Conversion of Human Bone Marrow-Derived Mesenchymal Stem Cells into Tendon Progenitor Cells by Ectopic Expression of Scleraxis

Abstract

Tendons and ligaments (T/L) are dense connective tissues of mesodermal origin. During embryonic development, the tendon-specific cells descend from a sub-set of mesenchymal progenitors condensed in the syndetome, a dorsolateral domain of the sclerotome. These cells are defined by the expression of the transcription factor scleraxis (Scx), which regulates tendon formation and several other characteristic genes, such as collagen type I, decorin, fibromodulin, and tenomodulin (Tnmd). In contrast to other mesenchymal progenitors, the genealogy and biology of the tenogenic lineage is not yet fully understood due to the lack of simple and efficient protocols enabling generation of progenitors in vitro. Here, we investigated whether the expression of Scx can lead to the direct commitment of mesenchymal stem cells (MSCs) into tendon progenitors. First, MSC derived from human bone marrow (hMSC) were lentivirally transduced with FLAG-Scx cDNA to establish 2 clonal cell lines, hMSC-Scx and hMSC-Mock. Subsequent to Scx transduction, hMSC underwent cell morphology change and had significantly reduced proliferation and clonogenicity. Gene expression analysis demonstrated that collagen type I and several T/L-related proteoglycans were upregulated in hMSC-Scx cells. When stimulated toward 3 different mesenchymal lineages, hMSC-Scx cells failed to differentiate into chondrocytes and osteoblasts, whereas adipogenic differentiation still occurred. Lastly, we detected a remarkable upregulation of the T/L differentiation gene Tnmd in hMSC-Scx. From these results, we conclude that Scx delivery results in the direct programming of hMSC into tendon progenitors and that the newly generated hMSC-Scx cell line can be a powerful and useful tool in T/L research.

Tendon development and musculoskeletal assembly: emerging roles for the extracellular matrix

“…This might involve feedback regulation of Scx and Mkx expression through the tendon ECM, and recent studies have suggested that Dcn and Fmod are regulated by Mkx (Fig. 2B) (Ito et al, 2010;Liu et al, 2010;Alberton et al, 2012). These results point to a system by which the dynamics of fibril ultrastructure feedback on ECM production to modify tendon strength continuously.…”

“…Scx is the earliest known marker of TPCs, and its overexpression transforms MSCs into tenocytes (Alberton et al, 2012), whereas Sox9 overexpression converts MSCs into cartilage ( Fig. 2A) (Takimoto et al, 2012).…”

“…In vitro, Scx overexpression is sufficient to transform mesenchymal stem cells (MSCs) and human embryonic stem cells (hESCs) into tenocytes ( Fig. 2A) (Alberton et al, 2012;Chen et al, 2012;Li et al, 2015). Thus, considerable effort has been made to elucidate the functions of Scx in the tenocyte lineage and to identify its downstream targets.…”

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

“…38 Ectopic expression of SCX in vitro has also been shown to induce the differentiation of MSCs into tendon progenitor cells. 39 Therefore, it is likely that SCX is expressed early in the differentiation of ESCs into tenocytes. Attempts were made to examine gene expression levels at 3 days, during the period of increased gel contraction in the presence of TGF-b3; however, the expression of all genes was low at this time point with large amounts of variability in their detection, which precluded getting any meaningful data.…”

Transcription factor EGR1 directs tendon differentiation and promotes tendon repair

“…In addition to the use of animal models to study tendon development, mesenchymal stem cells (MSCs) have been used to study tendon cell differentiation in vitro (15)(16)(17)(18). MSCs are multipotent progenitor cells that can be induced to differentiate into various tissues of mesodermal origin (19).…”