Figure 1.

Identification of the effect of HFS on cultured human astrocytes using single cell RNA-Seq and verification of the single cell analysis using qRT-PCR. (A) Experimental design. Culture media was changed one day before the stimulation. HFS (100 Hz, 100 μsec pulse width, 1.5–2.5 V) was applied for 24 hrs. (B) Schematic of the stimulation device. A pair of platinum wires were placed in parallel through the entire chamber of a 6-well plate. The wire was coated with silicon, and only the part across the wall was exposed. The bare wire nearly touched the bottom of the culture plate and the media fully covered the wire. (C) Heatmap of expression (Global Z-score) obtained by unsupervised two dimensional hierarchical clustering analysis using the most significant 100 genes. According to the gene expression profile, three clusters were identified. In C1, cells from both stimulated and control groups were evenly distributed. Cells in C2 and C3 were mainly from the stimulated and the control groups, respectively. (D) Principal component analysis (PCA) plot using all expressed genes of 111 cells (52 stimulated cells identified in C2 and 59 control cells identified in C3). PCA confirmed that gene expression profiling modulated by stimulation is strong enough to divide cells into two groups (stimulated vs. control). (E) qPCR assay to verify the data obtained by scRNA-Seq. Nine candidate genes were identified and confirmed by qPCR assay independently (***, p<0.001. **, p<0.01, *, p<0.05). Abbreviaions: GREM1, gremlin1; IGFBP, insulin like growth factor binding protein; CYR61, cysteine rich angiogenic inducer 61 (has been identified as CTGF-2 and also IGFBP10); STC2, stanniocalcin2; CTGF, connective tissue growth factor (has been identified as IGFBP8); TGFB2, Transforming growth factor beta 2; PAPPA, pregnancy-associated plasma protein A; THBS1, thrombospondin1.