Establishment of tendon-derived cell lines exhibiting pluripotent mesenchymal stem cell-like property

Abstract

Development of the musculoskeletal system requires coordinated formation of distinct types of tissues, including bone, cartilage, muscle, and tendon. Compared to muscle, cartilage, and bone, cellular and molecular bases of tendon development have not been well understood due to the lack of tendon cell lines. The purpose of this study was to establish and characterize tendon cell lines. Three clonal tendon cell lines (TT-E4, TT-G11, and TT-D6) were established using transgenic mice harboring a temperature-sensitive mutant of SV40 large T antigen. Proliferation of these cells was significantly enhanced by treatment with bFGF and TGF-β but not BMP2. Tendon phenotype-related genes such as those encoding scleraxis, Six1, EphA4, COMP, and type I collagen were expressed in these tendon cell clones. In addition to tendon phenotype-related genes, expression of osteopontin and Cbfal was observed. These clonal cell lines formed hard fibrous connective tissue when implanted onto chorioallantoic membrane in ovo. Furthermore, these cells also formed tendon-like tissues when they were implanted into defects made in patella tendon in mice. As these tendon cell lines also produced fibrocartilaginous tissues in tendon defect implantation experiments, mesenchymal stem cell properties were examined. Interestingly, these cells expressed genes related to osteogenic, chondrogenic, and adipogenic lineages at low levels when examined by RT-PCR. TT-G11 and TT-E4 cells differentiated into either osteoblasts or adipocytes, respectively, when they were cultured in cognate differentiation medium. These observations indicated that the established tendon cell line possesses mesenchymal stem cell-like properties, suggesting the existence of mesenchymal stem cell in tendon tissue.

Introduction

Musculoskeletal tissue formation requires coordinated development of distinct types of tissues during embryogenesis. To date, developmental biology on bone, cartilage, and muscle has been the major field of the study. In contrast, regulatory mechanisms involved in tendon and ligament formation have not been well understood. Tendon is a specific connective tissue that links skeletal elements and muscles. Like cartilage, tendon originates from lateral plate mesoderm. Drosophila studies suggest that mature tendon cells are singled out from a cluster of competent cells by the influences of approaching muscle cells [1]; however, whether this is also the case in vertebrae is still to be elucidated. In avians, development of proximal limb tendons is closely in association with muscle development [2]. In contrast, distal limb tendons develop from muscle and their morphogenesis is more correlated with that of skeletal elements [2]. The initial autonomous differentiation of tendon primordial cells precedes muscle binding, and it takes place even in limbs that are devoid of muscles. However, when muscles are experimentally removed in limbs, the tendon tissues cease their development and degenerate [2], [3], [4]. Signaling molecules in this process have not been fully understood.

Tendon and ligament cells have been reported to respond to a variety of growth factors [5], [6]. Signaling by TGF-β superfamily members, especially BMPs, has been thought to be a key regulator in the development of tendon during chick embryogenesis [7]. TGF-β and bFGF mRNAs have been reported to be expressed in both intrinsic tenocytes and extrinsic inflammatory cells in tendon after injury and repair, suggesting the significance of these molecules during tendon development and repair [8], [9], [10].

One of the intriguing features of tendon is its possible plasticity. Conversion of tendon into cartilage has been observed to occur as a consequence of surgical or nonsurgical trauma in human patients as well as animals [11], [12]. Thus, in addition to the molecular mechanisms underlying tendon-specific cell differentiation, certain signaling pathways may exist to alter the fate of tendon cells. Though tendons and ligaments are derived from mesenchymal cells, which also give rise to bone, cartilage, fat, and muscle, the presence of precursors or mesenchymal stem cells in tendon has not been studied. If there are such cells, development and repair of tendon would be under the control of certain types of extrinsic signals such as cytokines and intrinsic signals including transcription factors.

Scleraxis, a twist-related bHLH transcription factor, is specifically expressed in mesenchymal precursors of connective tissues in early development, while in later developmental stages in mice, scleraxis transcripts are selectively expressed in tendons and their progenitors [13], [14], [15]. On the other hand, scleraxis expression is downregulated in developing bones at the onset of ossification [16]. Other transcription factors such as two murine homeobox-containing genes, Six1 and Six2, are expressed in a complementary fashion during the development of limb tendons [17].

In addition to the transcription factors, several other molecules are expressed in tendon tissues. EphA4 is an Eph family member receptor tyrosine kinase and is expressed in response to regulatory signals during limb patterning. Later in limb development, EphA4 is expressed in cell condensations that form tendons and their attachments to cartilage rudiments [18], [19]. Another type of tendon-related molecule is a pentameric noncollagenous glycoprotein, COMP, which is a member of the thrombospondin gene family of extracellular calcium-binding proteins. COMP is also expressed in cartilage and ligament in addition to tendon tissues [20], [21], [22]. The restricted tissue distribution and expression of COMP in developing as well as adult tendon tissues suggest the involvement of this protein in the regulation of tendon formation [23]. The structural similarities between COMP and thrombospondin could imply that they may perform similar functions implicated in interactions with extracellular matrix components and cells [24].

Although fragmented information has been accumulated, the studies on tendon biology are still in an early stage. Tendon cell research has been hampered by the lack of appropriate cell lines. The purpose of this study is to establish and characterize tendon cell lines. We established three cell lines derived from the Achilles tendon of transgenic mice harboring a temperature-sensitive mutant of SV40 large T antigen [25], [26], [27].