单细胞水平解析人牙龈间充质干细胞异质性

doi:10.13560/j.cnki.biotech.bull.1985.2023-0253

摘要/Abstract

摘要：

利用单细胞转录组测序解析牙龈干细胞（gingival mesenchymal stem cells, GMSCs）内部功能亚群及异质性。体外分离培养、鉴定人GMSCs，利用10×scRNA-seq平台对GMSCs进行单细胞测序，根据细胞的基因表达差异进行分群，分析各亚群表面标记物表达情况，比较各亚群的细胞周期差异，成骨、成脂、成软骨分化基因表达差异以及基因表达谱差异，并分析各亚群细胞间的相互作用。成功提取到人GMSCs，呈旋涡状贴壁生长。单细胞测序分析结果显示GMSCs由9个细胞亚群构成，高表达表面标记物ENG、NT5E、THY1、ITGB1和CD44，阴性表达CD34、CD14、PECAM1、PTPRC。GMSCs总体G2/M期所占比例为41.59%；亚群6的G2/M期比例最低（16.44%）；而亚群2最高（99.08%）。成脂、成软骨、成骨相关基因在9个细胞亚群中的表达不完全相同；9个亚群具有不同的基因表达谱，亚群2/4具有较高的相似性，相对高表达TOP2A、UBE2C、KPNA2、CENPF等基因；亚群5高表达DKK1、KRT7、ID1等基因；亚群9高表达MALAT1、FN1、NEAT1等基因。GO分析显示各个亚群富集不同的通路。亚群9与其他亚群间的通讯最为活跃。单细胞水平研究提示GMSCs存在不同的功能亚群，且各个亚群的增殖能力、基因组表达谱具有异质性。

关键词: 牙龈干细胞, 单细胞测序, 细胞亚群, 功能异质性

Abstract:

Gingival mesenchymal stem cells（GMSCs）cell clusters and functional heterogeneity were analyzed through single cell RNA sequencing technology. Human GMSCs were isolated and cultured in vitro, GMSCs were sequenced by 10× scRNA-Seq platform. The cells were grouped according to the gene expression differences, and analyze the expression of surface markers and cell cycle differences in each cluster. Differences in gene expression and gene expression profile of osteogenic, lipogenic and chondrogenic differentiation were compared，and the interactions between different clusters of cells were also analyzed. Human GMSCs were successfully extracted and grew in a vortex like manner. The GMSCs were composed of 9 cell clusters. ENG, NT5E, THY1, ITGB1 and CD44 were highly expressed, and CD34, CD14, PECAM1 and PTPRC were negatively expressed. The proportion of G2/M phase of GMSCs was 41.59%. The G2/M phase ratio of cell cluster 6 was the lowest（16.44%）, while the cell cluster 2 was the highest with 99.08%. The expressions of adipogenic, chondrogenic and osteoblastic differentiation related genes were not identical in the 9 cell clusters. Nine clusters had different gene expression profiles, and 2/4 cell clusters showed high similarity, with relatively high expressions of TOP2A, UBE2C, KPNA2, CENPF genes. DKK1, KRT7 and ID1 genes were highly expressed in the cell cluster 5. MALAT1, FN1 and NEAT1 genes were highly expressed in the cell cluster 9. Moreover, GO analysis showed that each cell cluster enriched different pathways. The communication between cell cluster 9 and other cell clusters were the most active. The single-cell level study suggests that there are different functional clusters of GMSCs, and the proliferation capacity and genomic expression profile of each cluster were heterogeneous.

Key words: gingival mesenchymal stem cells, single cell RNA sequencing, cell clusters, functional heterogeneity

吴昊, 刘紫微, 郑颖, 戴雅文, 时权. 单细胞水平解析人牙龈间充质干细胞异质性[J]. 生物技术通报, 2023, 39(7): 325-332.

WU Hao, LIU Zi-wei, ZHENG Ying, DAI Ya-wen, SHI Quan. Study on the Heterogeneity of Human Gingival Mesenchymal Stem Cells at Single Cell Level[J]. Biotechnology Bulletin, 2023, 39(7): 325-332.

图/表 7

图1 GMSCs培养与鉴定 A: P3代GMSCs形态观察，scale bar：200 μm；B: GMSCs增殖曲线;C：GMSCs表面标记物检测

Fig. 1 Culture and identification of GMSCs A: Morphological observation of P3 generation GMSCs, scale bar: 200 μm. B: GMSCs proliferation curve. C: GMSCs surface marker detection

图2 数据总体质量 A：t-SNE可视化GMSCs的细胞亚群；B: GMSCs亚群表面标记物分析

Fig. 2 Overall data quality A: t-SNE visualizing the cell clusters of GMSCs. B: Analysis of surface markers of GMSCs clusters

图3 单细胞测序分析GMSCs功能亚群与标记物 A: GMSCs细胞亚群细胞周期分析； B: GMSCs成脂相关基因表达小提琴图；C: GMSCs成软骨相关基因表达小提琴图；D: GMSCs成骨相关基因表达小提琴图

Fig. 3 Single cell sequencing analysis of functional clusters and markers of GMSCs A: Cell cycle analysis of GMSCs cell clusters. B: Violin map of gene expression related to lipid formation in GMSCs. C: Violin map of gene expression related to chondrogenesis in GMSCs. D: Violin diagram of gene expression related to osteogenesis in GMSCs

图4 GMSCs亚群细胞周期与三系分化基因表达分析 A: GMSCs 的count 数，基因数，线粒体比率的小提琴图；B: GMSCs的count数与基因数和线粒体基因比例之间的关系图

Fig. 4 Analysis of cell cycle and three-line differentiation gene expression in GMSCs clusters A: Violin chart of count, gene number, and mitochondrial ratio of GMSCs.B: Relationship between the count number of GMSCs and the number of genes and the ratio of mitochondrial genes

图5 GMSCs亚群差异表达top基因heatmap图

Fig. 5 Heatmap of differentially expressed top genes in GMSCs clusters

图6 GMSCs亚群差异基因 GO 通路富集分析

Fig. 6 Analysis of GO pathway enrichment of GMSCs clusters differential genes

图7 GMSCs各亚群之间受体配体互作情况

Fig. 7 Interactions of receptors and ligands between different clusters of GMSCs

参考文献 17

[1]	Mabuchi Y, Okawara C, Méndez-Ferrer S, et al. Cellular heterogeneity of mesenchymal stem/stromal cells in the bone marrow[J]. Front Cell Dev Biol, 2021, 9: 689366. doi: 10.3389/fcell.2021.689366 URL
[2]	Olmedo-Moreno L, Aguilera Y, Baliña-Sánchez C, et al. Heterogeneity of in vitro expanded mesenchymal stromal cells and strategies to improve their therapeutic actions[J]. Pharmaceutics, 2022, 14(5): 1112. doi: 10.3390/pharmaceutics14051112 URL
[3]	Misra P, Jadhav AR, Bapat SA. Single-cell sequencing: a cutting edge tool in molecular medical research[J]. Med J Armed Forces India, 2022, 78: S7-S13.
[4]	Ke M, Elshenawy B, Sheldon H, et al. Single cell RNA-sequencing: a powerful yet still challenging technology to study cellular heterogeneity[J]. BioEssays, 2022, 44(11): 2200084. doi: 10.1002/bies.v44.11 URL
[5]	Zheng G, Xie ZY, Wang P, et al. Recent advances of single-cell RNA sequencing technology in mesenchymal stem cell research[J]. World J Stem Cells, 2020, 12(6): 438-447. doi: 10.4252/wjsc.v12.i6.438 pmid: 32742561
[6]	Zhang QZ, Shi SH, Liu Y, et al. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis[J]. J Immunol, 2009, 183(12): 7787-7798. doi: 10.4049/jimmunol.0902318 pmid: 19923445
[7]	Kim D, Lee AE, Xu QL, et al. Gingiva-derived mesenchymal stem cells: potential application in tissue engineering and regenerative medicine-a comprehensive review[J]. Front Immunol, 2021, 12: 667221. doi: 10.3389/fimmu.2021.667221 URL
[8]	Shi Q, Qian ZY, Liu DH, et al. GMSC-derived exosomes combined with a chitosan/silk hydrogel sponge accelerates wound healing in a diabetic rat skin defect model[J]. Front Physiol, 2017, 8: 904. doi: 10.3389/fphys.2017.00904 pmid: 29163228
[9]	Liu ZW, Yang S, Li XM, et al. Local transplantation of GMSC-derived exosomes to promote vascularized diabetic wound healing by regulating the Wnt/β-catenin pathways[J]. Nanoscale Adv, 2023, 5(3): 916-926. doi: 10.1039/d2na00762b pmid: 36756513
[10]	Wang ST, Liu ZW, Yang S, et al. Extracellular vesicles secreted by human gingival mesenchymal stem cells promote bone regeneration in rat femoral bone defects[J]. Front Bioeng Biotechnol, 2023, 11: 1098172. doi: 10.3389/fbioe.2023.1098172 URL
[11]	Chen T, Li JW, Jia YC, et al. Single-cell sequencing in the field of stem cells[J]. Curr Genom, 2020, 21(8): 576-584. doi: 10.2174/1389202921999200624154445 URL
[12]	Yang H, Gao LN, An Y, et al. Comparison of mesenchymal stem cells derived from gingival tissue and periodontal ligament in different incubation conditions[J]. Biomaterials, 2013, 34(29): 7033-7047. doi: 10.1016/j.biomaterials.2013.05.025 pmid: 23768902
[13]	Tomar GB, Srivastava RK, Gupta N, et al. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine[J]. Biochem Biophys Res Commun, 2010, 393(3): 377-383. doi: 10.1016/j.bbrc.2010.01.126 URL
[14]	郑玉婷, 朱晓帆, 杨宇霞, 等. 基于单细胞RNA测序的人羊水间充质干细胞异质性分析[J]. 郑州大学学报: 医学版, 2022, 57(4): 455-461.
	Zheng YT, Zhu XF, Yang YX, et al. Heterogeneity analysis of human amniotic fluid mesenchymal stem cells based on single cell RNA sequencing[J]. J Zhengzhou Univ Med Sci, 2022, 57(4): 455-461.
[15]	刘伟江. 单细胞水平解析围产期胎盘组织来源MSC生物学特性[D]. 北京: 军事科学院, 2022.
	Liu WJ. Analysis of biological characteristics of MSC from perinatal placenta tissue at single cell level[D]. Beijing: Academy of Military Sciences, 2022.
[16]	Zhang SY, Wang JY, Li BJ, et al. Single-cell transcriptome analysis of uncultured human umbilical cord mesenchymal stem cells[J]. Stem Cell Res Ther, 2021, 12(1): 1-5. doi: 10.1186/s13287-020-02006-w
[17]	Wang Z, Chai CY, Wang R, et al. Single-cell transcriptome atlas of human mesenchymal stem cells exploring cellular heterogeneity[J]. Clin Transl Med, 2021, 11(12): e650.