诱导的多潜能干细胞类胚体分化过程中残留未分化细胞的研究

目的：探讨诱导的多潜能干细胞（induced pluri potent stem cells,iPS cells）通过类胚体长期分化后残留未分化细胞的特性。方法：小鼠iPS细胞株,体外类胚体分化20天后消化打散,重新给予i PS细胞常规培养液培养。观察扩增的残留细胞形态;流式细胞仪和免疫荧光染色检测和观察残留细胞表面标志物及体外再次分化能力。将残留细胞扩增后注射入裸鼠背部皮下,6周后注射部位取材进行大体和组织学检查。结果：分化20天的类胚体中存在残留未分化的细胞,呈克隆样生长,高度表达SSEA-1、CD-9和OCT-4等多潜能性标志。残留细胞能反复传代,并可在体外再次分化和残留。残留细胞注射部位形成畸胎瘤,瘤体组织中存在成熟的内胚层、中胚层和外胚层组织。结论：iPS细胞分化为类胚体后残留部分未分化细胞,残留细胞在体内、外可再次分化,并能在体外分化中再次残留。

Residual undifferentiated cells during embryoid bodies differentiation of induced pluripotent stem cells

Objective To explore the residual undifferentiated cells during embryoid bodies differentiation of induced pluripotent stem cells.Methods Mouse iPS were firstly cultured in suspension to form embryoid bodies（EBs）.Twenty days later,EBs were digested into single cells and then re-plated in standard iPS cells culture condition.The morphology of residual undifferentiated cells in EBs was observed,while surface makers and in vitro redifferentiation potency of residual cells were examined by flow cytometry and immunofluorescent staining.The residual cells were expanded and subcutaneously injected into nude mice,and the specimens were harvested from the injection site for histological analysis 6 weeks after injection.Results A certain amount of residual undifferentiated cells in EBs differentiated for 20 days,which displayed clonal morphology and expressed undifferentiated cell markers including SSEA-1,CD-9 and OCT-4.Amplified cells could form secondary EBs in votro again,from which secondary undifferentiated cells could be re-expanded.Teratomas formed after amplified undifferentiated cells being injected into nude mice,which contain mature endoderm,mesoderm and ectoderm tissues.Conclusion A certain amount of undifferentiated cells exists in differentiated EBs of iPS cells.The residual cells can differentiate again in vitro and vivo,and can residue again in the in vitro differentiation.