从异常核型人胚胎干细胞中筛选不同X染色体失活状态的亚系

目的:从异常核型人胚胎干细胞系中分离两种不同X染色体失活(XCI)状态的细胞,建立亚系,并进行对其XCI状态特征和多能性标记进行鉴定。方法:G显带鉴定人胚胎干细胞系ch HESC-3早晚期代数细胞的核型,H3K27me3免疫荧光染色鉴定早晚期ch HESC-3表观遗传差异,RT-PCR检测早晚期ch HESC-3中XIST基因的表达。利用单细胞克隆的培养分选亚系,H3K27me3、RNA polymeraseⅡ以及DAPI三种标记的共染后每种表观标记各选两株进行RT-PCR,检测两种亚系中XIST基因的表达。并对这四株细胞进行干细胞标记鉴定。结果:G显带结果证明早期ch HESC-3为正常核型,晚期代数核型为异常核型,牵涉到8条染色体的复杂结构变异。H3K27me3免疫荧光染色证明异常核型ch HESC-3中有部分细胞出现了H3K27me3凝集点,而正常核型细胞中未发现。正常核型细胞(ch HESC-3N)没有XIST基因表达,异常核型细胞(ch HESC-3C)中有表达。在RNA polymeraseⅡ着色缺口中发现H3K27me3凝集点的细胞亚株XIST基因表达阳性,polymeraseⅡ着色缺口中未发现H3K27me3凝集点的细胞亚株XIST基因表达阴性,XIST阳性和阴性细胞各选两株进行多能性标记免疫荧光染色均为阳性。结论:成功从异常核型人胚胎干细胞系中分离两种不同XCI状态的细胞并建立亚系,两种表观类型的亚系均保持多能性标记并能在长期培养中保持各自特性。

Derivate Sub-lines with/without XCI Markers from hESCs with Abnormal Karyotype

Objective: To derivate and establish sub-lines with/without X chromosome inactivation (XCI) markers from hESCs with abnormal karyotype.Methods:Karyotype of chHESC-3 from early and late passages was tested by G-banding karyotypic analysis.Epigenetic heterogeneous of chHESC-3 were tested by immunofluorescence of H3K27me3.XIST expression in chHESC-3 was tested by RT-PCR.Epigenetic heterogeneous sub-lines were derived by single cell colony formation,and distinguished by triple immunofluorescence of H3K27me3,RNA polymeraseⅡand DAPI.XIST expression and pluripotent markers (AP、OCT4、TRA-1-60) were examined in four sub-lines.Results:G-banding results showed normal karyotype in early passage and abnormal karyotype concerned with 8 chromosomes in late passage of chHESC-3.H3K27me3 foci were found in part of the abnormal karyotypic chHESC-3 (chHESC-3C),no H3K27me3 foci were found in normal chHESC-3 (chHESC-3N).XIST gene was only expressed by chHESC-3C.H3K27me3 foci in RNA polymeraseⅡ holes that located in DAPI areas were found in those sub-lines with XIST expression.No H3K27me3 foci were found in those sublines without XIST expression.Four sub-lines (two with XIST expression and two without) showed undifferentiated status by pluripotent marker staining.Conclusions:Two kinds of sub-lines with different X-chromosome inactive markers were derived from chHESC-3C,both kinds of sub-lines kept epigenetic characters and pluripotent markers in continual long term culture.