RNA-seq And ATAC-seq Analyses Of Multilineage Differentiating Stress-enduring（Muse） Cells

Objective: To investigate the difference of gene transcriptomics and open chromatin between dermal fibroblasts（FBs）and multilineage differentiation stress-enduring（Muse）cells which derived from those FBs.Methods:（1）Healthy human dermal FBs were obtained from foreskin tissues after circumcisions.The primary FBs obtained were subcultured,and were confirmed by immunofluorescence staining with the antibody against vimentin.The FBs at passage fourth were subjected to long-time trypsinization（LTT）in order to obtain Muse cells.Single Muse cells proliferated into pupative Muse cell clusters a low-adherence suspension culture system.Immunofluorescence staining with the antibody against stage-specific embryonic antigen-3（SSEA-3）and cluster of differentiation 105（CD105）were performed on these cell clusters to confirm that these clusters enriched Muse cells.（2）Both FBs and Muse cells were lysed by Trizol lysed to extract RNA for further sequencing.Firstly,the RNA libraries were constructed,and the quality of raw data was assessed using fastqc software.The raw data must be aligned to the reference genome using STAR software and bowtie2 software.The cuffdiff software was used to calculate the reads of these samples and to annotate their genes.The differential gene were further analyzed by the R package（DEseq2）,and the target genes were subjected to gene ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis.The Tn5 transposon enzymes were used to recognize open chromatin fragments in cells.Peak calling was performed on each sample by genomic location annotation and motif analysis of the peaks regions.The enriched genes and pathways were evaluated by GO and KEGG enrichment analysis.Venn intersection analysis was performed on the two sequencing results.The expression of differential genes in sequencing by GO and KEGG was verified using real-time quantitative PCR and western blot analysis.Results:（1）A large number of pure FBs can be obtained from the foreskin tissue of healthy people.These cells showed positive using immunofluorescence staining with an antibody against vimentin.Muse cells expressing SSEA-3 and CD105 can be successfully sorted from cultured dermal fibroblasts by LTT,and proliferated and formed cell clusters in a low-adherence suspension culture system.（2）RNA sequencing detected 2355 significantly differential expressed genes（DEGs）regulating the transcriptome,including 1222 upregulated and 1133 down-regulated DEGs.Panorama of RNA-seq confirmed differences in transcription factors and open chromatin regions between fibroblasts and Muse cells.ATAC sequencing analysis showed that Muse cells had more repetitive and meaningful peaks than fibroblasts,and the peak signals were concentrated near the TSS promoter-transcription start site.More than 200 transcription factors had binding motif sequences in both sets of cellular open chromatin regions.Venn analysis identified the 5 types of DEGs.q RT-PCR analysis AKT1/2 and other genes was performed,and the expression of AKT gene in Muse cells was much higher than that in fibroblast group.AKT1/2 inhibitor was added to the culture medium of Muse cells for 10 days,and the homologous fibroblast stock solution was extracted for q RT-PCR and western blot analysis,and it was found that the expression of phosphorylatedAKT gene changed.RNA-seq and ATAC-seq analyses elucidated the genetic basis for differences in biological properties between Muse cells and fibroblasts.Conclusion:（1）Muse cells and FBs had significant gene changes at the level of transcription and open chromatin.（2）The results of RNA-seq and ATAC-seq analyses clarified the genetic basis of the different biological properties of Muse cells and fibroblasts.These results suggested that the cell cycle transition and the AKT genes may affect the morphology and biological characteristics of Muse cells.