Despite the recent identification of the transcriptional regulatory circuitry involving SOX2, NANOG, and OCT-4, the intracellular signaling networks that control pluripotency of human embryonic stem cells (hESCs) remain largely undefined. Here, we demonstrate an essential role for the serine/threonine protein kinase mammalian target of rapamycin (mTOR) in regulating hESC long-term undifferentiated growth. Inhibition of mTOR impairs pluripotency, prevents cell proliferation, and enhances mesoderm and endoderm activities in hESCs. At the molecular level, mTOR integrates signals from extrinsic pluripotency-supporting factors and represses the transcriptional activities of a subset of developmental and growthinhibitory genes, as revealed by genome-wide microarray analyses. Repression of the developmental genes by mTOR is necessary for the maintenance of hESC pluripotency. These results uncover a novel signaling mechanism by which mTOR controls fate decisions in hESCs. Our findings may contribute to effective strategies for tissue repair and regeneration.differentiation ͉ pluripotency ͉ OCT-4 ͉ long-term undifferentiated growth

SummaryThe gap in knowledge of the molecular mechanisms underlying differentiation of human pluripotent stem cells (hPSCs) into the mesenchymal cell lineages hinders the application of hPSCs for cell-based therapy. In this study, we identified a critical role of muscle segment homeobox 2 (MSX2) in initiating and accelerating the molecular program that leads to mesenchymal stem/stromal cell (MSC) differentiation from hPSCs. Genetic deletion of MSX2 impairs hPSC differentiation into MSCs. When aided with a cocktail of soluble molecules, MSX2 ectopic expression induces hPSCs to form nearly homogeneous and fully functional MSCs. Mechanistically, MSX2 induces hPSCs to form neural crest cells, an intermediate cell stage preceding MSCs, and further differentiation by regulating TWIST1 and PRAME. Furthermore, we found that MSX2 is also required for hPSC differentiation into MSCs through mesendoderm and trophoblast. Our findings provide novel mechanistic insights into lineage specification of hPSCs to MSCs and effective strategies for applications of stem cells for regenerative medicine.

Stem cells derived from adult tissues or from the inner cell mass of blastocyst-stage embryos can self-renew in culture and have the remarkable potential to undergo lineage-specific differentiation. Extensive studies have been devoted to achieving a better understanding of the soluble factors and the mechanism(s) by which they regulate the fate decisions of these cells, but it is only recently that a critical role has been revealed for physical and mechanical factors in controlling self-renewal and lineage specification. This review summarizes selected aspects of current work on stem cell mechanics with an emphasis on the influence of matrix stiffness, surface topography, cell shape and mechanical forces on the fate determination of mesenchymal stem cells and embryonic stem cells.