title: Generating high-purity cardiac and endothelial derivatives from patterned mesoderm using human pluripotent stem cells.
abstract: Human pluripotent stem cells (hPSCs) provide a valuable model for the study of human development and a means to generate a scalable source of cells for therapeutic applications. This protocol specifies cell fate efficiently into cardiac and endothelial lineages from hPSCs. The protocol takes 2 weeks to complete and requires experience in hPSC culture and differentiation techniques. Building on lessons taken from early development, this monolayer-directed differentiation protocol uses different concentrations of activin A and bone morphogenetic protein 4 (BMP4) to polarize cells into mesodermal subtypes that reflect mid-primitive-streak cardiogenic mesoderm and posterior-primitive-streak hemogenic mesoderm. This differentiation platform provides a basis for generating distinct cardiovascular progenitor populations that enable the derivation of cardiomyocytes and functionally distinct endothelial cell (EC) subtypes from cardiogenic versus hemogenic mesoderm with high efficiency without cell sorting. ECs derived from cardiogenic and hemogenic mesoderm can be matured into >90% CD31+/VE-cadherin+definitive ECs. To test the functionality of ECs at different stages of differentiation, we provide methods for assaying the blood-forming potential and de novo lumen-forming activity of ECs. To our knowledge, this is the first protocol that provides a common platform for directed differentiation of cardiomyocytes and endothelial subtypes from hPSCs. This protocol yields endothelial differentiation efficiencies exceeding those of previously published protocols. Derivation of these cell types is a critical step toward understanding the basis of disease and generating cells with therapeutic potential.
authors.affiliation: The Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Palpant
authors.affiliation: Department of Pathology, University of Washington School of Medicine, Seattle, Washington, USA.|Center for Cardiovascular Biology, University of Washington School of Medicine, Seattle, Washington, USA.|Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Lil
authors.initials: L
authors.lastname: Pabon
authors.affiliation: The Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia.|Center for Cardiovascular Biology, University of Washington School of Medicine, Seattle, Washington, USA.|Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Clayton E
authors.initials: CE
authors.lastname: Friedman
authors.affiliation: Center for Cardiovascular Biology, University of Washington School of Medicine, Seattle, Washington, USA.|Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington, USA.|Department of Bioengineering, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Meredith
authors.initials: M
authors.lastname: Roberts
authors.affiliation: The Clinical Research Division, Fred Hutchinson Cancer Research Center, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Brandon
authors.initials: B
authors.lastname: Hadland
authors.affiliation: Center for Cardiovascular Biology, University of Washington School of Medicine, Seattle, Washington, USA.|Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington, USA.|Department of Bioengineering, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Zaunbrecher
authors.affiliation: The Clinical Research Division, Fred Hutchinson Cancer Research Center, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Irwin
authors.initials: I
authors.lastname: Bernstein
authors.affiliation: Center for Cardiovascular Biology, University of Washington School of Medicine, Seattle, Washington, USA.|Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington, USA.|Department of Bioengineering, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Department of Pathology, University of Washington School of Medicine, Seattle, Washington, USA.|Center for Cardiovascular Biology, University of Washington School of Medicine, Seattle, Washington, USA.|Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington, USA.|Department of Bioengineering, University of Washington School of Medicine, Seattle, Washington, USA.|Division of Cardiology, Department of Medicine, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Murry
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D042783:Endothelial Cells / Q000166:cytology*; D006801:Humans; D008648:Mesoderm / Q000166:cytology*; D009206:Myocardium / Q000166:cytology*; D039904:Pluripotent Stem Cells / Q000166:cytology*