Abstract
Wnt signalling has been implicated in the regulation of stem cell self-renewal and differentiation; however, the majority of in vitro studies are carried out using monolayer 2D culture techniques. Here, we used mesenchymal stromal cell (MSC) EGFP reporter lines responsive to Wnt pathway activation in a 3D spheroid culture system to mimic better the in vivo environment. Endogenous Wnt signalling was then investigated under basal conditions and when MSCs were induced to undergo osteogenic and adipogenic differentiation. Interestingly, endogenous Wnt signalling was only active during 3D differentiation whereas 2D cultures showed no EGFP expression throughout an extended differentiation time-course. Furthermore, exogenous Wnt signalling in 3D adipogenic conditions inhibited differentiation compared to unstimulated controls. In addition, suppressing Wnt signalling by Dkk-1 restored and facilitated adipogenic differentiation in MSC spheroids. Our findings indicate that endogenous Wnt signalling is active and can be tracked in 3D MSC cultures where it may act as a molecular switch in adipogenesis. The identification of the signalling pathways that regulate MSCs in a 3D in vivo-like environment will advance our understanding of the molecular mechanisms that control MSC fate.
Highlights
Mesenchymal stromal cells (MSCs) are a heterogeneous cell population first isolated from the bone marrow, with other sources of related cell types present in other tissues, for example, in dental pulp and adipose tissue [1,2,3]
These cells were transduced using the Cignal Lenti T-cell factor (TCF)/lymphoid enhancer factor (LEF) Reporter (EGFP) Kit where EGFP is expressed under the control of a minimal (m) CMV promoter and repeats of the TCF/LEF transcriptional response element (TRE)
We have generated two EGFP reporter MSC lines for the real-time study of Wnt signalling. Using these tools we have demonstrated that Wnt signalling is activated in basal and osteogenic conditions only when examined in 3D culture and not in conventional 2D conditions
Summary
Mesenchymal stromal cells (MSCs) are a heterogeneous cell population first isolated from the bone marrow, with other sources of related cell types present in other tissues, for example, in dental pulp and adipose tissue [1,2,3]. The MSC population contains stem cells able to differentiate into osteoblasts, adipocytes, and chondrocytes both in vitro and in vivo [4]; the term “mesenchymal stem cell” is often used, referring to nonclonal undefined cell types. Despite this limitation, MSCs are widely viewed to have a range of therapeutic regenerative applications, in a musculoskeletal setting [5]. Discrepancies exist between in vivo data using mouse models and differentiation studies performed in vitro where, in multiple instances, the stimulation of Wnt signalling has been shown to inhibit differentiation [13,14,15,16,17]
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