This study aimed to determine whether TSCs and tenocytes, the two types of cells in tendons, share common properties in their phenotypes. Towards this aim, TSCs and tenocytes were isolated from rabbit patellar and Achilles tendons, and their differentiation potential, cell marker expression, morphology, and proliferative potential were examined. We found that TSCs were able to differentiate into specific lineages of cells (adipocytes, chondrocytes, and osteocytes), which was verified by detection of specific marker gene expression. The multi-differentiation potential of TSCs was further confirmed by an in vivo experiment, which demonstrated that implantation of TSCs in vivo resulted in the formation of tendon-like, fibrocartilage-like, and bone-like tissues. We also showed that TSCs expressed Oct-4, SSEA-4, and nucleostemin, which are known stem cell markers. In contrast, tenocytes from both the patellar and Achilles tendons essentially lacked trans-differentiation potential; moreover, tenocytes did not express Oct-4, SSEA-4, or nucleostemin. Morphologically, TSCs in culture differ from tenocytes in that the former exhibited a cobble-stone shape whereas the latter spread out and were highly elongated, a characteristic shape of fibroblasts in confluent conditions. Finally, TSCs proliferated significantly faster than tenocytes in culture.
The finding that TSCs, but not tenocytes, were capable of differentiating into non-tenocyte lineages of cells suggests that TSCs may play a key role in tendinopathy. While the pathogenesis of tendinopathy is still not clear, previous studies have identified typical histological features including accumulation of lipid cells, glycosaminoglycan accumulation, and tissue calcification, either alone or in combination [17
]. Therefore, by virtue of their ability to differentiate into adipocytes, chondrocytes, and osteocytes, TSCs could be responsible for the production of abnormal matrix components (e.g. fatty degeneration, glycosaminoglycan accumulation, and calcifications) seen in tendinopathic tendons. This is an intriguing hypothesis that should be tested in future studies. In addition to TSCs, however, tenocytes may well be involved in the development of tendinopathy through production of inflammatory mediators [18
] and tissue degradative enzymes (MMPs)[21
A few comments are in order regarding the stem cell markers identified on TSCs but not on tenocytes in this study. Oct-4 is a transcription factor that is typically expressed in embryonic stem cells during development and is essential for establishing and maintaining undifferentiated pluripotent stem cells [22
]. Like previous studies that showed Oct-4 expression in human and mouse BMSCs [23
], we also found that TSCs expressed Oct-4, encouraging future examination of whether the multi-potency of TSCs demonstrated in this study depends on Oct-4 expression.
In addition to Oct-4 expression, we found that SSEA-4 was consistently expressed in TSCs at low (< 2) and high passages (~12) even after long term culturing. It is known that SSEA is developmentally regulated during early embryogenesis and is widely used as a marker to monitor the differentiation of both mouse and human embryonic stem cells [26
]. Therefore, SSEA-4 may be used as one of TSC markers. Finally, we found that nucleostemin was highly expressed in rabbit TSCs. Nucleostemin is only expressed in the nucleoli of stem cells and cancer cells, but not in those of committed and terminally differentiated cells [28
]. Thus, the high levels of nucleostemin expression in rabbit TSCs indicate that TSCs were an actively proliferating, self-renewing population of cells in our culture conditions. On the other hand, the lack of expression of nucleostemin in tenocytes suggests that tenocytes are terminally differentiated cells without further differentiation potential, as indicated by our data. Finally, as this study shows that TSCs, but not tenocytes, express Oct-4, SSEA-4, and nucleostemin, they may be used as markers to detect these tendon stem cells in situ
A few comments are also necessary regarding cell cultures used in this study. First, TSCs are most likely a mixture of stem cells and progenitor cells, which are heterogeneous in clonogenicity, multi-differentiation potential, and self-renewal. The evidence for this include: 1) colony size of both PTSCs and ATSCs varied greatly (Figure ); 2) PTSCs and ATSCs did not undergo complete differentiation into adipocytes, chondrocytes, and osteocytes when grown in respective induction media (Figure ); and 3) not all individual cells were noted to express stem cell markers, including Oct-4 and SSEA-4 (Figure ). Future studies should look into the properties (e.g. gene expression profiles) of TSCs at the individual cell level rather than at the population level as in this study. Second, when tenocytes were exposed to induction medium for adipogenesis, a small percentage of cells (< 5%) formed lipid drops (Figure ). These positively-stained cells were likely those remaining TSCs and/or their committed progenitor cells, as it is difficult to isolate pure tenocytes from TSCs using the separation procedures used in this study.
Tendons are commonly considered to contain only tenocytes or tendon fibroblasts [18
]. While it was suggested that there might be a special cell population within tendons that possesses multiple differentiation potential [30
], the existence of stem cells in human and mouse tendons was not definitively shown until a recent study [9
]. Our finding that rabbit patellar and Achilles tendons contain stem cells is consistent with this study. However, our finding that tenocytes do not have multi-differentiation potential is inconsistent with an earlier study, which showed that so called tendon-derived fibroblasts can differentiate into adipocytes, chondrocytes, and osteocytes [10
]. Different tissue culture techniques may account for this discrepancy. Specifically, we used a tissue digestion method to isolate tendon cells directly from tendon tissues. Once tendon cells in culture attached to culture plates, we separated colony-forming cells, which were TSCs, from those cells that spread out, which were considered to be tenocytes. In the study by de Mos et al., tendon explant cultures were used in which cells that migrated out from tendon samples were collected and sub-cultured. Therefore, we suspect that such cultures contained a mixed population of cells including tenocytes and TSCs or their progeny cells. Consequently, those cells that were shown to differentiate into non-tenocytes could actually be TSCs.
While this study shows that TSCs from both patellar and Achilles tendons exhibit similar patterns in directed differentiation and gene expression, they display marked differences in colony formation and cell proliferation rate. In particular, PTSCs formed more and larger colonies (Figure ) and proliferated more rapidly than ATSCs (Figure ). The reasons for these differences are not clear but may reflect inherent differences between the two tendons in vivo
. The patellar tendon is similar to a ligament as it connects the patella and tibia, and the structure and composition of the patellar and Achilles tendons are different. Our findings of biological differences between patellar and Achilles TSCs support the notion that characteristics of adult stem cells such as TSCs are tissue origin-dependent [31
]. Furthermore, the differential proliferative properties of TSCs found in patellar and Achilles tendons may reflect differences in their function and regenerative potential in vivo