Human Mesenchymal Stromal Cells (hMSCs)-based tissue engineering is regarded as a very promising approach for cartilage regeneration. Our work is aimed at identifying new molecules having a crucial role in determining MSCs fate, and targeting such regulators for the guidance of chondrogenesis in the absence of differentiating agents, such as TGF-β. Recently, miR-221 and Slug transcription factor have emerged as anti-chondrogenic regulators. We investigated if inhibition of these factors by specific antagomiR or siRNA molecule could be sufficient to address hMSCs from Whartons Jelly (WJMSCs) towards chondrogenesis, in the absence of TGF-β. We demonstrated by immunocytochemistry assays that miR-221 or Slug silencing increased the expression of the major cartilage protein Col2A1 and the master chondrogenic regulator Sox9, while decreased Col1A1 expression. Only Slug silenced WJMSCs were able to increase the expression of TRPS1, a positive regulator of chondrocyte differentiation. In addition, Slug inhibition determined a reduction in the levels of miR-221, and we identified by chromatin immunoprecipitation assay a specific region of the miR-221 promoter that is involved in the in vivo recruitment of Slug. By embedding miR-221 or Slug depleted bone marrow MSCs in alginate constructs, we also confirmed the stability of gene silencing for 28 days after combination with the scaffold. Taken together, our data demonstrate that miR-221 and Slug are functionally correlated in MSCs and that the silencing of these regulators is sufficient to induce differentiation towards the chondrogenic lineage, in the absence of TGF-β. The combination of engineered hMSCs with alginate preserved the efficiency of gene silencing, demonstrating the feasibility of this approach for the generation of tissue engineering constructs. On-going experiments are aimed at evaluating the ability of the engineered hMSCs to trigger cartilage reparative processes in vitro or in vivo, by using an experimental model of osteochondral defect.
Disclosure: The authors declared no competing interests.