title: A Multi-Lineage Screen Reveals mTORC1 Inhibition Enhances Human Pluripotent Stem Cell Mesendoderm and Blood Progenitor Production.
abstract: Human pluripotent stem cells (hPSCs) exist in heterogeneous micro-environments with multiple subpopulations, convoluting fate-regulation analysis. We patterned hPSCs into engineered micro-environments and screened responses to 400 small-molecule kinase inhibitors, measuring yield and purity outputs of undifferentiated, neuroectoderm, mesendoderm, and extra-embryonic populations. Enrichment analysis revealed mammalian target of rapamycin (mTOR) inhibition as a strong inducer of mesendoderm. Dose responses of mTOR inhibitors such as rapamycin synergized with Bone Morphogenetic protein 4 (BMP4) and activin A to enhance the yield and purity of BRACHYURY-expressing cells. Mechanistically, small interfering RNA knockdown of RAPTOR, a component of mTOR complex 1, phenocopied the mesendoderm-enhancing effects of rapamycin. Functional analysis during mesoderm and endoderm differentiation revealed that mTOR inhibition increased the output of hemogenic endothelial cells 3-fold, with a concomitant enhancement of blood colony-forming cells. These data demonstrate the power of our multi-lineage screening approach and identify mTOR signaling as a node in hPSC differentiation to mesendoderm and its derivatives.
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3G9, Canada.
authors.forename: Emanuel Joseph Paul
authors.initials: EJP
authors.lastname: Nazareth
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3G9, Canada; Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada.
authors.forename: Nafees
authors.initials: N
authors.lastname: Rahman
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3G9, Canada.
authors.forename: Ting
authors.initials: T
authors.lastname: Yin
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3G9, Canada; Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada; Medicine by Design, University of Toronto, Toronto, ON M5S 3G9, Canada. Electronic address: peter.zandstra@utoronto.ca.
authors.forename: Peter William
authors.initials: PW
authors.lastname: Zandstra
mesh_terms: D028341:Activins / Q000235:genetics; D055415:Bone Morphogenetic Protein 4 / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects*; D019070:Cell Lineage / Q000187:drug effects*; D060833:Cellular Microenvironment / Q000187:drug effects; D004707:Endoderm / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005326:Fetal Proteins / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006801:Humans; D000076222:Mechanistic Target of Rapamycin Complex 1 / Q000037:antagonists & inhibitors* / Q000235:genetics; D054258:Neural Plate / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010770:Phosphotransferases / Q000037:antagonists & inhibitors; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000076223:Regulatory-Associated Protein of mTOR / Q000235:genetics; D020123:Sirolimus / Q000494:pharmacology; D054852:Small Molecule Libraries / Q000494:pharmacology; D020825:T-Box Domain Proteins / Q000235:genetics; D000098908:Brachyury Protein
chemical_list: D028341:Activins; D055415:Bone Morphogenetic Protein 4; D004791:Enzyme Inhibitors; D005326:Fetal Proteins; D000076222:Mechanistic Target of Rapamycin Complex 1; D010770:Phosphotransferases; D000076223:Regulatory-Associated Protein of mTOR; D020123:Sirolimus; D054852:Small Molecule Libraries; D020825:T-Box Domain Proteins; D000098908:Brachyury Protein; C525809:BMP4 protein, human; C422405:activin A