EmbryoidBodiesFromEmbryonicStemCellsInvitro胚状体从胚胎干细胞体外培养PPT学习教案

1、会计学1EmbryoidBodiesFromEmbryonicStemCellsInvitro胚状体从胚胎干细胞体外胚状体从胚胎干细胞体外培养培养Koichiro Abe, Hitoshi Niwa, Katsuro Iwase, Masaki Takiguchi, Masataka Mori, Shin Ichi Abe, Kuniya Abe, and Ken- Ichi Yamamura第1页/共17页Ectodermal tissuesMesodermal tissuesEndodermal tissues EBs resemble the embryo of the egg- cyl

2、inder 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 s

3、uggested 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 developme

4、nt 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ES cell line, D3, culturedIncubated 3 days in Dulbeccos modified eagles medium (DMEM)15 % fetal calf serum0.1 mM 2-mercaptoethanol110 g/ml

5、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 undiffe

6、rentiated 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, 15, and 18RNA electrophoresed and transferre

7、d to Hybond-N membranesHybridizations performed and signals detected第6页/共17页RT-PCRTotal RNA from various stages, and adult mouse liver was treated with RNase-free DNase cDNA synthesisParallel reaction performed without reverse transcriptase to assess presence of DNA contamination第7页/共17页Whole-mount

8、in situ hybridization & sectioning of EBsThese experiments were carried out as described by Sasaki and HoganStained EBs were refixed in paraformaldehyde and glutaraldahyde for 30 minutesThen washed twice with PPT and placed in molten agarose, solidified, then sectioned第8页/共17页Molecular ProbesXho

9、 I fragment of R1 used as -fetoprotein (AFP) probeA fragment of Sma I Sac I fragment of pHF22.1 used as a hepatocyte nuclear factor (HNF) probe A 660-bp Pst I Pvu II fragment of pmPA1 was subcloned and used as a transthyretin (TTR) cDNA probeA variant form of HNF1 (vHNF1) probe was synthesized by PC

10、R using adult mouse liver cDNA as a templateSerum albumen (ALB) probe was also made by PCR第9页/共17页vHNF1 and HNF4 transcript levels were very low in the undifferentiated, preaggregation and day 0 EBs, then abruptly incresed at day 1HNF3 began weak, but the expression level increased after day 1TTR ex

11、pression was still low at day 1, but increased rapidly thereafter These results demonstrate that the activation of vHNF1, and HNF4 or HNF3 preceded the rise in TTR expression, beginning at day 1At day 1, ES cells formed small compact cell aggregates, lacking morphologically distinct cell populations

12、Elevation of TTR levels around day 3 coincide with the appearance of primitive endodermal cells第10页/共17页These data show that all the serum protein genes were activated at different stages, followed by a strong increase in expression, and all the transcription factor genes were not active in the undi

13、fferentiated stem cells, and began to be expressed in early phase differentiation.第11页/共17页The results of the northern blot and RT-PCR analyses demonstrated that endodermal cell differentiation occurred during the EB developmentTwo types of populations were expressed by day 5Yolk-sac-like structures

14、Hematopoietic cellsThe TTR messages were found only in the population of the EBs which began to form yolk-sac-like structures and were only found in the outer endodermal layer of the yolk-sac-like structures第12页/共17页This suggests that at least one of the endoderm marker genes is predominantly expres

15、sed in the outer layer of the yolk-sac-like structure during EB development第13页/共17页The order of gene expression for the in vivo studies is similar to that found in EB formation in vitro第14页/共17页ConclusionThese data as a whole strongly suggest that development of EBs in vitro closely resembles the sequence of in vivo (normal)