Wnt Signaling Behaves as a “Master Regulator” in the Osteogenic and Adipogenic Commitment of Human Amniotic Fluid Mesenchymal Stem Cells

Abstract

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.FigureSchematic 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

Identification of Novel EZH2 Targets Regulating Osteogenic Differentiation in Mesenchymal Stem Cells

“…Conversely, osteogenic differentiation can be inhibited by the activation of protein kinase A, which upregulates peroxisome proliferatoractivated receptor gamma 2 (PPARg2) and in turn inhibits runtrelated transcription factor 2 (RUNX2) and osteopontin (OP), promoting adipogenesis [8]. RUNX2, a key lineage specific transcription factor, and signaling pathway molecules such as WNT and bone morphogenic protein (BMP) play a critical role in dictating MSC osteogenic differentiation [9][10][11][12][13].…”

Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential

“…Some studies have reported the great potential of ckit selected amniotic fluid cells prior to osteogenic differentiation [40] , while others have demonstrated that unselected cells are also able to properly differentiate into osteospecific cells [13,41,44,60] . In this review we mainly discuss the in vitro osteogenic differentiation of AFMSCs with occasional mention of papers in which authors use ckit selected cells, named AFSCs (Table 1).…”

“…In this context, there is some evidence to suggest a role by the canonical Wnt signal pathway in bone formation as activation of this pathway stimulates osteoprogenitor proliferation and osteogenesis of human MSCs [73,74] . Wnt signaling is also involved in AFMSC commitment toward osteogenesis [13] . Recently, we identified for the first time, the presence of Calcium Sensing Receptor (CaSR) in ovine and human AFMSCs [44,68] .…”

“…Several cell signaling cascades are involved in this cell fate decision between osteo and adipogenesis [11] . The master regulator of osteogenesis is Runx2, the gene target of many signaling pathways, including but not limited to transforming growth factorbeta 1 (TGFβ1), BMP [12] , Wingless type (Wnt) [13] , Hedgehog (HH) [14] , and (NEL) like protein type 1 (NELL1) [15] . Thus, considering the crucial role of MSCs in bone healing, the strategy of using the osteogenic potential of such cells transplanted into the bone defect seems promising [7,16] .…”

Gold nanoparticles promote osteogenic differentiation in human adipose-derived mesenchymal stem cells through the Wnt/β-catenin signaling pathway

“…[46][47][48] Wnt/β-catenin signaling inhibits adipogenic differentiation and alters the cell fate from adipocyte to osteoblast in hADMSCs. 49 To confirm the signaling pathway related to osteogenic differentiation in hADMSCs, we examined nonphosphorylated β-catenin in protein levels.…”