| BACKGROUND:Human umbilical cord-derived mesenchymal stem cells(hUC-MSCs)have attracted much attention in the fields of regenerative medicine,tissue engineering and immunomodulation.It is an ideal cell therapy product for their absence of ethical controversy,high sternness and enhanced proliferative capacity.Due to high demand of hUC-MSCs for therapy,it is a necessary to expand the cells in vitro before application.In addition,because hUC-MSCs are a highly heterogeneous population of cells with widely varying clinical applications,quality control of the cells after culture is also critical.OBJECTIVE:At present,there is no highly specific and sensitive screening marker as the assessment standard of high-quality cells.This study focused on the multi-omics changes of hUC-MSCs after culture at clinical application level,and tries to find suitable markers to be used as the quality control standard of hUC-MSCs.The reasons for the heterogeneity of hUC-MSCs were also analyzed,in order to provide the theoretical basis for clinical selection of suitable hUC-MSCs products.METHODS:1.Different passages of hUC-MSCs from the same umbilical cord cultured in clinical application grade(serum-free engineered culture)were collected,and sequenced with genome,transcriptome,protein microarray and methylation microarray in bulk level.2.Genes that may cause senescence during cell passaging were integrated and validated with qPCR,Western blot in cultured hUC-MSCs.Then lentivirus overexpression and knockout were used to explore the function of genes.β-galactosidase staining,CCK8 and cytokine microarray were used for validation of cell function.3.The bulk transcriptome of hUC-MSCs from different culture passages of the two umbilical cords was analyzed by WGCNA to reveal the transcriptome commonality of cellular dynamic changes during culture.4.Single-cell sequencing of hUC-MSCs from different passages of the two umbilical cords was performed to reveal the subpopulation changes,cellular functional heterogeneity,and quality control markers.RESULTS:1.Genomic changes in hUC-MSCs during passaging are mainly missense mutations and copy number increase,which affect cell development,differentiation,and DNA damage repair,but do not involve oncogenes;chromosomal fragment deletions increase in incidence and fragment size with increasing passaging,which may lead to partial loss of cell function.Transcriptome changes mainly involve cell cycle pathways.Changes in methylated genes are mainly involved in differentiation,development and damage response.Pathways of proteomic changes are mainly involved in immune regulation.2.The transcriptome,metabolome and proteome sequencing data were integrated,and five up-regulated genes and five down-regulated genes varying in the course of passaging were selected for functional validation,and finally DGKA was found to cause cellular senescence,because the rate of βgalactosidase positivity was increased in hUC-MSCs with DGKA overexpressed.Furthermore,CCK8 showed significantly reduced cell proliferation viability,qPCR assay showed increased expression of cell cycle blocking genes p21 and p 16 in hUC-MSCs with DGKA overexpressed.Cytokine assay of the culture supernatant indicated that DGKA caused a significant senescence associated secretory phenotype with increased expression of immunosuppression-related genes.3.WGCNA analysis of the common gene changes of the two umbilical cords at different stages of culture showed that the cells in the early stage of culture mainly activated immune signaling pathways and signal transduction pathways.The main activation pathways of cells in metaphase culture stage involve proliferation,differentiation and inflammatory activation.Ossification,apoptosis and growth arrest were involved in cell activation pathways in late culture stage.The hub genes of the cells in different culture stages were selected,and it was found that the cells in the early culture stage may contain more non-MSCs cells.4.The results of single-cell sequencing analysis showed that the hUC-MSCs could be divided into 14 subsets after batch effect was removed.The comprehensive analysis the functions(GSVA analysis and gene set scoring)and sternness(CytoTRACE)of different subsets shows the C8 with FOXA2+MLPH+LPXN+is the better choice for its well sternness and function.Moreover,the aging C7(SEPT7+LPRC75A+CXCL6-)and the potential markers representing sternness of hUC-MSCs(MIF and CD59)were found.Pseudotime analysis(Monocle)and functional scoring were used to identify the cell subsets at different progenitor stages.The activation of transcription factor KLF11 was identified in aging subgroup C7 by SCENIC transcription factor analysis,and DGKA is one of its downstream regulon.Transcription factors associated with lipogenic differentiation was significantly activated in G1 phase cell subsets.CONCLUSION:1.In this study,we analyzed the functional differences of cells in different passages at the molecular level.The genes that increase and decrease with the number of generations can be used as a measure of the degree of cellular senescence.2.For the first time,DGKA was found and confirmed to be the cause of senescence in hUC-MSCs.3.Combined hUC-MSCs from different donor sources were searched for core genes representing different culture stages,which are expected to be used as biomarkers for hUC-MSCs products in different culture stages for different indications of disease treatment4.The functions of different subsets were predicted at the single cell transcriptome level;the subsets with better function and stemness and senescent subpopulations were found,as well as potential markers representing stemness of hUC-MSCs,which are of great significance for clinical application of different generations and subsets of cells in the targeted treatment of different diseases. |
