EmbryoidBodiesFromEmbryonicStemCellsInvitro胚状体从胚胎干细胞体外培养.pptxVIP

会计学1EmbryoidBodiesFromEmbryonicStemCellsInvitro胚状体从胚胎干细胞体外培养 Endoderm–Specific Gene Expression in Embryonic Stem–Cells Differentiated To Embryoid BodiesExperimental Cell Research 229 (1999): pp 27-34Koichiro Abe, Hitoshi Niwa, Katsuro Iwase, Masaki Takiguchi, Masataka Mori, Shin– Ichi Abe, Kuniya Abe, and Ken- Ichi Yamamura第1页/共17页 IntroductionCell lineages arise during developmentEctodermal tissuesMesodermal tissuesEndodermal tissues EBs resemble the embryo of the egg- cylinder stageNeuronal cellsCardiac muscle cellsHematopoietic cellsYolk sac cells ES cells have full developmental potential EBs from ES cells EBs consist of: Later stages EBs are composed of:第2页/共17页 Past studies showed specific changes in expression of endoderm marker genes during EB development and suggested that EB formation could be considered an in-vitro model for endoderm differentiation.RNA blotReverse transcription polymerase chain reaction (RT-PCR)In-situ hybridizationThe nature of endoderm differentiation in-vitro is discussed in relation to normal embryonic and extraembryonic development in-vivo.This paper extended their analysis and systematically characterized temporal expression patterns of endoderm marker genes during EB formation.第3页/共17页 Materials and MethodsCell culturesES cell line, D3, culturedIncubated 3 days in Dulbecco’s modified eagles medium (DMEM)15 % fetal calf serum0.1 mM 2-mercaptoethanol110 μg/ml sodium pyruvate4.5 mg/ml D-glucoseIs supplemented with 1000 units/ml recombinant murine leukemia inhibitory factor (LIF)第4页/共17页 LIF was withdrawnThe cells were allowed to aggregateThe DMEM was changed every other day throughout the culture第5页/共17页 Northern Blot AnalysisTotal RNA prepared from undifferentiated ES cells, preaggregation ES cells, and EBs of various stages1 whole dish of EBs was used for isolation of total RNASamples were collected from undifferentiated ES cells, preaggregation phase cells, and EBs at days 0, 1, 2, 3, 4, 5, 7, 9, 11, 13,