title: Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells.
abstract: Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization, but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line, where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter, we screened for > 60 bioactive small molecules that would promote endothelial differentiation, and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated > 50% conversion of hPSCs to endothelial cells (ECs), specifically VEC(+)CD31(+)CD34(+)CD14(-)KDR(high) endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling, in combination with VEGF-A treatment, resulted in efficient formation of EPs via KDR(+) mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods, which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs.
authors.affiliation: 1] Department of Stem Cell and Regenerative Biology, 7 Divinity Avenue, Cambridge, MA 02138, USA [2] Harvard Stem Cell Institute, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA [3] Center for Regenerative Medicine, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 021141, USA [4] Department of Medicine-Cardiology/Cell and Molecular Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
authors.forename: Makoto
authors.initials: M
authors.lastname: Sahara
authors.affiliation: 1] Department of Stem Cell and Regenerative Biology, 7 Divinity Avenue, Cambridge, MA 02138, USA [2] Harvard Stem Cell Institute, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA.
authors.forename: Emil M
authors.initials: EM
authors.lastname: Hansson
authors.affiliation: Department of Anesthesiology and Intensive Care Medicine, Charité-University Medicine Berlin, Campus Charité Mitte, Charitéplatz 1, 10117 Berlin, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Wernet
authors.affiliation: 1] Department of Stem Cell and Regenerative Biology, 7 Divinity Avenue, Cambridge, MA 02138, USA [2] Harvard Stem Cell Institute, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA [3] Center for Regenerative Medicine, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 021141, USA.
authors.forename: Kathy O
authors.initials: KO
authors.lastname: Lui
authors.affiliation: 1] Department of Stem Cell and Regenerative Biology, 7 Divinity Avenue, Cambridge, MA 02138, USA [2] Harvard Stem Cell Institute, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA [3] Center for Regenerative Medicine, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 021141, USA.
authors.forename: Daniela
authors.initials: D
authors.lastname: Später
authors.affiliation: 1] Department of Stem Cell and Regenerative Biology, 7 Divinity Avenue, Cambridge, MA 02138, USA [2] Harvard Stem Cell Institute, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA [3] Department of Medicine-Cardiology/Cell and Molecular Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Chien
mesh_terms: D000818:Animals; D015703:Antigens, CD; D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology; D015820:Cadherins; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology*; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D051379:Mice; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D051880:Receptors, Notch / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000502:physiology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000502:physiology; D000099152:Cadherin 5
chemical_list: D015703:Antigens, CD; D055415:Bone Morphogenetic Protein 4; D015820:Cadherins; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D051880:Receptors, Notch; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; D000099152:Cadherin 5; C000605506:GSK3B protein, human; C000605507:Gsk3b protein, mouse