A Single-Cell Transcriptomic Atlas Characterizes Mesenchymal Stem Cells From Human Perinatal Placenta Tissue

Mesenchymal stem cells(MSCs)are a class of adult stem cells with self-renewal,multipotential differentiation and unique immunomodulatory ability.As the most widely used stem cells in clinical applications,perinatal placental tissue is the main source of MSC.However,the safety and efficacy of using MSCs in clinical applications are limited by the absence of specific surface markers and the variation in biological functions,especially immunomodulatory functions.How to solve the above problems has become the main stream of research in the field of MSC.The emergence of single-cell sequencing technology offers the possibility to address these issues.As an important tool for exploring unknown scientific problems,single-cell sequencing technology could let us to recognize and understand cell heterogeneity and cell function from the level of individual cells in multiple dimensions.Thus,in this study,single-cell sequencing technology was applied to systematically analyze and verify the development and biological characteristics of perinatal placental tissue-derived MSC,and decipher the mapping of MSCs development and differentiation.These will provide theoretical and experimental basis for comprehensively understanding the biological characteristics of perinatal tissue-derived MSCs.First,MSCs from different perinatal tissue sources,including placental(hpMSC),amniotic membrane(hmMSC),and umbilical cord(huMSC)sources,were isolated and prepared.The inflammatory cytokines IFN-γ and TNF-α were used to stimulate above three kinds of MSC,named as S_hpMSC,S_hmMSC,and S_huMSC respectively.Furthermore,we performed single-cell transcriptome high-throughput sequencing on untreated MSC from perinatal placenta tissue(hpMSC,hmMSC,huMSC)and inflammatory cytokines pre-treated perinatal placenta tissue derived-MSC.The results showed that three normal state MSCs consisted 10 subclusters and the cells of the subpopulations all matched the criteria for the definition of MSC,while the three MSCs after inflammatory factor treatment appeared to have 11 subclusters.Second,different gene function enrichment analysis revealed heterogeneity between the biological functions of the 10 subclusters.The results of cell cycle analysis showed that the 10 subclusters,had different proliferative capacities.AddModuleScore analysis of the differences in tri-lineage differentiation function and immunomodulatory function of MSC subpopulations showed that the 10 subclusters had similar adipocyte differentiation potential,but the chondrogenic and osteoblastic differentiation abilities differed significantly.The subclusters 4,6,8,and 9 haves showed significant osteogenic differentiation potential,and subcluster 4 also showed significant chondrogenic differentiation ability.The results of immunomodulatory function analysis showed that subclusters 4,6,8 and 9 had significantly higher immunomodulatory ability than other clusters.In addition,the cell surface markers of different subclusters were defined.RGMB and SERLNC2 were expressed in subclusters 1,2,5,7 and 10,whereas LYPD1,CSPG4,and PROCR were in subclusters 4,6,and 9.The specific markers of CADM1,SEZ6L2,LRIG1,and TMEM255B were enriched in subcluster 4,while subcluster 3 and 8 co-expressed CRIM1.These surface marker genes have made it possible to obtain different subpopulations of MSCs.The above findings suggest that heterogeneity exists among MSCs derived from different tissue.Third,the composition ratio of the 10 subclusters in hpMSC,hmMSC and huMSC revealed significant differences.Analysis of different genes and transcription factors revealed that hpMSC and hmMSC had highly similar genetic phenotypes,while huMSC presented a completely different genetic phenotype.GSEA analysis of different gene function showed that huMSC had a significant skeletal regenerative potential and was highly expressed in genes and transcription factors that regulate osteogenesis and chondrogenesis.hpMSC and hmMSC expressed MHC class I molecules,which are associated with immune rejection.Pseudo-time analysis of the single cell trajectory revealed that there was no difference between the development trajectory of MSC,and all MSCs had three development state.Further analysis of the surface markers of the three MSCs showed that the surface markers of hmMSC and hpMSC were RGMB,PCDH18,SERINC2,and TMEM204,whereas the surface markers of huMSC were PCDH10,PROCR,and LYPD1.These results indicated that MSCs from different sources had variability.Fourth,hpMSC,hmMSC and huMSC were confirmed to have immunomodulatory properties at the single-cell level,and some cell subpopulations showed high expression of immunomodulatory-related genes.To further explore the immunomodulatory properties of hpMSC,hmMSC and huMSC,those three kinds of MSCs were stimulated with inflammatory factors IFN-γ and TNF-α.Their various gene expression and related functions were compared before and after stimulation.The results showed that the three MSCs in the inflammatory state had high similarity,i.e.high expression of the same immune negative regulatory molecules such as IDO,CD274 and chemokines.Gene enrichment analysis demonstrated that the function of all cells was related to immune response,showing strong immune chemotactic function and immune regulation characteristics.Relevant validation was performed using in vitro and in vivo experiments,and the results indicated that S_hpMSC,S_hmMSC and S_huMSC were highly expressed in immune negative regulatory molecules at the mRNA level,and the same results were discovered using activated T cell supernatant to stimulate the three MSCs.Further,we established a mouse acute graft-versus-host disease(aGvHD)model to evaluate the difference immunomodulatory functions of huMSC and S_huMSC in vivo.The results showed that S_huMSC performed better than huMSC at the early-stage therapy,including better efficacy and strong inhibiting T cells proliferation.Their effect was tended to the same in the late stage of treatment.These results suggest that inflammatory factor pretreatment could significantly improve the immunomodulatory capacity of huMSC and effectively improve the therapeutic effect.The mechanism of immunomodulatory effects of MSC has been a research challenge in the field of MSC.We analyzed the immune characteristics of hpMSC,hmMSC and huMSC before and after inflammatory factor stimulation at the level of single cell genes by using pseudo-time analysis.The results showed that immunoregulation related genes were highly expressed in S_hpMSC,S_hmMSC,S_huMSC,which experienced inflammatory factors stimulation,while the expression level of different gene was varied.Further analysis revealed that MSC could be divided into two kinds of cell subpopulation,including cells that expressing constant immunomodulatory genes themselves(autoimmune regulatory cell subpopulation)and cells that expressing immunomodulatory genes stimulated by inflammatory factors(immunomodulatory plasticity cell subpopulation).The inflammatory microenvironment regulates the high expression of immunomodulatory-related genes in plastic cell subpopulations,which in turn amplifies the auto-immunomodulatory effect of MSC in a cascade,and this process is the mechanism by which MSC exerts immunomodulatory effects.Further,single-cell regulatory network inference and clustering(SCENIC)analysis transcription factor analysis showed that the transcription factor IRF1 was the highest expressed transcription factor in hpMSC,hmMSC and huMSC under the inflammatory microenvironment.In vitro qPCR,Western blot and immunofluorescence experiments showed that IRF1 expression was significantly elevated in S_hpMSC,S_hmMSC and S_huMSC and regulated the expression of immunosuppression-related genes.In vitro co-culture experiments between huMSC and T cells showed that the inhibitory ability of T cell proliferation was significantly reduced in huMSC after IRF1 knockdown,suggesting that IRF1 is a key transcription factor for the immunomodulatory effects of MSC in the inflammatory microenvironment.The above experimental results confirmed that the immunomodulatory mechanism of perinatal placenta-derived MSC is achieved by the combination of immunomodulatory functions of a subpopulation of cells expressing immunomodulatory genes by themselves and a subpopulation of cells expressing immunomodulatory genes regulated by the inflammatory microenvironment,in which IRF1 is a key transcription factor regulating the expression of immunomodulatory-related genes.In addition,analysis presented for single cell transcriptome data of hpMSC,hmMSC,and huMSC and RNA transcriptome data of BM-MSC,hpMSC,hmMSC,and huMSC revealed that Chi311 was highly expressed in huMSC and correlated with T cell proliferation and activation.Thus,we performed relevant experiments to verify that the expression of Chi311 was significantly increased at both mRNA and protein levels after stimulation of huMSC with inflammatory factors IFN-γ and TNF-α,and that the secreted Chi311 protein in cell supernatants was also significantly increased.The effect of Chi311 on the immunomodulatory function of huMSC was investigated using a mouse aGvHD model.The results showed that the effect of Chi311 knockdown huMSC was significantly reduced in the treatment of aGvHD,and its ability to inhibit Th 17-mediated inflammatory response was also significantly decreased.In vitro co-culture experiments showed that the ability to inhibit T cell proliferation of Chi311 knockdown huMSC was significantly declined.This phenomenon consistent with the latest reported findings.Study on the mechanism of Chi311 regulating huMSC immunity showed that Chi311 is a potential immunomodulatory mechanism of huMSC by inhibiting of STAT3 phosphorylation and Th17 differentiation.These findings suggested that Chi311 is an important regulatory molecule in the immunomodulatory role of huMSC.In conclusion,we systematically analyzed the biological properties of perinatal placental tissue-derived MSC at the single-cell level,and confirmed that hpMSC,hmMSC,and huMSC have heterogeneity and showed differences in multilineage differentiation and immunomodulatory functions.We also found that huMSC have significant skeletal regenerative potential and lower immunogenicity,revealed that immunomodulation is an inherent biological function of MSC,which the inflammatory microenvironment can cascade to amplify this immunomodulatory property.The gene expression profiles of hpMSC,hmMSC,and huMSC in the inflammatory microenvironment were mapped for the first time.The mechanism of the immunoregulatory function of the three kinds of MSCs was found to be a combination of a subpopulation of cells with constant expression of immunomodulatory genes and a subpopulation of cells with expression of immunomodulatory genes regulated by the inflammatory microenvironment.In addition,IRF1 was found to be a key transcription factor regulating hpMSC,hmMSC,and huMSC immunoregulatory functions in the inflammatory microenvironment,and Chi311 was an important huMSC immune negative regulatory molecule.The above findings are the first comprehensive and systematic studies on the biological properties of perinatal placental tissue-derived MSC,especially immunomodulatory properties at the single-cell level.Our study revealed the heterogeneity and variability of perinatal placental tissue-derived MSC and the mechanism of immunomodulatory functions,which provide an important theoretical basis and experimental basis for the future precision treatment of MSC.