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Multipotent neural stem cells originating from neuroepithelium exist outside of the mouse central nervous system [scRNA-seq]

ABSTRACT: Multipotent neural stem cells originating from neuroepithelium exist outside of the mouse central nervous system [scRNA-seq]

PROVIDER: PRJNA879225 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR21526562_1.fastq.gz	Fastqsanger.gz
	SRR21526562_2.fastq.gz	Fastqsanger.gz
	SRR21526563_1.fastq.gz	Fastqsanger.gz
	SRR21526563_2.fastq.gz	Fastqsanger.gz
	SRR21526564_1.fastq.gz	Fastqsanger.gz

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Project description:It has been widely accepted that mammalian neural stem cells (NSCs) exist only in the central nervous system. Here, we report that peripheral NSCs (pNSCs) exist outside of the CNS and can be isolated—from mouse embryonic limb, postnatal lung, tail, dorsal root ganglia, and adult lung tissues. pNSCs are from Sox1+ cells and originate from neuroepithelial cells. Many pNSCs that migrate out of the neural tube can differentiate into neurons and limited glia cells during embryo development. We found the existence of pNSCs provides previously unidentified insight of the mammalian nervous system development and presents an alternative potential strategy for neural regenerative therapy. Our study demonstrates that directly converted cells can be a promising source for generating transplantable assembloids. Mice and Cells: RNA-seq : RNA quality was verified on an Agilent Bioanalyzer Nano Eukaryote chip. 1 mg of total RNA was used for poly-A selection with NEBNext PolyA mRNA magnetic isolation module and subsequent cDNA synthesis, Illumina Tru-seq adaptor ligation and library preparation were performed with NEBNext Ultra RNA Library Prep Kit (NEB) according to the manufacturer’s instructions. Library quality and concentration were determined using an Agilent Bioanalyzer DNA1000 chip and a Qubit fluorometer. Libraries were sequenced on Illumina NextSeq500 in single-end mode. HISAT24 was used to align the RNA‐seq reads for each of the samples to the mouse reference genome GRCm38, and Cufflinks to annotate them. The counts of aligned reads to each gene were calculated with HTSeq. In‐house software was used to merge the expression results into a single text file used in the downstream analysis in Matlab (MathWorks). We performed quantile normalization to equalize the data and stabilized them through the log2 transform of the data plus one. The heatmap of the most highly variable transcripts, the hierarchical clustering dendrograms (calculated using the unweighted pair group method with arithmetic mean and Euclidean distance measure), and the PCAs were performed using in‐home functions developed in Matlab (MathWorks).

Project description:It has been widely accepted that mammalian neural stem cells (NSCs) only exist in central nervous system (CNS). Here, we challenge this concept and show that peripheral NSCs (pNSCs) could be isolated out of CNS from mouse embryonic limbs, postnatal tail and adult lung tissues. Derived-pNSCs express multiple NSC-specific markers, exhibit cell morphology, self-renewing capacity, genome-wide transcriptional profile, epigenetic features similar to those of brain control NSCs. Derived-pNSCs can differentiate into astrocytes, electrophysiologically functional neurons, and oligodendrocytes in vitro and in vivo, indicating multipotency. Lineage-tracing results reveal that pNSCs are progeny cells of neural epithelial cells, but not neural crest cells. Our finding of pNSCs out of CNS expands the field of developmental neuroscience and presents an alternative potential strategy for neural regenerative therapy. Generation of pNSCs: To generate pNSCs, embryonic limbs cells, adult lung cells and postnatal tail cells were cultured in simple defined NSC medium: DMEM/F-12 [1:1] with 2% B27 w/o Vitamin A, 1% N2 (both Gibco), 1x Glutmax (Gibco), 1x penicillin-streptomycin (Sigma), supplemented with 10 ng/ml EGF and 10 ng/ml human bFGF (both from Invitrogen). Medium was changed every 24h. After pNSCs clusters were observed, each single cluster colony was manually picked and cultured in 96 well individually. Microarray and data analysis: RNA samples to be analysed by microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion) that involved synthesis of T7-linked double-stranded cDNA and 12 h of in vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was then hybridized for 18 h onto MouseRef-8 v2 expression BeadChips (Illumina) following the manufacturer’s instructions. After washing as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were exclusively hybridized as biological replicates. The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using the log2 scaling and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor.

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Multipotent neural stem cells originating from neuroepithelium exist outside of the mouse central nervous system [scRNA-seq]

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