Synthesis of embryonic tendon-like tissue by human marrow stromal/mesenchymal stem cells requires a three-dimensional environment and transforming growth factor β3

Abstract

Tendon-like tissue generated from stem cells in vitro has the potential to replace tendons and ligaments lost through injury and disease. However, thus far, no information has been available on the mechanism of tendon formation in vitro and how to accelerate the process. We show here that human mesenchymal stem cells (MSCs) and bone marrow-derived mononuclear cells (BM-MNCs) can generate tendon-like tissue in 7 days mediated by transforming growth factor (TGF) β3. MSCs cultured in fixed-length fibrin gels spontaneously synthesized narrow-diameter collagen fibrils and exhibited fibripositors (actin-rich, collagen fibril-containing plasma membrane protrusions) identical to those that occur in embryonic tendon. In contrast, BM-MNCs did not synthesize tendon-like tissue under these conditions. We performed real-time PCR analysis of MSCs and BM-MNCs. MSCs upregulated genes encoding type I collagen, TGFβ3, and Smad2 at the time of maximum contraction of the tendon-like tissue (7 days). Western blot analysis showed phosphorylation of Smad2 at maximum contraction. The TGFβ inhibitor SB-431542, blocked the phosphorylation of Smad2 and stopped the formation of tendon-like tissue. Quantitative PCR showed that BM-MNCs expressed very low levels of TGFβ3 compared to MSCs. Therefore we added exogenous TGFβ3 protein to BM-MNCs in fibrin gels, which resulted in phosphorylation of Smad2, synthesis of collagen fibrils, the appearance of fibripositors at the plasma membrane, and the formation of tendon-like tissue. In conclusion, MSCs that self-generate TGFβ signaling or the addition of TGFβ3 protein to BM-MNCs in fixed-length fibrin gels spontaneously make embryonic tendon-like tissue in vitro within 7 days.

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

“…This has been previously seen in MSC-seeded 3D constructs, by which the exposure of MSCs to a uniaxial force alone reduced the ''stemness'' of the seeded cells and left them unable to differentiate into osteogenic lineages after retrieval from the construct. 8 The maximum amount of time that the constructs could be maintained for before tearing off the pins was 14 days, as has been previously reported. 34 This limitation makes this model unsuitable for studying the longer-term effects of tissue development, but instead can be used to further analyze early-onset ESC differentiation and the associated molecular pathways.…”

“…4 Multiple studies have shown that TGF-b is up-regulated after tendon injury [5][6][7] and after their injection into the injured tendon, pluripotent equine embryo-derived stem cells (ESCs) express SCX. 5 The addition of TGF-b3 induces tenogenic differentiation of in vitro cultures of mesenchymal stem cells (MSCs), 8 micromass cultures of undifferentiated limb mesenchyme, 9 and equine ESCs. 5 Taken together, this suggests that TGF-b signaling plays a key role in triggering the initiation of tenocyte differentiation from stem cells and tendon progenitors.…”

“…Three-dimensional constructs have also been utilized to manipulate and direct certain pathways of differentiation in stem cells, including those of tenocyte lineages from MSCs. These include growth under ''static tension'' (produced by anchoring the gel), 8,[12][13][14] ''uniaxial applied tension'' (an externally applied constant force), 15 ''cyclical tension'' (exposure to externally applied and released forces), 12,16 and seeding cells on electrochemically aligned collagen. 17 When cells are cultured in anchored gels, they are under tension in what has been described as a ''static'' culture system.…”

“…17 When cells are cultured in anchored gels, they are under tension in what has been described as a ''static'' culture system. 12 This tension has also been termed ''uniaxial'' 8 to indicate that the tension is applied in one direction between the anchors. However, during the culture and contraction of the matrix, the actual force applied to the cells will change and, until a construct reaches its maximum contraction, the direction of force will be variable.…”

Transcription factor EGR1 directs tendon differentiation and promotes tendon repair

“…Consequently, we investigated the role of Egr1 in the formation of in vitro-engineered tendons. We used a 3D fibrin-based cell culture system that promotes tendon differentiation from primary chick tendon cells and human MSCs (30)(31)(32). C3H10T1/2-EGR1 cells cultured under uniaxial tension led to thicker tendon-like structures compared with C3H10T1/2 cells ( Figure 7, A and B).…”