建立植入前遗传学检测人胚胎来源的无动物源性干细胞系

Established a Preimplantation Genetic Testing-derived and Xeno-free Human Embryonic Stem Cell Line

【目的】在无动物源性的干细胞培养体系中建立植入前遗传学检测(PGT)胚胎来源的人胚胎干细胞系,为医学研究提供疾病模型。【方法】在我们的研究中,对无动物源性的人类包皮成纤维细胞饲养层(XF-HFF)与成分确定的培养基(CDM)构建的无动物源性培养体系进行了评估。在该培养体系中,利用染色体平衡易位患者PGT来源的废弃胚胎建立一个新的人胚干细胞(hESC)细胞系。【结果】新建立的人胚胎干细胞系在无动物源性培养系统中可长期培养传代(>45个代次)。核型分析显示,在传代45代次后,新建立的人胚胎干细胞仍保持一致的核型。XF-HFF/CDM中的hESC仍保持多能性。通过荧光免疫染色检测到SSEA-3、SSEA-4、SSEA-1、TRA-1-60、TRA-1-81等多能标志物的表达;RT-PCR分析显示,在XF-HFF/CDM培养体系上生长的hESC中存在干细胞标记。小鼠体内实验也证实了hESC在体内维持其多能性。【结论】本研究建立了PGT来源的人胚胎干细胞系,为进一步建立携带遗传疾病基因的hESC系并为临床研究和治疗提供疾病模型打下了基础。

【Objective】A human embryonic stem cell line derived from Preimplantation genetic testing(PGT) embryos was established in a xeno-free stem cell culture system to provide disease models for medical research.【Methods】The xeno-free culture system using xeno-free human foreskin fibroblast feeder layers(XF-HFF)mixed with commercially available chemically-defined medium(CDM)was assessed.In the culture system,a new hESC cell line was established using discarded embryos derived from PGT in patients with chromosomal balance translocation.【Results】The new availa bled stem cell line was successfully cultured in the xeno-free culture system for a long time(> 45 passages). The karyotype analysis revealed that the new line kept the same karyotype over 45 passages.Moreover,the expression of pluripotent markers was detected by fluorescent immunostaining including SSEA-3,SSEA-4,SSEA-1,TRA-1-60, and TRA-1-81.RT-PCR analysis showed that the stem cell markers were present in hESC grown on XF-HFF-CDM.In addition,the teratoma formation analysis demonstrated that the cells cultured in XF-HFF/CDM maintained their pluripotency in vivo.【Conclusions】Our study may provide the possibility to establish embryonic stem cells with certain pathogenic genes,which could be applied for clinical research and treatment.