上海交通大学学报（医学版）

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2025 , Vol. 45 >Issue 4: 387 - 403

DOI: https://doi.org/10.3969/j.issn.1674-8115.2025.04.001

论著 · 基础研究

人胚胎干细胞向神经前体细胞分化的转录组与染色质可及性变化分析研究

  • 李林颖 ,
  • 蔡晓东 ,
  • 童冉 ,
  • 杨晨 ,
  • 王志明 ,
  • 贺潇宇 ,
  • 马子越 ,
  • 张丰 ,
  • 李令杰 ,
  • 周君梅
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  • 1.上海市儿童医院，上海交通大学医学院附属儿童医院中心实验室，上海 200062
    2.上海交通大学基础医学院组织胚胎学与遗传发育学系，上海市生殖医学重点实验室，教育部细胞分化与凋亡重点实验室，上海 200025
李林颖（1998—），女，硕士生；电子信箱：lin-ying@sjtu.edu.cn。
周君梅，研究员，博士；电子信箱：zhou_junmei@qq.com
李令杰，研究员，博士；电子信箱：lingjie@shsmu.edu.cn
张 丰，副研究员，博士；电子信箱：fzhang@shsmu.edu.cn。

收稿日期: 2024-09-02

  录用日期: 2024-11-11

  网络出版日期: 2025-04-28

基金资助

国家自然科学基金(32070867)

收起

Analysis of transcriptome and chromatin accessibility changes during the differentiation of human embryonic stem cells into neural progenitor cells

  • LI Linying ,
  • CAI Xiaodong ,
  • TONG Ran ,
  • YANG Chen ,
  • WANG Zhiming ,
  • HE Xiaoyu ,
  • MA Ziyue ,
  • ZHANG Feng ,
  • LI Lingjie ,
  • ZHOU Junmei
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  • 1.Department of Central Laboratory, Shanghai Children's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200062, China
    2.Department of Histoembryology, Genetics and Developmental Biology, Shanghai Jiao Tong University College of Basic Medical Sciences; Shanghai Key Laboratory of Reproductive Medicine; Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai 200025, China
ZHOU Junmei, E-mail: zhou_junmei@qq.com
LI Lingjie, E-mail: lingjie@shsmu.edu.cn
ZHANG Feng, E-mail: fzhang@ shsmu. edu.cn.

Received date: 2024-09-02

  Accepted date: 2024-11-11

  Online published: 2025-04-28

Supported by

National Natural Science Foundation of China(32070867)

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摘要

目的·利用人胚胎干细胞（human embryonic stem cell，hESC）体外分化模型和高通量多组学测序技术研究hESC分化成神经前体细胞（neural progenitor cell，NPC）过程中转录组和染色质可及性的变化情况。方法·首先在体外利用拟胚体形成法诱导hESC分化成NPC，并收集这2个阶段的细胞；通过反转录-实时荧光定量PCR（reverse transcription-quantitative real-time PCR，RT-qPCR）和免疫荧光染色（immunofluorescence，IF）鉴定细胞表型。应用转录组测序（transcriptome sequencing，RNA-seq）检测并分析hESC和NPC的差异表达基因（differentially expressed gene，DEG）。应用染色质可及性测序（assay for transposase-accessible chromatin with high throughput sequencing，ATAC-seq）技术获取hESC和NPC的染色质可及性变化情况，并对差异的染色质开放区域进行基序富集分析以发现具有潜在调控作用的转录因子。最后对RNA-seq和ATAC-seq多组学数据进行联合分析，并构建蛋白质互作（protein-protein interaction，PPI）网络，寻找体外神经早期分化过程中的关键基因和调控通路。结果·RT-qPCR与IF均显示多能性标志物（NANOG、POU5F1）在hESC阶段表达量高而在NPC阶段表达量明显降低；同时神经早期分化标志物（PAX6、SOX1、NES）在hESC阶段基本不表达而在NPC阶段表达量显著升高。RNA-seq分析结果显示，与hESC阶段相比，NPC阶段中有5 597个基因的表达水平呈现上调，而3 654个基因的表达水平下降，基因功能富集分析显示NPC阶段上调的基因富集至神经发育相关的功能。ATAC-seq分析结果显示，共27 491个基因组区域在hESC向NPC分化过程中染色质可及性发生了显著改变，其中有12 381个区域染色质可及性增强，15 110个区域染色质可及性减弱；基序富集分析揭示DLX1、LHX2等转录因子基因可能在hESC向NPC分化过程中发挥重要作用。RNA-seq和ATAC-seq的多组学数据联合分析结果显示，在NPC阶段高表达的重叠基因主要富集在轴突导向、前脑发育、神经元迁移等。神经分化后CTNND2、LHX2基因表达水平升高，且相关基因区域染色质可及性也增加。PPI网络分析发现，PRKACA、CDH2、ERBB4等是下游候选基因。结论·利用hESC体外分化模型结合高通量多组学测序技术可用于揭示hESC向NPC分化过程中的转录组及染色质可及性的变化规律；该过程中轴突导向、前脑发育、神经元迁移等通路的相关基因表达水平升高，染色质可及性增强。

关键词： 人胚胎干细胞; 神经前体细胞; 染色质可及性测序; 转录组测序

本文引用格式

李林颖 , 蔡晓东 , 童冉 , 杨晨 , 王志明 , 贺潇宇 , 马子越 , 张丰 , 李令杰 , 周君梅 . 人胚胎干细胞向神经前体细胞分化的转录组与染色质可及性变化分析研究[J]. 上海交通大学学报（医学版）, 2025 , 45(4) : 387 -403 . DOI: 10.3969/j.issn.1674-8115.2025.04.001

Abstract

Objective ·To investigate the changes in transcriptome and chromatin accessibility during the differentiation of human embryonic stem cells (hESCs) into neural progenitor cells (NPCs) using in vitro differentiation models and high-throughput multi-omics sequencing technologies. Methods ·hESCs were first induced to differentiate into NPCs in vitro using the embryoid body formation method, and cells at both stages were collected. The cell phenotypes were identified by reverse transcription-quantitative real-time PCR (RT-qPCR) and immunofluorescence (IF) staining. Transcriptome sequencing (RNA-seq) was conducted to detect and analyze the differentially expressed genes (DEGs) between hESCs and NPCs. The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) was employed to assess chromatin accessibility changes between hESCs and NPCs. Motif enrichment analysis was performed on differentially accessible chromatin regions to discover potential regulatory transcription factors. Finally, an integrated analysis of RNA-seq and ATAC-seq data and the protein-protein interaction (PPI) network were performed to identify key genes and regulatory pathways involved in the early stages of neural differentiation in vitro. Results ·Both RT-qPCR and IF results indicated that the expression levels of pluripotency markers (NANOG and POU5F1) were high at the hESC stage but significantly decreased at the NPC stage, while early neural differentiation markers (PAX6, SOX1, and NES) were minimally expressed at the hESC stage but markedly upregulated at the NPC stage. RNA-seq analysis revealed that compared to the hESC stage, there were 5 597 genes upregulated and 3 654 genes downregulated at the NPC stage. Gene function enrichment analysis showed that the upregulated genes at the NPC stage were enriched in the functions related to neural development. ATAC-seq analysis demonstrated a total of 27 491 genomic regions had significant changes in chromatin accessibility during the differentiation from hESC to NPC, with 12 381 regions showing increased accessibility and 15 110 regions showing decreased accessibility. Motif enrichment analysis revealed that transcription factor genes such as DLX1 and LHX2 might play an important role in the differentiation process from hESCs into NPCs. Integrated analysis of RNA-seq and ATAC-seq data revealed that overlapping genes with high expression at the NPC stage were mainly enriched in axon guidance, forebrain development, and neuron migration. After neural differentiation, the expression levels of CTNND2 and LHX2 genes increased, and the chromatin accessibility of related genomic regions also increased. PPI network analysis indentified candidate downstream genes including PRKACA, CDH2, and ERBB4. Conclusion ·The in vitro differentiation model of hESCs combined with high-throughput multi-omics sequencing technologies can be used to depict the changes in transcriptome and chromatin accessibility during the differentiation of hESCs into NPCs. In this process, the expression levels of genes related to axon guidance, forebrain development, and neuronal migration pathways increase and related chromatin accessibility is enhanced.

Key words： human embryonic stem cell (hESC); neural progenitor cell (NPC); assay for transposase-accessible chromatin with sequencing (ATAC-Seq); RNA sequencing (RNA-seq)

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