成纤维细胞生长因子支持人胚胎干细胞在无饲养层中的生长(英文)

Feeder-free growth of human embryonic stem cells supported by basic fibroblast growth factor

背景:人胚胎干细胞是一种全能型细胞,可以分化为3个胚层的组织,目前国内对其无饲养层生长的研究较少。成纤维细胞生长因子是维持胚胎干细胞不分化状态的重要因子。目的:探讨长期培养过程中不同质量浓度成纤维细胞生长因子对人胚胎干细胞未分化状态和全能性维持的影响。方法:两株人胚胎干细胞在鼠胚胎成纤维细胞条件培养基中培养3代,分别转移到含100,160,250μg/L成纤维细胞生长因子的鼠胚胎成纤维细胞条件培养基中培养8代。从培养皿中移出胚胎干细胞,用IV胶原酶消化聚集成团的细胞,观察细胞分化状态和全能性情况。收集传8代后的胚胎干细胞,种植于SCID小鼠体内。对所得细胞做形态学评估,并进行碱性磷酸酶染色、表面标记免疫组化检测、RT-PCR检测OCT-4的表达、体内致瘤情况。结果与结论:在含160,250μg/L成纤维细胞生长因子的鼠胚胎成纤维细胞条件培养基中,两株人胚胎干细胞可以保持原有性状,即细胞克隆呈圆形,核质比较高,中间大片区域为未分化细胞,周围为分化细胞;呈碱性磷酸酶强阳性表达;表达OCT-4转录因子蛋白;细胞表面标志SSEA-4,TRA-1-60,TRA-1-81均呈阳性表达;聚集成团的胚胎干细胞培养10d后形成拟胚体;种植于SCID小鼠体内可得含3个胚层组织的畸胎瘤。含100μg/L成纤维细胞生长因子的鼠胚胎成纤维细胞条件培养基不足以维持人胚胎干细胞的长期增殖,4代以后大部分细胞分化死亡。提示成纤维细胞生长因子质量浓度达160μg/L以上时,可以单独支持人胚胎干细胞的体外稳定增殖,且不影响细胞分化状态和全能性。

BACKGROUND：Human embryonic stem cells （hESCs） are pluripotent cells which may differentiate into tissues of all three germ layers.Such research as the feeder-free growth of hESCs is few in China.Fibroblast growth factor （FGF） is a major factor to maintain the undifferentiated state of hESCs.OBJECTIVE：To evaluate the ability of FGF at different concentrations in maintaining the undifferentiated state and pluripotency of hESC lines in the long-term culture.METHODS：Two cell lines of hES-8 and hES-18 were cultured with mouse embryonic fibroblast condition medium for 3 passages and then transferred into mouse embryonic fibroblast condition medium containing different concentrations of FGF：100,160,250 μg/L for 8 passages.The hESCs were removed from the petri dish,cell clusters were digested with collagenase IV and gathered.Cell differentiation and pluripotency were observed.The eighth generation of the hESCs were collected and incubated into severe combined immunodeficiency mice,so as to observe teratoma formation.Morphologies of the cells were evaluated.Alkaline phosphatase staining,surface labeling immunocytochemical analysis and RT-PCR assay method were utilized to determine the OCT-4 expression and tumorigenesis in vivo.RESULTS AND CONCLUSION：Cultured in mouse embryonic fibroblast condition medium containing 160 and 250 μg/L FGF,two cell lines of hESC could maintain undifferentiated state：Clones were round with a high ratio of nucleus to cytoplasm.Large areas in the center of clones were undifferentiated cells,while surrounding the clones were differentiated cells;Strong positive expression for alkaline phosphatase staining was observed;Two cell lines showed high levels of OCT-4 transcription factor protein;The surface markers SSEA-4,TRA-1-60,TRA-1-81 were all positive on both two lines;The hESC clusters could form embryoid body in vivo 10 days later;3 germ layers of teratomas were also obtained after implanted into severe combined immunodeficiency mice.Mouse embryonic fibroblast condition medium containing 100 μg/L FGF was not sufficient to maintain the long-term proliferation of hESCs,and most of the cells differentiated and died after 4 passages.Alone with concentration 160 μg/LbFGF or more could maintain two hESC lines undifferentiated stably in vitro,has no influence on the differentiation and totipotency of two cell lines.