In vitro characterization of stem/progenitor cells from semitendinosus and gracilis tendons as a possible new tool for cell-based therapy for tendon disorders

Purpose: this study was conducted to characterize tendon stem/progenitor cells (TSPCs) isolated from human semitendinosus and gracilis tendons in terms of stemness properties and multi-differentiation potential. Methods: TSPCs were isolated from waste portions of semitendinosus and gracilis tendons from three donors who underwent anterior cruciate ligament reconstruction. TSPCs were plated in culture until passage 4, when experiments to assess cell proliferation, viability and clonogenic ability were performed. The immunophenotype of TSPCs was evaluated by cytofluorimetric analysis. The in vitro osteogenic, chondrogenic, adipogenic and tenogenic potential was evaluated using biochemical, histological and gene expression analysis to detect specific differentiation markers. Statistical analysis was performed using Student’s t-test. Results: after a few passages in culture the cell populations showed a homogeneous fibroblast-like morphology typical of mesenchymal stem cells. The average doubling time of TSPCs increased from 52.4±4.8 at passage 2 to 100.8±23.4 hours at passage 4. The highest percentage of colonies was also found at passage 4 (4.7±2.3%). TSPCs showed the typical mesenchymal phenotype, with high expression of CD73, CD90 and CD105 and no expression of CD34 and CD45. Cells induced to differentiate toward osteogenic lineage showed significant upregulations of ALP activity (+189%, p<0.05) and calcified matrix deposition (+49%, p<0.05) compared with undifferentiated cells; culture in chondrogenic medium also provoked a significant increase in glycosaminoglycan levels (+108%, p<0.05). On the other hand, TSPCs were not able to respond to adipogenic stimuli. Scleraxis gene expression and decorin gene expression, considered tenogenic markers, were already very high in control cells, and culture in tenogenic medium further increased these values although not significantly. Conclusions: our data show that it is possible to isolate TSPCs from very small fragments of tissue and that they show the typical features of MSCs and multi-differentiation potential, above all toward osteogenic and chondrogenic lineages. Clinical relevance: this study can be considered one of the first attempts to clarify the biology of tendon cell populations, focusing in particular on the potential applicability of this cell source for future regenerative medicine purposes.