小鼠胚胎及人包皮成纤维细胞按比例制成混合饲养层上的人胚胎干细胞生长状态

临床医学
中国组织工程研究与临床康复 第 12 卷 第 3 期 2008–01–15 出版
Journal of Clinical Rehabilitative Tissue Engineering Research January 15, 2008 Vol.12, No.3
小鼠胚胎及人包皮成纤维细胞按比例制成混合饲养层上的人胚胎干细胞 生长状态★
李 斌1,2，彭秋平2，卢伟英1,2，徐 雯1,2，金应霞2，黄元华1,2
Growth state of human embryonic stem cells on mixed feeder layers with mouse embryonic fibroblasts and human foreskin fibroblasts at different ratios
Li Bin1,2, Peng Qiu-ping2, Lu Wei-ying1,2, Xu Wen1,2, Jin Ying-xia1, Huang Yuan-hua1,2
1
Abstract
AIM: The key of the human embryonic stem cell culture is to guarantee the totipotency and inhibit spontaneous differentiation. There are the problems in the traditional methods that human embryonic stem cells were cultured on the mouse embryonic fibroblasts or human foreskin fibroblasts as feeder layer. We aimed to solve the problems, observe growth state of human embryonic stem cells when it was cultured on the mixed feeder layers of different proportion. METHODS：Experiments were completed in Reproductive Medical Center of Hainan Medical College from April 2006 to July 2007. ①The foreskin was from the boy who was circumcised, provided by Department of Urinary Surgery of Hospital Affiliated to Hainan Medical College. Child guardian signed an informed consent of the treatment and experiment and the experiment was approved by the hospital medical ethics committee. Human embryonic stem cell line (HN-1) was isolated from human blastocysts and identified. Eleven clean fetal mice of 12.5-14.5 d were collected and the disposal of animal conformed to the animal ethics standards during the experiments. ②The fetal mice whose heads, limbs and internal organs were removed were repeatedly digested by the trypsin to obtain cells, then cells were cultured. Parts of the original cells were cryopreserved after confluence. It was the mouse embryonic fibroblast feeder layer that cells which were treated for 2.0-3.0 h with mitomycin C were cultured in the center plate coated by gelatin at 1×108 L-1. Isolation, culture and preparation of feeder layer of human foreskin fibroblast were the same as above. Mixed feeder layer was that cells which were mixed according to 1∶0，3∶1，1∶1，1∶3，0∶1 were cultured in the center plate coated by gelatin at 1×108 L-1. ③Growth states of human embryonic stem cells were observed in three different feeder layers and undifferentiated phenotypes were detected, including expression of alkaline phosphatase, and presence of OCT-4, Tert and cell marker (OCT-4). Without feeder layer, human embryonic stem cell differentiation was observed in vitro. RESULTS：①Comparison of human embryonic stem cell growth state on different feeder layers: Colonies of human embryonic stem cells on the mouse embryonic fibroblast feeder layer and human foreskin fibroblast feeder layer was both flat and thin, but those on the mixed cells feeder layer were full and thick. The clonal morphology of the mixed feeder layer, overall, was significantly better than others. ②Comparison of human embryonic stem cell growth state on the mixed feeder layers prepared by different ratios: When mouse embryonic fibroblasts and human foreskin fibroblasts were mixed at a ratio of 1∶1 human embryonic stem cells accumulated significantly and cloning edge was clear, obvious and full. No significant changes were found at 1∶3. It was significantly better than others. ③Detection of human embryonic stem cell undifferented phenotypes on mixed feeder layer: Expression of alkaline phosphatase and OCT-4 antigen were strongly positive. Specific bands of OCT-4 and telomerase mRNA appeared respectively on 200-300 bp and 300-400 bp. ④Differentiation experiment in vitro: Human embryonic stem cells on mixed feeder layer was able to form embryoid bodies and differentiate into a variety of cells. CONCLUSION： ①Comparison with conventional feeder layers prepared by mouse embryonic fibroblasts or human foreskin fibroblasts, the mixed cells feeder layer may be better suitable for human embryonic stem cell culture in vitro and obtain better human embryonic stem cell clonal morphology. ②The feeder layer mixed at a ratio of 1∶1 can get better effect. Li B, Peng QP, Lu WY, Xu W, Jin YX, Huang YH.Growth state of human embryonic stem cells on mixed feeder layers with mouse embryonic fibroblasts and human foreskin fibroblasts at different ratios.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12 (3):424-428(China) [/zglckf/ejournal/upfiles/08-3/3k-424(ps).pdf] 摘要
目的：人胚胎干细胞传代培养的关键是抑制其自发分化、保证细胞的全能性。欲解决以小鼠胚胎成纤维细胞或人包皮成纤维 细胞为常规饲养层培养人胚胎干细胞时存在的问题，观察两者按一定比例制成的混合饲养层上人胚胎干细胞的生长状态。 方法：实验于 2006-04/2007-07 在海南医学院附属医院生殖医学中心完成。①对象：包皮来自于行包皮环切的儿童，由海南 医学院附属医院泌尿外科提供，儿童家属对治疗及实验均签署知情同意书，实验经医院医学伦理委员会批准。人胚胎干细胞 系 HN-1 由本实验室从人类囊胚中分离培养并鉴定。清洁级孕 12.5~14.5 d 的胎鼠 11 只，实验过程中对动物的处置符合动物 伦理学标准。②实验方法：将去除头、四肢和内脏的胎鼠按常规胰蛋白酶反复消化法获得细胞悬液进行接种培养，待生长汇 合后冻存部分原代细胞，用丝裂霉素 C 处理 2.0~3.0 h 后，按 1×108 L-1 密度接种于明胶包被的中心皿内，即小鼠胚胎成纤维 细胞饲养层。人包皮成纤维细胞的分离培养与饲养层制备同上。上述两种成纤维细胞分别计数后，按 1∶0，3∶1，1∶1， 1∶3，0∶1 比例混合，然后以 1×108 L-1 密度接种于明胶包被的中心皿内，即混合饲养层。③实验评估：观察体外传代培养 的人胚胎干细胞在 3 种不同饲养层上的生长状态。并对生长在混合饲养层上的人胚胎干细胞进行碱性磷酸酶检测、OCT-4 表 达免疫组化检测、OCT-4 及端粒酶 mRNA 表达 RT-PCR 检测。撤除饲养层，观察人胚胎干细胞体外分化情况。 结果：①不同饲养层上人胚胎干细胞的生长状态比较：生长在小鼠胚胎成纤维细胞和人包皮成纤维细胞上的人胚胎干细胞克隆 扁平、不饱满，而生长在混合饲养层上的人胚胎干细胞克隆饱满、厚实，其克隆形态显著好于其他两种饲养层。②人胚胎干细 胞在不同比例混合饲养层上的生长状态比较：小鼠胚胎成纤维细胞：人包皮成纤维细胞按 1∶1 混合时，人胚胎干细胞显著堆积 生长，克隆边缘清晰、隆起明显且饱满，按 1∶3 混合时无明显变化，优于其余 3 种混合比例。③混合饲养层上人胚胎干细胞未 分化状态的检测：碱性磷酸酶染色及 OCT-4 抗原表达均呈强阳性，分别在 200~300 bp 和 300~400 bp 处可见 OCT-4 和端粒酶
Hainan Medical College, Haikou 571101, Hainan Province, China; 2 Reproductive Medical Center, Affiliated Hospital of Hainan Medical College, Haikou 570102, Hainan Province, China Li Bin ★ , Master, Assistant, Hainan Medical College, Haikou 571101, Hainan Province, China；Reproductive Medical Center, Affiliated Hospital of Hainan Medical College, Haikou 570102, Hainan Province, China libinliccc@ Correspondence to: Huang Yuan-hua, Master, Professor, Hainan Medical College, Haikou 571101, Hainan Province, China; Reproductive Medical Center, Affiliated Hospital of Hainan Medical College, Haikou 570102, Hainan Province, China huang_yuanhua@ Received:2007-08-22 Accepted:2007-11-22
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