Influence of continuous passaging in vitro on human umbilical cord-derived mesenchymal stem cell
Objective To analyze the transcriptome differences between different generations of human umbilical cord-derived mesenchymal stem cell(hUC-MSC),and to investigate the effects of continuous passaging in vitro on the hUC-MSC transcriptome.Methods The Wharton's jelly was isolated from the procured umbilical cord and cultured in cell culture bottle.Fibroblast-like cells emerged from the tissue block after culture for 1 week,and low generation(P3)and high generation(P15)of hUC-MSC were obtained after digestion and passaging.First,immunophenotype and trilineage differentiation potential were identified in P3 and P15 hUC-MSCs and their RNAs were extracted to construct sequencing libraries for transcriptome sequencing.The transcriptome gene expression matrix of P3 and P15 hUC-MSCs were obtained.This matrix was screened for P3 and P15 hUC-MSCs differentially expressed genes(DEGs)with adjusted P＜0.05 and | 10g2FC | ≥2,and analyzed for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).To verify the sequencing results,real-time fluorescence quantitative PCR was used to detect the relative expressions of tumor necrosis factor receptor superfamily member,tumor necrosis factor receptor superfamily member 1 1B(TNFRSF11B)mRNA of P3 and P15 hUC-MSCs.The levels of TNFRSF11B proteins in supernatant of P3 and P15 hUC-MSCs were detected by ELISA.Results P3 and P15 hUC-MSCs all expressed CD73,CD90 and CD105,but did not express CD14,CD34,CD45 and HLA-DR.All were differentiated into adipogenic,osteogenic and chondrogenic cells.The expressions of 60 656 genes were compared,and 497 DEGs were obtained.Compared with P3 hUC-MSC,335 genes were up-regulated such as TNFRSF11B,FGFR2,CCND2 and WT1,and 162 genes were down-regulated such as NFE2,IL-7R and C1QL4 in P15 hUC-MSC.GO enrichment analysis revealed that DEGs were enriched in 204 GO entries,including 183 biological processes,19 cellular components,and 2 molecular functions,which were mainly enriched in biological processes such as system and organ development,protein phosphorylation,and cellular adhesion;cellular component analysis showed that the differentially expressed proteins were mainly enriched in the cell surface and plasma membrane.KEGG pathway enrichment analysis showed that DEGs were mainly enriched in PI3K-Akt and RAP1 signaling pathways,axon guidance,ECM-receptor interactions,and increase of proteoglycan in cancer.The relative expression of TNFRSF11B mRNA and the TNFRSF1 1B protein level in supernatant were higher in P15 hUC-MSC[4.484±0.018,(118.925±0.475)μg/L]than those in P3 hUC-MSC[1.000±0.021,(22.046±0.116)μg/L](t=49.490,P＜0.001;t=343.300,P＜0.001).Conclusions Continuous passaging of hUC-MSC in vitro leads to the changes in the transcriptome and increase of heterogeneity.The expression of some genes in high-generation hUC-MSC is up-regulated and mainly enriched in cancer-related pathways.
human umbilical cord-derived mesenchymal stem cellcontinuous passaging in vitrotranscriptome sequencingtumor necrosis factor receptor superfamily member 11B
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