Three-Dimensional Culture and Transforming Growth Factor Beta3 Synergistically Promote Tenogenic Differentiation of Equine Embryo-Derived Stem Cells

Abstract

The natural reparative mechanisms triggered by tendon damage often lead to the formation of biomechanically inferior scar tissue that is prone to re-injury. Before the efficient application of stem cell-based regenerative therapies, the processes regulating tenocyte differentiation should first be better understood. Three-dimensional (3D) growth environments under strain and the exogenous addition of transforming growth factor beta3 (TGFb3) have separately been shown to promote tendon differentiation. The aim of this study was to determine the ability of both of these factors to induce tendon differentiation of equine embryo-derived stem cells (ESCs). ESCs seeded into 3D collagen constructs can contract the matrix to a similar degree to that of tenocyte-seeded constructs and histologically appear nearly identical, with no areas of cartilage or bone tissue deposition. Tendon-associated genes and proteins Tenascin-C, Collagen Type I, and COMP are significantly up-regulated in the 3D ESC constructs compared with tenogenic induction in monolayer ESC cultures. The addition of TGFb3 to the 3D cultures further up-regulates the expression of these genes and also induces the expression of mature tenocyte markers Tenomodulin and Thrombospondin-4. Our results show that when ESCs are exposed to the intrinsic forces exerted by a 3D culture environment, they express tendon-associated genes and proteins which are indicative of tenocyte lineage differentiation and that this effect is synergistically enhanced and accelerated by the addition of TGF-b3.

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

“…8) and mouse (Havis et al, 2014) limb explants. 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).…”

Use of biomimetic microtissue spheroids and specific growth factor supplementation to improve tenocyte differentiation and adaptation to a collagen-based scaffold in vitro

“…In support of this, studies investigating the tenogenic potential of primary canine MSCs revealed that combinations of TGF-b1 and IGF-1 (5 ng/ml of each) were sufficient to induce the expression of COL1, COL3, DCN, SCX and TNMD after 7 and 14 days in combination with high density 3D culture conditions [26]. Similarly, a more recent report by Barsby et al [21] has confirmed a synergistic effect between 3D growth environments and the addition of TGF-b3 (20 ng/ml) in the tenogenic differentiation of equine embryonic stem cells (ESCs). Due to its high homology to IGF-1, insulin has also been evaluated for its ability to influence tenogenic differentiation and has proven to be a potent inducer of tenocyte formation in human MSC cultures [42].…”

Effects of substrate stiffness on the tenoinduction of human mesenchymal stem cells

“…Cells seeded on decellularized tendon slices, engineered scaffold-free tendon tissue and collagen matrix showed upregulation of TSP-4 6263, 64 . The current study also showed TSP-4 upregulation both with modulus and alignment after day 21 [Figure 3E].…”