Activation of Wnt/beta-catenin pathway during osteogenic differentiation of adipose derived stem cells in monolayer cultures and 3D spheroids

摘要

Introduction: Wnt/p-catenin pathway activity is essential for osteoblast differentiation, skeletal development and bone homeostasis. Transcriptional activity of p-catenin is also involved in differentiation of stem cells and in maintenance of stem cells in an undifferentiated state. Interestingly, the Wnt signalling depends on tissue source of mesenchymal stem cells (MSC), as osteogenic differentiation is activated in bone marrow MSC but inhibited in periodontal ligament MSC in the presence of Wnt3a ligand. In this study we aimed to characterize the activity of Wnt/p-catenin pathway in osteogenic differentiation of human adipose-derived MSC (ADSC) cultured in standard monolayers or three-dimensional (3D) spheroids Materials and Methods: ADSC were seeded in osteogenic medium (OM) into 6-well plates for monolayer culture or into 96-well, round-bottom plates coated with Pluronic F127 for generation of spheroids. Two days of culture in coated plates on a rotary shaker resulted in spheroid formation. Cells were lysed at day 2 or 7 for RNA extraction and gene expression analysis or Western Blot. Expression of Runx2, Osterix and bone sialoprotein genes as well as Runx2 protein level was analysed to study osteogenic differentiation. Expression of Wnt3a, c-Myc genes and β-catenin protein level was determined to assess Wnt/p-catenin pathway activation. Expression of connexin 43 (Cx43) gene was analyzed to study the capability of gap junction formation. Results and Discussion: Expression of Runx2 and Osterix transcription factors was much higher in 3D spheroids than in monolayer cultures. Runx2 protein level followed a similar pattern. In addition, expression of bone sialoprotein gene was also elevated in spheroids but not in monolayers. This indicates more efficient osteogenic differentiation of ADSC in spheroids, which has been previously observed in bone marrow MSCs. Preliminary data demonstrated that the level of β-catenin increases in ADSC monolayers but not in ADSC spheroids upon addition of OM as compared to undifferentiated cells. Expression of a classical β-catenin target (c-Myc) was lower in ADSC spheroids than in monolayers at 2 days in OM. Surprisingly, expression of Wnt3a gene in any of the studied conditions was not detected. On the other hand, expression of Cx43 gene was elevated in spheroids compared to monolayers. Therefore osteogenic differentiation of ADSC in spheroids prevented activation of Wnt/β-catenin. This might be caused by increased expression of Cx43, as β-catenin binding to Cx43 protein can prevent nuclear translocatjon and activity of β-catenin. Conclusions: Osteogenic differentiation of ADSC is increased in 3D spheroids generated by Pluronic-coated plates and this phenomenon is associated with decreased β-catenin signalling but increased Cx43 expression. Functional studies will determine if increased osteogenesis of ADSC is caused by downregulated Wnt/β-catenin signalling in 3D spheroids.