EmbryoidBodiesFromEmbryonicStemCellsInvitro：胚状体从胚胎干细胞体外培养课件

1、Embryoid Bodies From Embryonic Stem Cells In-vitroRobert ChristensenBiology DepartmentEastern Connecticut State University第1页，共17页。EndodermSpecific Gene Expression in Embryonic StemCells Differentiated To Embryoid BodiesExperimental Cell Research 229 (1999): pp 27-34Koichiro Abe, Hitoshi Niwa, Katsu

2、ro Iwase, Masaki Takiguchi, Masataka Mori, Shin Ichi Abe, Kuniya Abe, and Ken- Ichi Yamamura第2页，共17页。IntroductionCell lineages arise during developmentEctodermal tissuesMesodermal tissuesEndodermal tissues EBs resemble the embryo of the egg- cylinder stageNeuronal cellsCardiac muscle cellsHematopoie

3、tic cellsYolk sac cells ES cells have full developmental potential EBs from ES cells EBs consist of: Later stages EBs are composed of:第3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-v

4、itro 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 syst

5、ematically characterized temporal expression patterns of endoderm marker genes during EB formation.第4页，共17页。Materials and MethodsCell culturesES cell line, D3, culturedIncubated 3 days in Dulbeccos modified eagles medium (DMEM)15 % fetal calf serum0.1 mM 2-mercaptoethanol110 g/ml sodium pyruvate4.5

6、mg/ml D-glucoseIs supplemented with 1000 units/ml recombinant murine leukemia inhibitory factor (LIF)第5页，共17页。LIF was withdrawnThe cells were allowed to aggregateThe DMEM was changed every other day throughout the culture第6页，共17页。Northern Blot AnalysisTotal RNA prepared from undifferentiated ES cell

7、s, 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 transferred to Hybond-N mem

8、branesHybridizations performed and signals detected第7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8页，共17页。Whole-mount in situ hybridi

9、zation & 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9页，共17页。Molecular ProbesXho I fragment of R1

10、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 PCR using adult mous

11、e liver cDNA as a templateSerum albumen (ALB) probe was also made by PCR第10页，共17页。ResultsvHNF1 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 expression

12、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 populationsElevation

13、 of TTR levels around day 3 coincide with the appearance of primitive endodermal cells第11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 undifferenti

14、ated stem cells, and began to be expressed in early phase differentiation.第12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 structuresHematop

15、oietic 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13页，共17页。This suggests that at least one of the endoderm marker genes is predominantly expressed in

16、 the outer layer of the yolk-sac-like structure during EB development第14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15页，共17页。ConclusionThese data as a whole strongly suggest that development of EBs in vitro closely resembles the sequence of in v