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

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

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.

MeSH terms

  • Activins / genetics
  • Bone Morphogenetic Protein 4 / genetics
  • Cell Differentiation / drug effects*
  • Cell Lineage / drug effects*
  • Cellular Microenvironment / drug effects
  • Endoderm / drug effects
  • Endoderm / metabolism
  • Enzyme Inhibitors / pharmacology
  • Fetal Proteins / genetics
  • Gene Expression Regulation, Developmental / drug effects
  • Humans
  • Mechanistic Target of Rapamycin Complex 1 / antagonists & inhibitors*
  • Mechanistic Target of Rapamycin Complex 1 / genetics
  • Neural Plate / cytology
  • Neural Plate / drug effects
  • Neural Plate / metabolism
  • Phosphotransferases / antagonists & inhibitors
  • Pluripotent Stem Cells / cytology
  • Pluripotent Stem Cells / drug effects*
  • Pluripotent Stem Cells / metabolism
  • Regulatory-Associated Protein of mTOR / genetics
  • Sirolimus / pharmacology
  • Small Molecule Libraries / pharmacology
  • T-Box Domain Proteins / genetics

Substances

  • BMP4 protein, human
  • Bone Morphogenetic Protein 4
  • Enzyme Inhibitors
  • Fetal Proteins
  • Regulatory-Associated Protein of mTOR
  • Small Molecule Libraries
  • T-Box Domain Proteins
  • activin A
  • Activins
  • Phosphotransferases
  • Mechanistic Target of Rapamycin Complex 1
  • Brachyury protein
  • Sirolimus

Grant support