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

doi:10.3969/j.issn.1674-8115.2025.04.001

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (4): 387-403.doi: 10.3969/j.issn.1674-8115.2025.04.001

• 论著 · 基础研究 • 下一篇

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¹, 蔡晓东², 童冉², 杨晨², 王志明², 贺潇宇², 马子越², 张丰²(), 李令杰²(), 周君梅¹()

^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

收稿日期:2024-09-02 接受日期:2024-11-11 出版日期:2025-04-28 发布日期:2025-04-28
通讯作者: 张丰,李令杰,周君梅 E-mail:fzhang@shsmu.edu.cn;lingjie@shsmu.edu.cn;zhou_junmei@qq.com
作者简介:李林颖（1998—），女，硕士生；电子信箱：lin-ying@sjtu.edu.cn。
基金资助:
国家自然科学基金(32070867)

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

LI Linying¹, CAI Xiaodong², TONG Ran², YANG Chen², WANG Zhiming², HE Xiaoyu², MA Ziyue², ZHANG Feng²(), LI Lingjie²(), ZHOU Junmei¹()

^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

Received:2024-09-02 Accepted:2024-11-11 Online:2025-04-28 Published:2025-04-28
Contact: ZHANG Feng, LI Lingjie, ZHOU Junmei E-mail:fzhang@shsmu.edu.cn;lingjie@shsmu.edu.cn;zhou_junmei@qq.com
Supported by:
National Natural Science Foundation of China(32070867)

摘要/Abstract

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

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)

中图分类号:

R321.5

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.

LI Linying, CAI Xiaodong, TONG Ran, YANG Chen, WANG Zhiming, HE Xiaoyu, MA Ziyue, ZHANG Feng, LI Lingjie, ZHOU Junmei. Analysis of transcriptome and chromatin accessibility changes during the differentiation of human embryonic stem cells into neural progenitor cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(4): 387-403.

图/表 11

表1 RT-qPCR引物序列

Tab 1 Primer sequences for RT-qPCR

Gene	Forward (5'→3')	Reverse (5'→3')
GAPDH	CTGAGAACGGGAAGCTTGT	GGGTGCTAAGCAGTTGGT
NANOG	GAATGAAATCTAAGAGGTGGCA	CCTGGTGGTAGGAAGAGTAAAGG
POU5F1	ACATCAAAGCTCTGCAGAAAGAACT	CTGAATACCTTCCCAAATAGAACCC
SOX1	GGAATGGGAGGACAGGATTT	ACTTTTATTTCTCGGCCCGT
PAX6	GCCTATGCAACCCCCAGT	TCACTTCCGGGAACTTGAAC
NES	TGCGGGCTACTGAAAAGTTC	AGGCTGAGGGACATCTTGAG

图1 利用双重SMAD抑制策略在体外将hESC分化成NPCNote：A. Schematic diagram of the NPC culture system and morphology of hESC-induced differentiation into NPCs. SMADi—SMAD inhibition. Scale bar=200 μm. B. The gene expression of typical genes related to pluripotency and NPCs during the induction process detected by RT-qPCR. ①P=0.002, ②P=0.001, ③P=0.002, ④P<0.001, ⑤P=0.006. NPCs were the cells induced for 27 d. C. Expression of hESC markers (NANOG and POU5F1) and NPC markers (PAX6 and SOX1), detected by immunofluorescence staining. NPCs were the cells induced for 27 d. Scale bar=25 μm.

Fig 1 Dual SMAD inhibition strategy for hESC differentiation into NPC in vitro

图2 体外神经早期分化过程中RNA-seq和ATAC-seq质控分析Note： A. Experimental flow chart. Cells were collected at the ESC stage and NPC stage; nuclei were extracted for standard ATAC-seq library preparation, while total RNA was extracted for RNA-seq library preparation. B. Box plot of gene expression levels. C. Pearson correlation analysis was performed on the expression levels between different samples. D. Transcription start site (TSS) enrichment scores of ATAC-seq data. E. Fragment size distribution of ATAC-seq data. F. Pearson correlation analysis of ATAC-seq data between different samples. G. Gene TSS enrichment heat map showing the reads from ATAC-seq data in the TSS±3.0 kb. The blue-to-red scale represents the transition from high to low in the reads in the specified area.

Fig 2 Quality control analysis of RNA-seq and ATAC-seq datasets obtained from the process of early neural differentiation in vitro

表2 在神经诱导过程中RNA-seq文库所获得的原始读段数和比对读段数

Tab 2 Number of raw reads and mapped reads obtained from RNA-seq libraries during neural induction

Index	Sample
Index	ESC_S1	ESC_S2	NPC_S1	NPC_S2
Total raw read pairs/n	45 792 974	53 251 136	50 419 620	52 973 024
Total raw bases/n	6 914 739 074	8 040 921 536	7 613 362 620	7 998 926 624
Mapped reads/n	44 506 927	51 742 966	48 948 795	51 574 332
Mapped ratio/%	97.95	97.92	97.87	98.13
High-quality mapped reads/n	43 139 843	50 148 322	47 576 740	50 093 517
High-quality mapped ratio/%	94.94	94.90	95.13	95.31

表3 在神经诱导过程中ATAC-seq文库所获得的原始读段数和比对读段数

Tab 3 Number of raw reads and mapped reads obtained by ATAC-seq libraries during neural induction

Index	Sample
Index	ESC_S1	ESC_S2	NPC_S1	NPC_S2
Total raw read pairs/n	63 713 180	32 001 236	88 523 604	61 924 682
Total raw bases/n	9 620 690 180	4 832 186 636	13 367 000 000	9 350 626 982
Mapped read pairs/n	61 240 240	30 684 142	84 944 932	59 439 388
Mapped ratio/%	98.36	98.52	98.24	97.98
High-quality mapped read pairs/n	54 624 252	27 152 448	75 936 202	53 201 562
High-quality mapped ratio/%	87.73	87.18	87.82	87.70
Percent duplication/%	23.70	20.59	22.18	29.05
Non-duplicate read pairs/n	41 677 046	21 562 952	59 092 184	37 745 230

图3 NPC分化过程中转录组的分析Note： A. Volcano plot shows the distribution of DEGs between the two distinct developmental stages (ESC and NPC). DEGs were defined as |log2FC|>1 and P<0.05. B. Gene expression levels were clustered across all samples. Different colors indicate the value of each gene expression level normalized with Z-scores. C. GO analysis was performed on the DEGs upregulated at the NPC stage. D. KEGG analysis of the DEGs upregulated at NPC stage. E. GSEA analysis identifies pathways upregulated during NPC differentiation.

Fig 3 Transcriptome analysis of NPC differentiation

图4 NPC分化过程中染色质可及性分析Note： A. Volcano plot shows the peak signals of differentially open chromatin obtained from the ATAC-seq dataset. Differential open areas were defined as |log2FC| >0.585 and P<0.05. B. The differential region enrichment plot shows the average peak signal between different samples in the differentially upregulated region (left) and the differentially downregulated region (right) at the NPC stage. C. Differential region enrichment heatmap reveals peak signals within genomic region. D. GO analysis of genes associated with differentially open chromatin regions at the NPC stage. E. KEGG analysis of genes associated with differentially open chromatin regions at the NPC stage.

Fig 4 Chromatin accessibility analysis during NPC differentiation process

表4 对在NPC差异上调的开放染色质信号进行基序富集分析

Tab 4 Motif enrichment analysis of open chromatin signals that were differentially upregulated at the NPC stage

E value	Motif ID	E value	Motif ID
2.38×10^-111	DLX1	1.05×10^-100	NKX6.1
6.26×10^-109	SHOX	2.79×10^-91	LHX2
6.18×10^-101	MSX1

图5 整合RNA-seq和ATAC-seq数据分析参与NPC分化的重要基因Note： A. Venn diagram showing the overlap between DEGs in the NPC cell population and related genes predicted from differentially open chromatin regions. In the ATAC-seq dataset, genes were annotated to regions with ±1 kb of the promoter. B. Scatter plot showing the correlation between the expression levels of the overlapping genes (A) obtained in RNA-seq and the associated peaks obtained from ATAC-seq. C. GO analysis of 1 344 upregulated genes. D. Expression levels of CTNND2 and LHX2 in RNA-seq during NPC differentiation. E. The Integrative Genomics Viewer showing ATAC-seq signals at the transcription start sites of CTNND2 and LHX2 loci during NPC differentiation.

Fig 5 Integration of RNA-seq and ATAC-seq data to analyze important genes involved in NPC differentiation

图6 PPI网络分析揭示下游候选中心基因Note： The thickness of the edge in the network diagram represents the interaction strength between the two proteins, and the size of the node represents the degree. The red circles represent the protein with degree≥10, and the others represent proteins that interact with the red proteins.

Fig 6 Candidate hub downstream genes detected by PPI network analysis

表5 PPI网络中度≥10的下游候选中心基因

Tab 5 Candidate downstream hub genes with the degrees≥10 in the PPI network

Gene	Degree
EZH2	22
PRKACA	18
PRKACB	18
JUN	18
FN1	16
CDH2	15
ERBB4	15
RUNX1	15
GSK3B	14
IFT88	13
TUBA1A	12
PDGFRA	11
GNG2	11
DYNC1I2	10
H2BC21	10
PIK3CA	10
PIK3R1	10
MLLT3	10

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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

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

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