Endogenous Wntβ-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells 英文参考文献.docVIP

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Endogenous Wntβ-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells 英文参考文献.doc

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Endogenous Wntβ-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells 英文参考文献

EndogenousWnt/b-CateninSignalingIsRequiredfor CardiacDifferentiationinHumanEmbryonicStemCells SharonL.Paige1,2,3,TomoakiOsugi1,2,3,OlgaK.Afanasiev1,2,3,LilPabon1,2,3,HansReinecke1,2,3 CharlesE.Murry1,2,3,4 , * 1Department of Pathology, University of Washington, Seattle, Washington, United States of America, 2Center for Cardiovascular Biology, University of Washington, Seattle, Washington, United States of America, 3Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, United States of America,4DepartmentofBioengineering,UniversityofWashington,Seattle,Washington,UnitedStatesofAmerica Abstract Background:Wnt/b-cateninsignalingisanimportantregulatorofdifferentiationandmorphogenesisthatcanalsocontrol stemcellfates.Ourgrouphasdevelopedanefficientprotocoltogeneratecardiomyocytesfromhumanembryonicstem (ES)cellsviainductionwithactivinAandBMP4. Methodology/Principal Findings: We tested the hypothesis that Wnt/b-catenin signals control both early mesoderm inductionandlatercardiacdifferentiationinthissystem.AdditionofexogenousWnt3aatthetimeofinductionenhanced cardiac differentiation, while early inhibition of endogenous Wnt/b-catenin signaling with Dkk1 inhibited cardiac differentiation, as indicated by quantitative RT-PCR analysis for b-myosin heavy chain (b-MHC), cardiac troponin T (cTnT), Nkx2.5, and flow cytometry analysis for sarcomeric myosin heavy chain (sMHC). Conversely, late antagonism of endogenously produced Wnts enhanced cardiogenesis, indicating a biphasic role for the pathway in human cardiac differentiation. Using quantitative RT-PCR, we show that canonical Wnt ligand expression is induced by activin A/BMP4 treatment, and the extent of early Wnt ligand expression can predict the subsequent efficiency of cardiogenesis. MeasurementofBrachyuryexpressionshowedthatadditionofWnt3aenhancesmesoderminduction,whereasblockadeof endogenously produced Wnts markedly inhibits mesoderm formation. Finally, we show that Wnt/b-caten