类胚体中残留胚胎干细胞数量与其致瘤性相关性的初步研究

目的 探讨类胚体(EBs)中残留未分化胚胎干细胞(ESCs)的数量与其致瘤性的相关性.方法 小鼠R1胚胎干细胞株,体外类胚体诱导分化10d,流式细胞仪检测残留未分化ESCs表面标志SSEA-1阳性率.将第10天EBs消化打散后重新给予ESCs常规培养体系培养,观察EBs中残留未分化ESCs形态,流式细胞仪检测残留细胞表面标志物;第10天EBs消化打散后以104～2×106细胞量分别注射至裸鼠四肢肌肉内,观察不同细胞数量与畸胎瘤形成的相关性.结果 ESCs分化为EBs 10 d后有(13.5±0.75)%的细胞表达SSEA-1,提示存在残留未分化ESCs.残留未分化细胞生长形态呈克隆样,高表达SSEA-1等未分化ESCs标志.EBs消化打散后仅在注射2×l06个细胞的部位形成畸胎瘤,瘤体组织中存在成熟的内胚层、中胚层和外胚层组织,其余各组均未见畸胎瘤的形成.结论 胚胎干细胞分化为类胚体后仍存在残留未分化胚胎干细胞,并需要一定细胞数量才具有致瘤性.

Correlation between the amount of residual undifferentiated embryonic stem cells in embryoid bodies andits tumorigenicity

Objective To explore the correlation between the amount of residual undifferentiated embryonic stem cells （ESCs） in embryoid bodies and its tumorigenicity. Methods Mouse R1 ESCs were cultured in suspension to form embryoid bodies （EBs）. Ten days later, EBs were digested into single cells and then re-plated in standard ESCs culture condition. The residual undifferentiated embryonic stem cells surface maker SSEA-1 was examined by flow cytometry in EBs. The morphology of residual undifferentiated cells in EBs were observed, meanwhile the surface marker SSEA-1 was examined by immunofluorescent staining.EBs were digested into single cells and grouped into 104, 105, 106, 2x106, and then injected into limb muscle of nude mice. The correlation of the amount of cells and its tumorigenicity was observed. Results Residual undifferentiated ESCs were observed after EBs differentiated for 10 days, which displayed clonal morphology and expressed undifferentiated cell markers of ESCs, such as SSEA-1. The expression rate of undifferentiated cells surface marker SSEA-1 was （13.5_＋0.75）% in EBs differentiated for 10 days. Only two millions single cells harvested from EBs were able to form teratoma after being injected into muscle of nude mice for 6 weeks. Mature endoderm,mesoderm and ectoderm tissues could be found in teratoma. No teratoma fom~ed in other groups. Conclusion A certain amount residual undifferentiated ESCs still exist after differentiation of ESCs into EBs.About 2.7x105 undifferentiated cells are able to form teratoma by injecting into muscle of nude mice.