Sigle-cell transcriptome analysis of EpiSC-derived neural stem cell lines derived from EpiSCs under different Wnt signal inputs
The four neural stem cell lines, XN4, XN1, KN1, and CN4, were derived from EpiSCs under different Wnt signal inputs (Nakamura et al., to be submitted). Microarray analysis of the transcripts indicated that these cells have different anteroposterior regional characteristics of the embryonic CNS from the forebrain to the thoracic spinal cord. However, each cell line has a relatively broad anteroposterior regional coverage. For instance, the XN1 line expressed midbrain-characteristic genes and hindbrain-characteristic genes. Single-cell transcriptome analysis of the four cell lines was performed to confirm that the broad anteroposterior specificity is intrinsic to individual cells of the cell line rather than the cell lines are composite of separable subpopulations with narrower anteroposterior specificities. Overall design: The NSC lines, XN4, XN1, KN1, and CN4, in a growing phase, were dissociated using Accutase, passed through a 20 µm cell strainer, suspended in 10x volumes of CellCover (Anacyte Laboratories), and stored at 4º C, up to 2 weeks, until cells were processed for the single-cell transcriptome library preparation. After washing in 50% DMEM /50% Neurobasal Medium with 0.004% BSA, the cells were loaded on a BD Rhapsody cartridge (BD Biosciences) to capture mRNAs of cells on isolated magnetic beads. Single-cell RNA sequencing libraries were prepared for each NSC line using a BD Rhapsody WTA Amplification kit (633801) using the manufacturer-supplied protocol. The libraries were sequenced using Hi-SeqX (Illumina) for 150 b paired-end reads to gain Fastq data. The sequence data were processed using the BD Rhapsody WTA pipeline on the Seven Bridge Genomics platform. R1 reads were used to gain transcript data in individual cells and R2 to align them on the mouse mm10 (GRCm38) genome sequence.The single-cell transcriptome analysis was performed for the two purposes: (1) to show that a significant fraction of cells simultaneously express genes representing different CNS regions and (2) to show that the cells in a cell line form an inseparable cell group in the UMAP projection of the data. For the first purpose, the reads per cell spreadsheet data (RPC.csv) were directly analyzed. For the second purpose, transcript reads of ribosomal and mitochondrial proteins were omitted from the analysis because the cells were nearly homogeneous, and the reads of these transcripts were found to erroneously contribute to sub-clustering the cells. Then 4000 cells for each line were randomly selected, normalized, and scaled according to the standard procedure of Seurat (v. 4.3.0) (Satija et al., 2015).