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目的:利用多囊卵巢综合征(PCOS)患者体外受精周期中废弃胚胎建立人胚胎干细胞(hESCs)系。方法:收集PCOS患者体外受精-胚胎移植周期中废弃的新鲜或冷冻胚胎,序贯培养至囊胚,机械分离内细胞团,接种于混合饲养层(小鼠胚胎成纤维细胞∶人包皮成纤维细胞为1∶1),传代培养。对稳定传代的hESCs进行形态学观察、碱性磷酸酶染色、阶段特异性胚胎抗原-4、转录因子OCT-4的表达、核型分析、体内、外分化潜能鉴定。结果:①收集到59枚新鲜废胚,培养后获得14枚囊胚,分离11个内细胞团,贴壁、传代后建立5株hESCs系,均具备hESC特性。②收集到解冻胚胎19枚,培养后获得6枚囊胚,分离5个内细胞团,形成5个原代克隆,目前传至3~4代。结论:建立了PCOS源性hESCs系,为研究PCOS发病机理、筛选候选基因、基因定位及基因治疗等提供良好的生物学模型;废弃的新鲜及冷冻胚胎均可作为hESC建系的材料来源。
OBJECTIVE: To establish human embryonic stem cells (hESCs) lines from abandoned embryos in the in vitro fertilization cycle of patients with polycystic ovary syndrome (PCOS). Methods: Fresh or frozen embryos discarded during the in vitro fertilization-embryo transfer cycle of PCOS patients were collected and sequentially cultured to blastocysts. The inner cell masses were mechanically separated and seeded on the mixed feeder layer (mouse embryonic fibroblasts: human foreskin fibroblasts 1: 1), subculture. Morphological observation, alkaline phosphatase staining, stage-specific embryonic antigen-4, expression of transcription factor OCT-4, karyotype analysis, and identification of in vitro and in vivo differentiation potential were performed on stable passaged hESCs. RESULTS: Fifty-nine fresh embryos were collected and 14 blastocysts were obtained. Eleven inner cell masses were isolated and adherent. Five hESCs lines were established after passage, all of which had hESC characteristics. (2) Nineteen thawed embryos were collected, six blastocysts were obtained after culturing, and five inner cell masses were separated to form five primary clones. At present, three to four generations were obtained. CONCLUSION: A PCOS-derived hESCs line was established to provide a good biological model for studying the pathogenesis of PCOS, screening candidate genes, gene mapping and gene therapy. Abandoned fresh and frozen embryos can be used as materials for hESC lineage.