Abstract
Studies revealed that PI3K/AKT/mTOR signaling is important in the regulation of human embryonic stem cell (hESC) self-renewal and differentiation. However, its action on osteogenic differentiation of hESCs is poorly understood. We tested the effects of pharmacological PI3K/AKT/mTOR inhibitors on their potential to induce osteogenic differentiation of hESCs. Under feeder-free culture conditions, rapamycin (an mTOR inhibitor) potently inhibited the activities of mTOR and p70S6K in undifferentiated hESCs; however, LY294002 (a PI3K inhibitor) and an AKT inhibitor had no effects. Treatment with any of these inhibitors down-regulated the hESC markers Oct4 and Nanog, but only rapamycin induced the up-regulation of the early osteogenic markers BMP2 and Runx2. We also observed that hESCs differentiated when treated with FK506, a structural analog of rapamycin, but did not exhibit an osteogenic phenotype. Increases in Smad1/5/8 phosphorylation and Id1-4 mRNA expression indicated that rapamycin significantly stimulated BMP/Smad signaling. After inducing both hESCs and human embryoid bodies (hEBs) for 2-3 weeks with rapamycin, osteoblastic differentiation was further characterized by the expression of osteoblastic marker mRNAs and/or proteins (osterix, osteocalcin, osteoprotegerin, osteonectin, and bone sialoprotein), alkaline phosphatase activity, and alizarin red S staining for mineralized bone nodule formation. No significant differences in the osteogenic phenotypes of rapamycin-differentiated hESCs and hEBs were detected. Our results suggest that, among these 3 inhibitors, only rapamycin functions as a potent stimulator of osteoblastic differentiation of hESCs, and it does so by modulating rapamycin-sensitive mTOR and BMP/Smad signaling.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Biomarkers
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Bone Morphogenetic Protein Receptors / genetics
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Bone Morphogenetic Protein Receptors / metabolism
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Cell Differentiation / drug effects*
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Cell Line
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Chromones / pharmacology
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Core Binding Factor Alpha 1 Subunit / genetics
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Embryonic Stem Cells / cytology*
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Embryonic Stem Cells / drug effects
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Embryonic Stem Cells / metabolism
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Enzyme Inhibitors / pharmacology
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Homeodomain Proteins / genetics
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Humans
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Immunosuppressive Agents / pharmacology
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Inhibitor of Differentiation Protein 1 / genetics
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Intracellular Signaling Peptides and Proteins / antagonists & inhibitors
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Intracellular Signaling Peptides and Proteins / metabolism
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Morpholines / pharmacology
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Nanog Homeobox Protein
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Octamer Transcription Factor-3 / genetics
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Osteoblasts* / cytology
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Osteoblasts* / metabolism
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Osteogenesis* / drug effects
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Osteogenesis* / genetics
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Phosphatidylinositol 3-Kinases / metabolism
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Phosphoinositide-3 Kinase Inhibitors
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Protein Serine-Threonine Kinases / antagonists & inhibitors
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Protein Serine-Threonine Kinases / metabolism
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Proto-Oncogene Proteins c-akt / antagonists & inhibitors
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Proto-Oncogene Proteins c-akt / metabolism
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RNA, Messenger
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Ribosomal Protein S6 Kinases, 70-kDa / metabolism
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Signal Transduction / drug effects
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Sirolimus / pharmacology*
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Smad Proteins / metabolism
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TOR Serine-Threonine Kinases
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Tacrolimus / pharmacology
Substances
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Biomarkers
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Chromones
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Core Binding Factor Alpha 1 Subunit
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Enzyme Inhibitors
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Homeodomain Proteins
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ID1 protein, human
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Immunosuppressive Agents
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Inhibitor of Differentiation Protein 1
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Intracellular Signaling Peptides and Proteins
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Morpholines
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NANOG protein, human
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Nanog Homeobox Protein
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Octamer Transcription Factor-3
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POU5F1 protein, human
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Phosphoinositide-3 Kinase Inhibitors
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RNA, Messenger
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Smad Proteins
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2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
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MTOR protein, human
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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Ribosomal Protein S6 Kinases, 70-kDa
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TOR Serine-Threonine Kinases
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Bone Morphogenetic Protein Receptors
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Sirolimus
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Tacrolimus