Rapamycin promotes the osteoblastic differentiation of human embryonic stem cells by blocking the mTOR pathway and stimulating the BMP/Smad pathway.
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.
Lee
Kyu-Won
KW
Development and Differentiation Research Center, KRIBB, Daejeon, Republic of Korea.
Yook
Jin-Yong
JY
Son
Mi-Young
MY
Kim
Min-Jeong
MJ
Koo
Deog-Bon
DB
Han
Yong-Mahn
YM
Cho
Yee Sook
YS
D015415:Biomarkers; D052004:Bone Morphogenetic Protein Receptors / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D051798:Inhibitor of Differentiation Protein 1 / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D012333:RNA, Messenger; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology*; D051785:Smad Proteins / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases; D016559:Tacrolimus / Q000494:pharmacology
D015415:Biomarkers; D052004:Bone Morphogenetic Protein Receptors; D002867:Chromones; D050659:Core Binding Factor Alpha 1 Subunit; D004791:Enzyme Inhibitors; D018398:Homeodomain Proteins; D007166:Immunosuppressive Agents; D051798:Inhibitor of Differentiation Protein 1; D047908:Intracellular Signaling Peptides and Proteins; D009025:Morpholines; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D020123:Sirolimus; D051785:Smad Proteins; D058570:TOR Serine-Threonine Kinases; D016559:Tacrolimus; C497453:ID1 protein, human; C486780:NANOG protein, human; C494173:POU5F1 protein, human; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C546842:MTOR protein, human