Embryonic mechanical and soluble cues regulate tendon progenitor cell gene expression as a function of developmental stage and anatomical origin

Abstract

Stem cell-based engineering strategies for tendons have yet to yield a normal functional tissue, due in part to a need for tenogenic factors. Additionally, the ability to evaluate differentiation has been challenged by a lack of markers for differentiation. We propose to inform tendon regeneration with developmental cues involved in normal tissue formation and with phenotypic markers that are characteristic of differentiating tendon progenitor cells (TPCs). Mechanical forces, fibroblast growth factor (FGF)-4 and transforming growth factor (TGF)-β2 are implicated in embryonic tendon development, yet the isolated effects of these factors on differentiating TPCs are unknown. Additionally, developmental mechanisms vary between limb and axial tendons, suggesting the respective cell types are programmed to respond uniquely to exogenous factors. To characterize developmental cues and benchmarks for differentiation toward limb vs. axial phenotypes, we dynamically loaded and treated TPCs with growth factors and assessed gene expression profiles as a function of developmental stage and anatomical origin. Based on scleraxis expression, TGFβ2 was tenogenic for TPCs at all stages, while loading was for late-stage cells only, and FGF4 had no effect despite regulation of other genes. When factors were combined, TGF 2 continued to be tenogenic, while FGF4 appeared anti-tenogenic. Various treatments elicited distinct responses by axial vs. limb TPCs of specific stages. These results identified tenogenic factors, suggest tendon engineering strategies should be customized for tissues by anatomical origin, and provide stage-specific gene expression profiles of limb and axial TPCs as benchmarks with which to monitor tenogenic differentiation of stem cells.

TGFβ and FGF promote tendon progenitor fate and act downstream of muscle contraction to regulate tendon differentiation during chick limb development

“…This is consistent with previous reports showing TNMD upregulation by TGFβ ligands in 3D-culture systems of human tendon cells (Bayer et al, 2014) and of equine embryo-derived stem cells (Barsby et al, 2014), and in high-density cultures of chick limb cells (Lorda-Diez et al, 2009). It is worth mentioning that TGFβ dramatically decreases Tnmd expression (and activates Scx) in 2D-culture systems of embryonic or adult mouse tendon progenitors and in mouse mesenchymal stem cells (Guerquin et al, 2013;Brown et al, 2014;Liu et al, 2015). We believe that the opposite effects of TGFβ on Tnmd expression are due to the different cell contact environments in 2D-culture versus 3D-culture systems.…”

“…Error bars indicate s.e.m. embryonic tendon progenitors (Brown et al, 2014) and stem cell culture systems Barsby and Guest, 2013;Goncalves et al, 2013;Guerquin et al, 2013;Havis et al, 2014). We find that the positive effect of TGFβ2 on chick limb SCX expression is independent of ERK MAPK signalling (Fig.…”

“…FGF positively regulates SCX expression in axial and foetal limb tendons during chick development (Edom-Vovard et al, 2002;Brent et al, 2003;Brent and Tabin, 2004). In contrast to the chick model, FGF has an anti-tenogenic effect in mouse embryonic tendon cells (Brown et al, 2014) and inhibition of the ERK MAPK pathway is sufficient to increase Scx expression in early mouse limb explants (Havis et al, 2014). Although the experimental situations in the reports described above differ between the chick and mouse models, they nevertheless suggest a differential regulation of Scx by FGF in the chick and mouse models.…”

Transcriptomic analysis of mouse limb tendon cells during development

“…6). This decrease in Scx expression is consistent with that observed in mouse tendon progenitor cells upon FGF4 treatment (Brown et al, 2014). Moreover, the inhibition of ERK MAPK signalling appeared to be sufficient for inducing Scx expression in E9.5 mouse limb mesodermal progenitors and in mesenchymal stem cells.…”

“…FGF2 treatment in mouse stem cells activated the expression of Scx (Ker et al, 2011), whereas FGF4 treatment in mouse tendon progenitor cells inhibits Scx expression (Brown et al, 2014). We assessed the efficiency of FGF4 and ERK inhibition (PD18) in C3H10T1/2 cells by Pea3 upregulation in the presence of FGF4 and Pea3 downregulation with PD18 (Fig.…”