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中华细胞与干细胞杂志(电子版) ›› 2021, Vol. 11 ›› Issue (01) : 1 -7. doi: 10.3877/cma.j.issn.2095-1221.2021.01.001

所属专题: 文献

论著

单细胞测序分析人类胚胎干细胞神经分化的分子机制
曹冰1, 张晓明2, 梁富龙3,()   
  1. 1. 361000 厦门市第五医院医务总监科
    2. 150000 哈尔滨医科大学附属第二医院卒中中心
    3. 361000 厦门市第五医院医务总监科;361000 厦门市第五医院神经内科
  • 收稿日期:2020-02-03 出版日期:2021-02-01
  • 通信作者: 梁富龙
  • 基金资助:
    厦门市第五医院科研课题(50193)

Molecular mechanism of neural differentiation in human embryonic stem cells by single-cell sequencing analysis

Bing Cao1, Xiaoming Zhang2, Fulong Liang3,()   

  1. 1. Department of Medical Director, Xiamen Fifth Hospital, Xiamen 361000, China
    2. Stroke Center, the Second Affiliated Hospital of Harbin Medical University, Harbin 150000, China
    3. Department of Medical Director, Xiamen Fifth Hospital, Xiamen 361000, China; Department of Neurology, Xiamen Fifth Hospital, Xiamen 361000, China
  • Received:2020-02-03 Published:2021-02-01
  • Corresponding author: Fulong Liang
引用本文:

曹冰, 张晓明, 梁富龙. 单细胞测序分析人类胚胎干细胞神经分化的分子机制[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(01): 1-7.

Bing Cao, Xiaoming Zhang, Fulong Liang. Molecular mechanism of neural differentiation in human embryonic stem cells by single-cell sequencing analysis[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(01): 1-7.

目的

探讨人类胚胎干细胞(ESCs)分化为神经细胞的关键性靶基因及分子机制,为临床靶向治疗神经康复患者提供分子理论依据。

方法

基于GEO数据平台芯片,采用单细胞测序方法(scRNA-seq),利用R语言从多分子维度(单细胞差异基因、蛋白互作网络和基因通路等)分析人类ESCs分化过程中的关键Marker基因并利用质控和数据过滤、PCA、TSNE分析、细胞轨迹分析、GO富集分析、KEGG富集分析、KEGG通路分析等论证Marker基因调控人类ESCs分化作用机制。

结果

GO功能富集分析结果为:Marker基因在胚层分化、细胞外基质和信号转导中作用显著;Marker基因互作网络及KEGG通路显示了特征性纤维连接蛋白1(FN1)、同源域蛋白(NANOG)、生长因子受体结合蛋白2(GRB2)为关键基因;KEGG通路分析显示:(1)FN1在调控细胞外基质通路中作用显著;(2)NANOG、GRB2在调控干细胞多能性信号通路作用显著。

结论

FN1可能通过调控细胞外基质通路介导人类ESCs基质环境的变化;NANOG、GRB2上调在干细胞多能性信号通路中高表达介导人类ESCs定向分化为神经组织。

Objective

To explore the key target genes and molecular mechanisms of human embryonic stem cell differentiation into neural cells, and provide molecular theoretical basis for clinical targeted treatment of neurological rehabilitation patients.

Methods

Based on the chip data from GEO platform, the key Marker genes in the differentiation process of human embryonic stem cells were analyzed from the multi-molecular dimension (single cell differential genes, protein interaction networks, gene pathways, etc.) by using R language as well as using single cell sequencing method. Quality control and data filtering, PCA, TSNE analysis, cell trajectory analysis, GO enrichment analysis, KEGG enrichment analysis, KEGG pathway analysis were used to show the mechanism of Marker gene regulating human embryonic stem cell differentiation.

Results

GO functional enrichment analysis showed that Marker genes played a significant role in germ layer differentiation, extracellular matrix and signal transduction. Marker gene interaction network and KEGG pathway showed that characteristic Fibronectin 1 (FN1) , Nanog homeobox (NANOG) and Growth factor receptor-bound protein 2 (GRB2) genes were the key genes. KEGG pathway analysis showed that FN1 played a significant role in regulating extracellular matrix pathway, and NANOG and GRB2 played significant roles in regulating stem cell pluripotency signaling pathway.

Conclusion

FN1 may mediate the matrix environment of human embryonic stem cells by regulating extracellular matrix pathways and up-regulation of NANOG and GRB2 in stem cell pluripotency signaling pathway could mediate directional differentiation of human embryonic stem cells into neural tissue.

图1 主成分细胞1~4中相关基因表达谱分析热图
图2 主成分分析曲线
图3 TSNE聚类分析散点图
图4 TSNE聚类分析小提琴图
图5 细胞轨迹分析
图6 GO功能富集柱状图
图7 Marker基因圈
图8 KEGG通路功能富集气泡图
图9 基因互作网络
1
Shi L, Cui Y, Zhou X, et al. Comparative transcriptomic analysis identifies reprogramming and differentiation genes differentially expressed in UiPSCs and ESCs[J]. Biosci Trends, 2017, 11(3):355-359.
2
Hernández R, Jiménez-Luna C, Perales-Adán J, et al. Differentiation of human mesenchymal stem cells towards neuronal lineage: clinical trials in nervous system disorders[J]. Biomol Ther (Seoul), 2020, 28(1):34-44.
3
Rostom R, Svensson V, Teichmann SA, et al. Computational approaches for interpreting scRNA-seq data[J]. FEBS Lett, 2017, 591(15):2213-2225.
4
Chu LF, Leng N, Zhang J, et al. Single-cell RNA-seq reveals novel regulators of human embryonic stem cell differentiation to definitive endoderm[J]. Genome Biol, 2016, 17(1):173.
5
Yu X, Li Z, Zheng H, et al. Protective roles of melatonin in central nervous system diseases by regulation of neural stem cells[J]. Cell Prolif, 2017, 50(2):e12323.
6
Heng JCD, Orlov YL, Ng HH. Transcription factors for the modulation of pluripotency and reprogramming[J]. Cold Spring Harb Symp Quant Biol, 2010, 75:237-244.
7
Li YQ. Master stem cell transcription factors and signaling regulation[J]. Cell Reprogram, 2010, 12(1):3-13.
8
Whyte WA, Orlando DA, Hnisz D, et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes[J]. Cell, 2013, 153(2):307-319.
9
Juengst E, Fossel M. The ethics of embryonic stem cells--now and forever, cells without end[J]. JAMA, 2000, 284(24):3180-3184.
10
McLaren A. Important differences between sources of embryonic stem cells[J]. Nature, 2000, 408(6812):513.
11
McLaren A. Ethical and social considerations of stem cell research[J]. Nature, 2001, 414(6859):129-131.
12
Bradley JA, Bolton EM, Pedersen RA. Stem cell medicine encounters the immune system[J]. Nat Rev Immunol, 2002, 2(11):859-871.
13
Drukker M, Katz G, Urbach A, et al. Characterization of the expression of MHC proteins in human embryonic stem cells[J]. Proc Natl Acad Sci U S A, 2002, 99(15):9864-9869.
14
Arnhold S, Klein H, Semkova I, et al. Neurally selected embryonic stem cells induce tumor formation after long-term survival following engraftment into the subretinal space[J]. Invest Ophthalmol Vis Sci, 2004, 45(12):4251-4255.
15
Bieberich E, Silva J, Wang G,et al. Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cell-derived neural transplants[J]. J Cell Biol, 2004, 167(4):723-734.
16
Wang Q, Matsumoto Y, Shindo T, et al. Neural stem cells transplantation in cortex in a mouse model of Alzheimer's disease[J]. J Med Invest, 2006, 53(1-2):61-69.
17
Kolodziejczyk AA, Kim JK, Svensson V, et al. The technology and biology of single-cell RNA sequencing[J]. Mol Cell, 2015, 58(4):610-620.
18
Wagner A, Regev A, Yosef N. Revealing the vectors of cellular identity with single-cell genomics[J]. Nat Biotechnol, 2016, 34(11):1145-1160.
19
Dooley TP, Reddy SP, Wilborn TW, et al. Biomarkers of human cutaneous squamous cell carcinoma from tissues and cell lines identified by DNA microarrays and qRT-PCR[J]. Biochem Biophys Res Commun, 2003, 306(4):1026-1036.
20
Xu TP, Huang MD, Xia R, et al. Decreased expression of the long non-coding RNA FENDRR is associated with poor prognosis in gastric cancer and FENDRR regulates gastric cancer cell metastasis by affecting fibronectin1 expression[J]. J Hematol Oncol, 2014, 7:63.
21
Rao S. Embryonic stem cells: a perfect tool for studying mammalian transcriptional enhancers[J]. J Stem Cell Res Ther, 2012, Suppl 10:10.
22
Li M, Belmonte JC. Ground rules of the pluripotency gene regulatory network[J]. Nat Rev Genet, 2017, 18(3):180-191.
23
Mitsui K, Tokuzawa Y, Itoh H, et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells[J]. Cell, 2003, 113(5):631-642.
24
Chambers I, Colby D, Robertson M, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells[J]. Cell, 2003, 113(5):643-655.
25
Inoue F, Kreimer A, Ashuach T, et al. Identification and massively parallel characterization of regulatory elements driving neural induction[J]. Cell Stem Cell, 2019, 25(5):713-727.e10.
26
Corti A, Sota R, Dugo M, et al. DNA damage and transcriptional regulation in iPSC-derived neurons from Ataxia Telangiectasia patients[J]. Sci Rep, 2019, 9(1):651.
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