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Stem Cell Reports. 2016 May 10;6(5):679-691. doi: 10.1016/j.stemcr.2016.04.003. Epub 2016 Apr 28.

A Multi-Lineage Screen Reveals mTORC1 Inhibition Enhances Human Pluripotent Stem Cell Mesendoderm and Blood Progenitor Production.

Author information

1
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3G9, Canada.
2
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.
3
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.

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.

PMID:
27132889
PMCID:
PMC4939733
DOI:
10.1016/j.stemcr.2016.04.003
[Indexed for MEDLINE]
Free PMC Article

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