are shown in CNON-CTRL, CNON-SCZ, MT-CTRL, and the embryonic brain (sample CS14_3). The size of the dot represents the percentage of cells expressing the gene, and the colors represent the average expression level of each gene. (B) Single-cell reference mapping of CNON datasets to the olfactory neuroepithelium, Patient 2. Central panel shows UMAP dimensionality reduction plot of cells from the olfactory neuroepithelium of Patient 2 (25) with 13 cell types annotated. Left panel: UMAP dimensionality reduction plot of 11,425 CNON cells (CNON-CTRL); all cells mapped to the mesenchymal cell type. Right panel: UMAP dimensionality reduction plot of 2547 CNON cells (CNON-SCZ). The majority of cells (2420 CNON-SCZ cells) are mapped to the mesenchymal cell type, and 127 CNON-SCZ cells are mapped to the vascular smooth muscle cell clusters. (C) Single-cell reference mapping of CNON datasets to olfactory neuroepithelium dataset (integration of four patient sample data). Central panel shows UMAP dimensionality reduction plot of cells from the olfactory neuroepithelium, integrated data from four patients (25) with 13 cell types annotated. Left panel: UMAP dimensionality reduction plot of 10,979 CNON cells (CNON-CTRL); 10901 cells are mapped to the mesenchymal cell type, while 78 cells are mapped to the vascular smooth muscle cell clusters. Right panel: UMAP dimensionality reduction plot of 2075 CNON cells (CNON-SCZ). The majority of cells (1868 CNON-SCZ cells) are mapped to the mesenchymal cell type, and 207 CNON-SCZ cells are mapped to the vascular smooth muscle cell clusters. (D) Single-cell reference mapping of CNON datasets to embryonic brain (CS14_3). Central panel: UMAP dimensionality reduction plot of CS14_3 with 13 clusters. Left panel: UMAP dimensionality reduction plot of 12,234 CNON cells (CNON-CTRL); 12,233 cells are mapped to Cluster 9, 1 cell is mapped to cluster 0. Right panel: UMAP dimensionality reduction plot of 3303 CNON cells (CNON-SCZ). 3297cells are mapped to cluster 9, and 6 cells are mapped to cluster 0.

Figure 3

(A) Average gene expression of selected cell markers in CNON. A gene is considered a MC marker gene if it is expressed in the MC cluster higher than in all other clusters with (a) statistical significance, (b) logFoldChange > 2, and (c) expressed in at least 50% of cells of MC. MC markers TAGLN, COL1A2, COL1A1, CALD1, TPM2, COL3A1, TPM1, and LGALS1; housekeeping markers GAPDH and ACTB; and the neural stem cell marker ITGB1 are shown in CNON-CTRL, CNON-SCZ, MT-CTRL, and the embryonic brain (sample CS14_3). The size of the dot represents the percentage of cells expressing the gene, and the colors represent the average expression level of each gene. (B) Single-cell reference mapping of CNON datasets to the olfactory neuroepithelium, Patient 2. Central panel shows UMAP dimensionality reduction plot of cells from the olfactory neuroepithelium of Patient 2 (25) with 13 cell types annotated. Left panel: UMAP dimensionality reduction plot of 11,425 CNON cells (CNON-CTRL); all cells mapped to the mesenchymal cell type. Right panel: UMAP dimensionality reduction plot of 2547 CNON cells (CNON-SCZ). The majority of cells (2420 CNON-SCZ cells) are mapped to the mesenchymal cell type, and 127 CNON-SCZ cells are mapped to the vascular smooth muscle cell clusters. (C) Single-cell reference mapping of CNON datasets to olfactory neuroepithelium dataset (integration of four patient sample data). Central panel shows UMAP dimensionality reduction plot of cells from the olfactory neuroepithelium, integrated data from four patients (25) with 13 cell types annotated. Left panel: UMAP dimensionality reduction plot of 10,979 CNON cells (CNON-CTRL); 10901 cells are mapped to the mesenchymal cell type, while 78 cells are mapped to the vascular smooth muscle cell clusters. Right panel: UMAP dimensionality reduction plot of 2075 CNON cells (CNON-SCZ). The majority of cells (1868 CNON-SCZ cells) are mapped to the mesenchymal cell type, and 207 CNON-SCZ cells are mapped to the vascular smooth muscle cell clusters. (D) Single-cell reference mapping of CNON datasets to embryonic brain (CS14_3). Central panel: UMAP dimensionality reduction plot of CS14_3 with 13 clusters. Left panel: UMAP dimensionality reduction plot of 12,234 CNON cells (CNON-CTRL); 12,233 cells are mapped to Cluster 9, 1 cell is mapped to cluster 0. Right panel: UMAP dimensionality reduction plot of 3303 CNON cells (CNON-SCZ). 3297cells are mapped to cluster 9, and 6 cells are mapped to cluster 0.

Figure 4

Diagram of study design.