1号染色体长臂扩增削弱人胚胎干细胞神经分化潜能

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。

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

ObjectiveTo explore the effects of long arm of chromosome 1 (1q) amplification on neural differentiation and to establish a cellular model for studying neural diseases. MethodsThe 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. ResultsOne 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) . ConclusionAmp (1q) hESCs maintained the ability of self-renewal, while the neural differentiation ability was impaired due to the extra copy of chromosome 1q.