Abstract
Summary
Mesenchymal stem cells from human semitendinosus and gracilis tendons (TSPCs) could be a promising MSCs resource for tissue-engineering application. In comparison to adipose-derived stem cells, TSPCs possess similar stem-cells properties and a higher chondrogenic differentiation potential.
Introduction
Mesenchymal stem cells (MSCs) isolated from bone marrow (BMSCs) or adipose tissue (ASCs) have been deeply characterised for their usefulness in musculoskeletal tissue regeneration. However, other potentially valuable MSCs sources have been recently proposed. The goal of this study was to isolate MSCs from human semitendinosus and gracilis tendons (TSPCs, tendon stem progenitor cells) and to compare their features with that of human ASCs.
Methods
Human TSPCs and ASCs were isolated from semitendinosus and gracilis tendons portions and adipose tissue of healthy donors undergoing ACL reconstruction (n=6) or liposuction (n=6), respectively. At early passages in culture (until passage 4) proliferation rate (expressed as doubling time) and CFU-F ability were observed for both cell populations. Immunophenotype profile was assessed by FACS analysis on ASCs and TSPCs at P4. At the same passage, TSPC and ASCs multi-differentiative potential was also assessed. Alkaline phosphatase (ALP) activity and extracellular calcified matrix deposition were analyzed after 14 and 21 days of osteogenic induction, respectively. Glycosaminoglycans (GAGs) production were quantified in pellet culture (DMMB assay) at 21 and 28 days of chondrogenic differentiation. Finally, the adipogenic differentiation was also evaluated quantifying the lipid vacuoles stained by Oil Red O (ORO) after 14 days of culture in adipogenic inductive medium.
Results
The yield of TSPCs per gram of tissue was significantly higher than that observed for ASCs (p<0.05); moreover until P3, TSPCs grow faster than ASCs, with a significantly lower doubling time (p<0.05). Both population showed a similar clonogenic ability and were highly positive for the typical MSCs surface markers, such as CD90, CD105, CD73 and CD44. Both osteo-differentiated TSPCs and ASCs showed a significant increase of ALP activity levels (+235%, +125%, respectively) and extracellular calcified matrix production respect to undifferentiated cells. However, OSTEO-ASCs were able to produce a higher amount of calcified matrix (10 fold higher) than OSTEO-TSPCs (p<0.001). Surprisingly, TSPCs possess a higher chondrogenic potential than ASCs: CHONDRO-TSPCs showed a significant increase of GAGs levels (+121%, p<0.05) respect to untreated cells, whereas ASCs were not positively affected by the chondro-inductive medium. On the other hand, after adipogenic differentiation, TSPCs showed a significant lower lipid vacuoles production than ASCs (p<0.05).
The evaluation of stemness (OCT4, NANOG) as well as tissue-specific markers (SCX, COLL III, RUNX2, PPAR? and SOX9) in TSPCs and ASCs is currently under investigation.
Discussion/Conclusion
Semitendinosus and gracilis tendons contain a subpopulation of cells that possesses the peculiar features of stem cells, including a great proliferation rate, clonogenic ability and the typical immunophenotype. Moreover TSPCs possess an osteogenic potential similar to ASCs, but a higher chondrogenic and a lower adipogenic potential. Further analysis such as gene expression evaluation of stem cells markers and specific differentiation markers, could explain this different behavior. Overall, tendon could be a new and promising source of MSCs for musculoskeletal-engineering applications.
