人脐带间充质干细胞的异质性探讨及基于单细胞RNA测序结果的亚群分析

Exploring the heterogeneity of human umbilical cord mesenchymal stem cells and analysing the subpopulation based on single-cell RNA sequencing results

目的探讨人脐带间充质干细胞(hUCMSCs)的异质性,并基于单细胞RNA测序结果进行亚群分析。方法选择两株由郑州奥博细胞医学实验室有限公司提供的hUCMSCs(分别命名为hUCMSCs-1和hUCMSCs-2),通过实时荧光定量聚合酶链反应(qPCR)进行干性基因[性别决定区Y-box 2(Sox2)、Nanog]检测。对hUCMSCs进行成骨诱导,3 d后检测成骨基因碱性磷酸酶(Alp)的表达,7 d后进行碱性磷酸酶染色,14 d后进行茜素红染色。对两株hUCMSCs进行成软骨诱导,21 d后检测成软骨基因二型胶原(COL2A1)的表达。用10 ng/mL白细胞介素-1β(IL-1β)刺激大鼠软骨细胞24 h,与hUCMSCs-1和hUCMSCs-2共培养后,检测软骨细胞合成代谢基因蛋白聚糖(ACAN)和分解代谢基因基质金属蛋白酶3(MMP3)的表达。对hUCMSCs-1进行单细胞RNA测序,并根据功能进行亚群分组,通过生物信息学分析方法描绘各个亚群标志基因热图、富集的信号通路。结果两株hUCMSCs的Sox2、Nanog、Alp、COL2A1表达水平差异有统计学意义(P<0.05)。在成骨诱导7 d后,hUCMSCs-1的碱性磷酸酶染色程度深于hUCMSCs-2。在成骨诱导14 d后,茜素红染色结果显示,hUCMSCs-1钙结节数量多于hUCMSCs-2。经IL-1β干预后,软骨细胞与两株hUCMSCs共培养后的MMP3表达水平差异有统计学意义(P<0.05),但ACAN表达水平差异不显著(P>0.05)。基于单细胞RNA测序结果,hUCMSCs-1可分为C1、C2、C3三个功能亚群。C1亚群的信号通路在细胞外基质受体相互作用上富集,与软骨细胞合成代谢相关;C2亚群信号通路在细胞衰老和凋亡上富集,与细胞的自我更新相关;C3亚群信号通路在DNA复制和减数分裂上富集,与细胞周期相关。结论不同个体来源的hUCMSCs存在异质性,其在成骨、成软骨和抗分解代谢能力方面可能存在差异。通过单细胞RNA测序可较好地根据hUCMSCs的功能特性对其进行分群,有助于提高间充质干细胞治疗的临床疗效。

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 typeⅡalpha 1(COL2A1)was detected 21 days later.Rat chondrocytes were stimulated with 10 ng/mL interleukin-1β(IL-1β)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 significant 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-1β,there were significant differences 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 sequencing,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.