naa a22 4500 51073958X CHVBK 20180411083023.0 cr unu---uuuuu 180411e20131001xx s 000 0 eng 10.1007/s12015-013-9436-5 doi (NATIONALLICENCE)springer-10.1007/s12015-013-9436-5 Wnt Signaling Behaves as a "Master Regulator” in the Osteogenic and Adipogenic Commitment of Human Amniotic Fluid Mesenchymal Stem Cells [Elektronische Daten] [Iolanda D'Alimonte, Angela Lannutti, Caterina Pipino, Pamela Di Tomo, Laura Pierdomenico, Eleonora Cianci, Ivana Antonucci, Marco Marchisio, Mario Romano, Liborio Stuppia, Francesco Caciagli, Assunta Pandolfi, Renata Ciccarelli] Human amniotic fluid mesenchymal stem cells (huAFMSCs) are emerging as a promising therapeutic option in regenerative medicine. Here, we characterized huAFMSC phenotype and multipotentiality. When cultured in osteogenic medium, huAFMSC displayed a significant increase in: Alkaline Phosphatase (ALP) activity and mRNA expression, Alizarin Red S staining and Runx2 mRNA expression; whereas maintaining these cells in an adipogenic culture medium gave a time-dependent increase in PPARγ and FABP4 mRNA expression, glycerol-3-phosphate dehydrogenase (GPDH) activity and positivity to Oil Red Oil staining. These results confirm that huAFMSCs can differentiate toward osteogenic and adipogenic phenotypes. The canonical Wnt/ßcatenin signaling pathway appears to trigger huAFMSC osteoblastogenesis, since during early phases of osteogenic differentiation, the expression of Dishevelled-2 (Dvl-2), of the non-phosphorylated form of ß-catenin, and the phosphorylation of glycogen synthase kinase-3ß (GSK3ß) at serine 9 were upregulated. On the contrary, during adipogenic differentiation Dvl-2 expression decreased, whereas that of ß-catenin remained unchanged. This was associated with a late increase in GSK3ß phosphorylation. Consistent with this scenario, huAFMSCs exposure to Dickkopf-1, a selective inhibitor of the Wnt signaling, abolished Runx2 and ALP mRNA upregulation during huAFMSC osteogenic differentiation, whereas it enhanced FABP4 expression in adipocyte-differentiating cells. Taken together, these results unravel novel molecular determinants of huAFMSC commitment towards osteoblastogenesis, which may represent potential targets for directing the differentiation of these cells and improving their use in regenerative medicine. Figure Schematic representation of Wnt pathway involved in the osteogenic and adipogenic differentiation of huAFMSCs. Our paper demonstrates that osteogenic commitment of these cells is linked to the stimulation of Wnt signal leading to the final transcriptional activation of early osteogenic markers such as RUNX-2 and ALP, mediated by β-catenin. DKK1 is a secreted Wnt antagonist that may be used as a drug to inhibit Wnt signal. In contrast, adipogenic commitment involves early inhibition of Wnt pathway leading to ubiquitination/degradation of β-catenin. This results in the transcription of PPARγ and FABP4, considered as the main initiators of adipogenesis. APC, adenomatous polyposis coli; βcat, β-catenin; CK1, casein kinase 1; DKK1, dickkopf 1; Dvl, Dishevelled; GSK3β, glycogen synthase kinase 3β; LRP5/6, low density lipoprotein receptor-related protein 5/6 The Author(s), 2013 Human amniotic fluids nationallicence Mesenchymal stem cells nationallicence Osteogenic/adipogenic differentiation nationallicence Wnt signaling nationallicence AF : Amniotic fluid nationallicence APC : Allophycocyanin-conjugated nationallicence ALP : Alkaline phosphatase nationallicence ARS : Alizarin Red S nationallicence DKK-1 : Dickkopf-1 nationallicence DMEM : Dulbecco-Modified Essential Medium nationallicence Dvl : Dishevelled nationallicence FABP4 : Fatty acid binding protein 4 nationallicence FBS : Foetal Bovine Serum nationallicence FITC : Fluorescein isothiocyanate-conjugated nationallicence GPDH : Glycerol-3-phosphate dehydrogenase nationallicence GSK-3β : Glycogen synthase kinase-3β nationallicence hu-AFMSCs : Human-amniotic fluid derived mesenchymal stem cells nationallicence IBMX : 3-isobutyl-1-methylxanthine nationallicence MEM : α-Minimal Essential Medium nationallicence MFI : Mean Fluorescence Intensity nationallicence MSCs : Mesenchymal stem cells nationallicence PE : Phycoerithrin nationallicence PPARγ : Peroxisome proliferator-activated receptor-γ nationallicence PBS : Phosphate saline buffer nationallicence RUNX-2 : Runt-related transcription factor-2 nationallicence RT : Room temperature nationallicence SDS : Sodium dodecyl sulphate nationallicence Wnt : Wingless nationallicence D'Alimonte Iolanda Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut Lannutti Angela Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut Pipino Caterina Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut Di Tomo Pamela Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut Pierdomenico Laura Department of Medicine and Aging Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut Cianci Eleonora Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut Antonucci Ivana Department of Psychological Sciences, Humanities and Territory, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut Marchisio Marco Department of Medicine and Aging Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut Romano Mario Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut Stuppia Liborio Department of Psychological Sciences, Humanities and Territory, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut Caciagli Francesco Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut Pandolfi Assunta Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut Ciccarelli Renata Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut Stem Cell Reviews and Reports Springer US; http://www.springer-ny.com 9/5(2013-10-01), 642-654 1550-8943 9:5<642 2013 9 12015 https://doi.org/10.1007/s12015-013-9436-5 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s12015-013-9436-5 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- D'Alimonte Iolanda Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut NATIONALLICENCE 700 1- Lannutti Angela Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut NATIONALLICENCE 700 1- Pipino Caterina Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut NATIONALLICENCE 700 1- Di Tomo Pamela Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut NATIONALLICENCE 700 1- Pierdomenico Laura Department of Medicine and Aging Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut NATIONALLICENCE 700 1- Cianci Eleonora Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut NATIONALLICENCE 700 1- Antonucci Ivana Department of Psychological Sciences, Humanities and Territory, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut NATIONALLICENCE 700 1- Marchisio Marco Department of Medicine and Aging Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut NATIONALLICENCE 700 1- Romano Mario Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut NATIONALLICENCE 700 1- Stuppia Liborio Department of Psychological Sciences, Humanities and Territory, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut NATIONALLICENCE 700 1- Caciagli Francesco Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut NATIONALLICENCE 700 1- Pandolfi Assunta Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut NATIONALLICENCE 700 1- Ciccarelli Renata Department of Experimental and Clinical Sciences, University "G. D'Annunzio” of Chieti-Pescara, Chieti, Italy aut NATIONALLICENCE 773 0- Stem Cell Reviews and Reports Springer US; http://www.springer-ny.com 9/5(2013-10-01), 642-654 1550-8943 9:5<642 2013 9 12015 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer