Abstract
To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and tissue engineering.
Highlights
Tendon degeneration and overuse in sports are common ailment encountered in the field of orthopaedic surgery
The results revealed an increase in candidate tenogenic markers protein expression in Human bone marrow stromal cell (hMSC) in day 4 and day 10 growth and differentiation factor 5 (GDF5)-induced hMSCs compared to control (Fig 1); and considerable similarity in the cellular distribution of candidate tenogenic markers in GDF5-induced hMSCs and tenocytes at day 4 and 10
Through the global gene expression profiles analysis, several pathways were identified as important pathways for tenogenic differentiation: (i) the glycolysis and gluconeogenesis signalling pathways were down regulated upon GDF5 induction in hMSC and in tenocytes; (ii) the cell cycle related signalling pathways were down-regulated in the day-10 GDF5-induced hMSCs; (iii) the activated pathways which may be crucial in tenogenic differentiation were agiopoietin-Tie2 signalling, TGF-beta-dependent induction if EMT via SMADS signalling, pigment epithelium-derived factor (PEDF) signalling and vascular endothelial growth factor (VEGF) signalling via VEGFR2; (iv)the cell adhesion and cytoskeleton remodelling signalling as well as EMT pathways were identified as important pathways at the late tenogenic differentiation stage or in mature tenocytes
Summary
Tendon degeneration and overuse in sports are common ailment encountered in the field of orthopaedic surgery. Current tendon tissue engineering research has been focused in the investigation of intrinsic and extrinsic factors that can induce bone marrow stromal cells (MSCs) into tenogenic lineage for use as an alternative cell source to replenish functional tendon cells at tendon injured site. In this regards, growth and differentiation factor 5 (GDF5) has been identified as one of the important factors in inducing tenogenic differentiation in MSCs [1,2,3]. These methods have successfully induced tenogenic differentiation in MSCs in vitro with the presence of GDF-5
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