人胚胎干细胞体外培养并分化为心肌细胞

目的 :体外培养并鉴定人胚胎干细胞 (EG细胞 ) ,在不添加细胞因子的条件下 ,观察细胞集落分化情况。方法 :取 4～ 6周人胚胎生殖嵴、肠背系膜和中肾嵴 ,进行组织块培养 ,利用组织化学和免疫组织化学技术对培养的细胞进行鉴定。结果 :培养 7～ 8小时 ,在组织块周围出现成纤维细胞 ;5～ 7天后 ,在成纤维细胞上出现EG细胞集落 ,集落内细胞表达阶段特异性胚胎表面抗原SSEA 1和SSEA 3,并表达碱性磷酸酶 (ALP)活性 ;培养 1 8天后 ,有6个集落分化为有节律跳动的心肌细胞 ,这些细胞集落在继续培养 1 5天后 ,仍能有节律收缩。此时集落内细胞ALP表达呈阴性 ,但仍有少量细胞表达SSEA 1和SSEA 3。结论 :体外培养人胚生殖嵴、肠背系膜和中肾嵴组织块 ,以内源性的成纤维细胞作为饲养层 ,可以分离得到EG细胞集落 ,一些EG细胞集落可以自发地分化成心肌细胞。

Cultivation of human embryonic stem cells and differentiation into cardiomyocytes

Objective:To culture human embryonic stem cell (EG cell) in vitro and to observe the differentiation of cultures without any cytokines in presence.Methods:Human embryonic (4-6 weeks postfertilization) genital ridges,mesenteries and mesonephros ridge were cultured.Histochemistry and immunohistochemistry were used to identify the cultures.Results:Cultured for 7-8 h,fibroblasts appeared around the tissue fragments.After 5-7 d,EG cluster grew on the fibroblasts and expressed the cell surface markers SSEA-1,SSEA-3 and alkaline phosphatase (ALP) was positive.After 18 d,there were 6 cell clusters differentiated into cardiomyocytes which could beat rhythmically even after another continuous culture of 15 d.ALP was negative in these cell clusters but there were still a few cells which could remain SSEA-1 and SSEA-3 positive.Conclusion:Culturing human genital ridges,mesenteries and mesonephros ridges in tissue using the endogenous fibroblasts as feeder cell layer,human stem cells (EG cells) clusters can be obtained,and some of the clusters can spontaneously differentiate into cardiomyocytes.