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中华细胞与干细胞杂志(电子版)

中华细胞与干细胞杂志(电子版) ›› 2022, Vol. 12 ›› Issue (03) : 129 -137. doi: 10.3877/cma.j.issn.2095-1221.2022.03.001

论著

1号染色体长臂扩增削弱人胚胎干细胞神经分化潜能
李佳一1, 张美丽1, 黄粤1,†()   
  1. 1. 100005　北京，中国医学科学院基础医学研究所　北京协和医学院基础学院
  • 收稿日期:2022-04-12 出版日期:2022-06-01
  • 通信作者: 黄粤
  • 基金资助:
    中国医学科学院医学与健康科技创新工程项目(2021-I2M-1-019)

Amplification of chromosome 1q impairs neural differentiation ability of human embryonic stem cells

Jiayi Li1, Meili Zhang1, Yue Huang1,†()   

  1. 1. State Key Laboratory of Medical Molecular Biology, Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing 100005, China
  • Received:2022-04-12 Published:2022-06-01
  • Corresponding author: Yue Huang
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李佳一, 张美丽, 黄粤. 1号染色体长臂扩增削弱人胚胎干细胞神经分化潜能[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(03): 129-137.

Jiayi Li, Meili Zhang, Yue Huang. Amplification of chromosome 1q impairs neural differentiation ability of human embryonic stem cells[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(03): 129-137.

目的

探讨1号染色体长臂（1q）的扩增对人胚胎干细胞（hESCs）神经分化的影响。

方法

通过对H9 hESCs克隆化培养的方法获得1q扩增的hESCs系。中期染色体计数的方法明确细胞内的染色体数目，核型分析鉴定染色体变异的情况，全基因组测序（WGS）分析基因组片段拷贝数变异的情况。使用碱性磷酸酶（AP）染色法检测细胞干性维持的情况，RT-qPCR和免疫荧光染色等方法检测胚胎干细胞（ESCs）标志物OCT4、SOX2、NANOG、REX1和SSEA4等的表达。拟胚体（EB）形成实验进行hESCs不定向分化、全反式视黄酸（RA）诱导hESCs向外胚层分化、使用STEMdiff? SMADi Neural Induction Kit诱导hESCs向神经祖细胞（NPCs）定向分化，并通过RT-qPCR、AP染色和免疫荧光染色等方法检测其分化能力。两组间比较采用独立样本t检验。

结果

分离获得一株1q发生2个拷贝扩增的细胞，核型分析发现额外获得的2个1q是等臂染色体，核型为[47，XX，+i （1q）]，将其命名为Amp （1q）。Amp （1q）AP染色呈阳性，且表达ESCs标志物OCT4、SOX2、NANOG、REX1和SSEA4，具备干细胞自我更新的特征。EB分化过程中，与H9细胞相比，Amp （1q）向外胚层的分化能力下降，MAP2 （29.67±1.53比66.67±1.15）和PAX6 （8001±567.09比28308.00±1692.50）的表达降低（P均< 0.05）；RA诱导分化实验进一步证明，与H9细胞相比，Amp （1q）存在向外胚层分化的缺陷，MAP2 （22.50±3.54比42.50±2.12）和PAX6 （5403.00±569.93比38756.00±1068.44）的表达降低（P均< 0.05）。当定向诱导向神经谱系分化时，Amp （1q）形成NPCs的能力降低，NPCs标志物PAX6的表达水平低于H9细胞（13.83±3.75比88.33±1.53） （P均< 0.05）。

结论

Amp （1q）具有ESCs自我更新的能力，但1q的扩增会削弱hESCs神经分化的能力。

关键词: 1号染色体长臂扩增, 人胚胎干细胞, 自我更新, 神经分化
Objective

To explore the effects of long arm of chromosome 1 (1q) amplification on neural differentiation and to establish a cellular model for studying neural diseases.

Methods

The cell line harboring chromosome 1q amplification was isolated from H9 human embryonic stem cells (hESCs) by subcloning. The number of chromosomes was determined by chromosome counting.Chromosome aberration was analyzed by karyotyping. Whole genome sequencing (WGS) was used to analyze genome copy number variations. Alkaline phosphatase (AP) staining was used to evaluate the state of stem cells. RT-qPCR and immunofluorescence staining were used to detect the expression of ESCs markers OCT4, SOX2, NANOG, REX1 and SSEA4. Embryoid body (EB) formation was used to detect the undirected differentiation abilities of hESCs. The ectoderm differentiation was induced by all-trans retinoic acid (RA) . Neural progenitor cell (NPC) differentiation was induced by using STEMdiff? SMADi Neural Induction Kit. RT-qPCR, AP staining and immunofluorescence were used to determine the differentiation abilities of hESCs. Student's t test was used for comparison between two groups.

Results

One aneuploid cell line harboring isochromosome 1q gain was isolated and named as Amp (1q) hESCs. The karyotype of Amp (1q) hESCs is 47, XX, +i (1q) . Amp (1q) cells showed positive AP staining and expressed hESCs markers OCT4, SOX2, NANOG, REX1 and SSEA4, and had the ability of self-renewal. During EB differentiation, Amp (1q) hESCs showed impaired ectoderm differentiation, and the expression of MAP2 (29.67±1.53 vs 66.67±1.15) and PAX6 (8001.00±567.09 vs 28308.00±1692.50) decreased compared with H9 cells (all P < 0.05) . The defective ectoderm differentiation of Amp (1q) hESCs were confirmed by RA-induced differentiation, and the expression of MAP2 (22.50±3.54 vs 42.50±2.12) and PAX6 (5403.00±569.93 vs 38756.00±1068.44) decreased compared with H9 cells (all P < 0.05) . The capacity of Amp (1q) hESCs to form NPCs was significantly decreased, and the expression level of NPCs marker PAX6 in Amp (1q) hESC-derived cells was significantly lower than H9 cells (13.83±3.75 vs 88.33±1.53, all P < 0 05) .

Conclusion

Amp (1q) hESCs maintained the ability of self-renewal, while the neural differentiation ability was impaired due to the extra copy of chromosome 1q.

Key words: Chromosome 1q amplification, Human embryonic stem cells, Self-renewal, Neural differentiation
表1 RT-qPCR引物序列
基因 引物序列（5'-3'） 预期扩增长度（bp）
GAPDH 上游TGATGACATCAAGAAGGTGGTGAAG 240
  下游TCCTTGGAGGCCATGTGGGCCAT  
OCT4 上游CGACCATCTGCCGCTTTGAG 573
  下游CCCCCTGTCCCCCATTCCTA  
SOX2 上游GGGAAATGGGAGGGGTGCAAAAGAGG 151
  下游TTGCGTGAGTGTGGATGGGATTGGTG  
NANOG 上游CAGTCCTGGAGCAACCACTC 188
  下游ACATTAAGGCCTTCCCCAGC  
REX1 上游ACATTTCCTGGTTCATACTGG 169
  下游ATTTGACTGAATAAACCTCCTG  
MAP2 上游AGGAAGGGAAGAGCATGTAA 190
  下游ACACCACTTCCTCAACCACAG  
PAX6 上游AACGATAACATACCAAGCGTGT 120
  下游GGTCTGCCCGTTCAACATC  
HAND1 上游CCATGCTCCACGAACCCTTC 170
  下游CCTGGCGTCAGGACCATAG  
T(Brachyury) 上游TATGAGCCTCGAATCCACATAGT 109
  下游CCTCGTTCTGATAAGCAGTCAC  
SOX17 上游CTGCCACTTGAACAGTTTGG 138
  下游GAGGAAGCTGTTTTGGGACA  
GATA4 上游GTGTCCCAGACGTTCTCAGTC 102
  下游GGGAGACGCATAGCCTTGT  
图1 人非整倍体胚胎干细胞Amp（1q）的分离鉴定注：a图为G显带后的核型分析，红圈中箭头指示2个扩增后发生着丝粒融合的1q；b图为使用1号染色体涂染探针进行的FISH，1号染色体探针用FITC标记（正置显微镜，×400）；c图为全基因组测序分析图，横坐标代表染色体编号，纵坐标代表拷贝数变化
图2 倒置显微镜下观察Amp（1q）高表达胚胎干细胞标志物注：a、b图为明场下观察的H9和Amp（1q）细胞克隆形态（×200）；c、d图为H9和Amp （1q）细胞AP染色图像（×100）
图3 显微镜下观察H9和Amp（1q）细胞中ESCs标志物OCT4、SOX2、NANOG和SSEA4的表达（免疫荧光染色，×200）
图4 RT-qPCR检测H9和Amp（1q）细胞中ESCs标志物OCT4、SOX2、NANOG和REX1的表达注：与H9比较，aP < 0.05；ns为差异无统计学意义
图5 显微镜下观察H9和Amp（1q）细胞的EB形成过程（×50）注：分别记录了第1、4、8、12天的EB形态
图6 Amp（1q）细胞在EB形成时外胚层分化异常注：a ~ b图为检测EB形成的第0、6、12天时外胚层标志物MAP2和PAX6的表达；c ~ d图为检测EB形成的第0、6、12天时中胚层标志物HAND1和T的表达；e ~ f图为检测EB形成的第0、6、12天时内胚层标志物SOX17和GATA4的表达；与H9比较，aP < 0.05；ns为差异无统计学意义
图7 RA诱导细胞分化的方案流程
图8 倒置显微镜下观察的细胞形态变化和染色结果注：a、b、e、f图为RA诱导下H9和Amp （1q）细胞在明场下观察的形态变化（×100）；c、d、g、h图为RA诱导下H9和Amp （1q）细胞的AP染色结果（×100）
图9 RT-qPCR检测细胞在诱导外胚层分化后多能性标志物和外胚层标志物表达水平注：检测H9和Amp （1q）细胞在诱导外胚层分化后多能性标志物OCT4（a图）、NANOG（b图）以及外胚层标志物MAP2（c图）、PAX6（d图）的表达；与H9+RA比较，aP < 0.05；ns为差异无统计学意义
图10 NPCs分化流程
图11 H9和Amp（1q）细胞诱导为NPCs的过程注：分别在第0、6、9天记录形态变化，红圈中指示"玫瑰花环"结构（×100，d、h图×450）
图12 免疫荧光染色检测H9和Amp（1q）细胞诱导NPCs分化后标志物PAX6的表达（×200）
图13 PAX6表达的定量统计结果注：与H9比较，aP < 0.05
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