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

• Cell patterning enables quantitative yield and frequency analysis of hPSC fate
• A kinase inhibitor library is used to identify key PSC differentiation pathways
• mTOR inhibition enhances mesendoderm, hemogenic endothelium, and blood progenitors
• siRNA knockdown of RAPTOR, an mTORC1 component, phenocopies mTOR inhibition

SummaryHuman 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.

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