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Next Generation Sequencing based mRNA sequencing of human mesenchymal stem cells from hair follicle outer root sheath

ABSTRACT: Next Generation Sequencing (NGS) based mRNA sequencing (RNA-seq) has provided high-throughput measurements to analyze the transcriptome of cells. We report the comprehensive transcriptome profiling of primarily isolated human mesenchymal stem cells from hair follicle outer root sheath (MSCORS) in series cultivation passages using NGS-based RNA-seq method. Human anagen hair follicles were non-invasively plucked from scalp of healthy donors, cultivated on a Transwell membrane, from which the MSCORS were isolated and subcultured onto normal culture flask. Attached and proliferating MSCORS were cultivated and characterized as mesenchymal stem cells (MSC) according to the minimum criteria for defining multipotent MSC from the International Society for Cellular Therapy (ISCT). mRNA profiles of MSCORS in p0, p1 and p2 were quality controlled, and generated by deep sequencing, in triplicate, using Illumina PE150. Paired-end raw data were pre-processed using a published protocol. HISAT2 (Hierarchical Indexing for Spliced Alignment of Transcripts) was used to align reference genome. HTSeq was used to calculate Reads Count, and gene abundence was determined by analyzing FPKM (fragments per kilobase of exon model per million reads mapped).

ORGANISM(S): Homo sapiens

PROVIDER: GSE159139 | GEO | 2020/10/07

REPOSITORIES: GEO

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Project description:Mesenchymal stem cell (MSC) heterogeneity can lead to variable treatment outcomes. Genetically modifying MSCs to express erythropoietin could confer additional therapeutic benefits that may enhance standalone MSC treatments. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) between healthy, retinal degenerated, and stem cell-treated rat retinal degeneration models for high-throughput data analysis. MSC, Wharton's jelly Mesecnhymal stem cells; MSCEPO, EPO-expressing MSC; DPSC, dental pulp MSC stem cells Methods: Retinal mRNA profiles of 31-day-old healthy, sodium iodate-administered (retinotoxic inducer), DPSC-treated, MSC-treated, and MSCEPO-treated Sprague Dawley rats were generated by deep sequencing, in triplicate, using Illumina Nextseq 500. The sequence reads that passed quality filters were analyzed at the transcript isoform level with Salmon, and then converted to the gene level count matrices for quantification with DESeq2. The quant assay kit was used for qPCR amplification. Results: Using the Salmon-DESeq2 pipeline, we mapped about 25-35 million sequence reads per sample to the rat genome (build Rnor 6.0) and identified 20,376 transcripts in the retinas of the control and experimental groups. Approximately 500-1000 genes were found to be differentially expressed using the arbitrary cutoff of Padj<.05 and fold change>±2.0. Hierarchical clustering of differentially expressed genes revealed profiles involved in cellular regeneration,cell death, and phototransduction, which indicated the therapeutic effectiveness of the stem cells on retinal survival. Gene set enrichment analysis and pathway topology analysis showed enrichment in the aforementioned processes, Padj<.05. Conclusions: Our study represents the first detailed analysis of MSCEPO-treated retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for further investigations in MSC enhancement methods. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within the retina. We conclude that RNA-seq based retinal transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions, especially in regards to MSC-mediated tissue repair and phototransduction.

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Next Generation Sequencing based mRNA sequencing of human mesenchymal stem cells from hair follicle outer root sheath

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