Exploring the heterogeneity of human umbilical cord mesenchymal stem cells and analysing the subpopulation based on single-cell RNA sequencing results
LIU Kai
QI Yuxin
BI Yifei
ZHANG Bingjun
DAI Liming
ZHANG Xiaoling
Abstract：Objective To explore the heterogeneity of human umbilical cord mesenchymal stem cells(hUCMSCs)and to analyse the subpopulation based on single-cell ribonucleic acid(RNA)sequencing results.Methods Two strains of hUCMSCs(named hUCMSCs-1 and hUCMSCs-2,respectively)presented by Zhengzhou Aobo Cell Medicine Laboratory Co.,Ltd were selected,and the stemness genes[sex-determining region Y-box 2(Sox2)and Nanog]were detected by real-time fluorescence quantitative polymerase chain reaction(qPCR).The two strains of hUCMSCs were induced into osteoblasts.After 3 days,the expression of osteogenic gene alkaline phosphatase(Alp)was detected.After 7 days,alkaline phosphatase staining was performed,and after 14 days,alizarin red staining was performed.The two strains of hUCMSCs were induced into chondrocytes,and the expression of chondrogenic gene collagen type Ⅱ alpha 1(COL2A1)was detected 21 days later.Rat chondrocytes were stimulated with 10 ng/mL interleukin-1 β(IL-1 β)for 24 hours.After co-culture with hUCMSCs-1 and hUCMSCs-2,the expressions of anabolic gene aggrecan(ACAN)and catabolic gene matrix metalloproteinase 3(MMP3)in the cartilage cells were detected.Single-cell RNA sequencing of hUCMSCs-1 was performed,and subpopulations were grouped according to their function.Bioinformatics analysis methods were used to depict heat maps of marker genes and enriched signaling pathways of each subpopulation.Results There were signif-icant differences in the levels of Sox2,Nanog,Alp and COL2A1 expressions between the two strains of hUCMSCs(P＜0.05).After 7 days of osteogenic induction,the alkaline phosphatase staining of hUCMSCs-1 was darker than that of hUCMSCs-2.After 14 days of osteogenic induction,the results of alizarin red staining showed that the number of calcium nodules in hUCMSCs-1 was more than that in hUCMSCs-2.After the intervention of IL-1 β,there were significant differ-ences in MMP3 expression levels between the cartilage cells co-cultured with the two strains of hUCMSCs(P＜0.05),but there was no significant difference in ACAN expression(P＞0.05).Based on the results of single-cell RNA sequen-cing,hUCMSCs-1 could be divided into three functional subpopulations:C1,C2 and C3.The signal pathway of C1 subpopulation was enriched in the interaction of extracellular matrix receptors,which was related to the anabolism of chondrocytes.The signal pathway of C2 subpopulation was enriched in cell senescence and apoptosis,which was related to cell self-renewal.The signal pathway of C3 subpopulation was enriched in DNA replication and meiosis,which was related to the cell cycle.Conclusion Heterogeneity exists in hUCMSCs coming from different individual sources and these hUCMSCs may differ in their osteogenic,chondrogenic,and anti-catabolic abilities.Single-cell RNA sequencing can better classify hUCMSCs according to their functional characteristics,which helps to improve the clinical efficacy of mesenchymal stem cell therapy.
Keywords：Human umbilical cord mesenchymal stem cells(hUCMSCs)Single-cell ribonucleic acid(RNA)sequencingFunctional subpopulation
Publication Date：2024-05-28
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：8( 523-530 )