RESUMO
The transcription factor scleraxis is required for tendon development and is upregulated during embryonic stem cell (ESC) differentiation into tenocytes. However, its role beyond early embryonic development is not defined. We utilized a short hairpin RNA to knock down scleraxis expression in ESCs and adult and fetal tenocytes. No effect on growth or morphology was observed in two-dimensional cultures. However, scleraxis knockdown in fetal tenocytes significantly reduced COL1A1, COMP, and SOX9 gene expression. Scleraxis knockdown in adult tenocytes had no effect on the expression of these genes. Strikingly, differentiating ESCs and fetal tenocytes without scleraxis failed to reorganize a three-dimensional (3D) matrix and generate artificial tendons. This was associated with a significantly reduced survival. In contrast, there was no effect on the survival and remodeling capacity of adult tenocytes following scleraxis knockdown. Overexpression of scleraxis in fetal tenocytes rescued gene expression, cell survival in 3D, and subsequent matrix contraction. Together, these results demonstrate that scleraxis is not only essential for ESC differentiation into tenocytes but that it also has an active role in maintaining fetal tenocytes, which is then redundant in adult tenocytes.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Feto/citologia , Tendões/citologia , Tenócitos/citologia , Envelhecimento/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína de Matriz Oligomérica de Cartilagem/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , DNA Complementar/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Géis/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Cavalos , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Tenócitos/efeitos dos fármacos , Tenócitos/metabolismoRESUMO
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 (TGF-ß3) 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 TGF-ß3 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-ß3.