Purpose: Human Mesenchymal Stromal Cells (hMSCs)-based tissue engineering is regarded as an extremely promising approach for the repair of cartilage defects either due to fractures or chronic diseases, such as osteoarthritis (OA). Nevertheless, there is a need for reliable tools that can induce reproducible and stable chondrogenesis of hMSCs. While current strategies normally make use of growth factors, mainly TGF-βs, from a translational standpoint the use of such molecules raises issues related to their side effects in vivo and involvement in OA. Accordingly, much effort is put into characterizing the chondrogenic process at a molecular level and identifying critical factors to be targeted to optimize the chondrogenic properties of hMSCs. In this context, we investigated the role of two molecules that have recently emerged as negative chondro-regulators, miR-221 and the transcription factor Slug. We evaluated whether the inhibition of these molecules could be sufficient to guide hMSCs towards a chondrocyte-like phenotype and stimulate their chondrogenic potential, in the absence of TGF-β. Methods: miR-221 and Slug expression was transiently inhibited using specific antagomiR and siRNA molecules in monolayered hMSCs derived from the Wharton's Jelly of umbilical cords or bone marrow. The effect of miR-221 and Slug silencing on the expression of cartilage extracellular matrix proteins and chondro-transcription factors was evaluated by qRT-PCR and immunocytochemistry. Chromatin immunoprecipitation (ChIP) assays were employed to investigate a functional correlation between miR-221 and Slug. The engineered hMSCs were then cultured as pellets or in alginate constructs and the efficiency of gene silencing was monitored up to 28 days of 3D-culture. Results: We first showed that chondrogenic differentiation of hMSCs induced by TGF-β is not sufficient to abolish the expression of the anti-chondrogenic regulators miR-221 and Slug. Then, in search of alternative differentiation protocols, we demonstrated that both miR-221 and Slug silencing increased the protein expression of the major cartilage matrix marker Col2A1 and the pro-chondrogenic transcription factor Sox9, while decreased Col1A1, in the absence of TGF-β (Figure A). Slug inhibition determined a reduction in the levels of miR-221 (Figure B) and, consistently, we identified by ChIP assay a specific region of the miR-221 promoter that is transcriptionally active and involved in the in vivo recruitment of Slug (Figure C). Interestingly, the levels of Slug protein were much lower in antagomiR-221-treated hMSCs than in TGF-β-treated cells. By culturing miR-221 and Slug depleted hMSCs in 3D culture systems (such as pellets or in alginate), we demonstrated that the efficiency of gene silencing is well maintained for at least 28 days. Conclusions: miR-221 or Slug silencing in hMSCs induces differentiation towards the chondrogenic lineage, in the absence of conventional chondrogenic inducers, such as TGF-β. Our data demonstrate that miR-221 expression is, in part, regulated by Slug, and support the hypothesis that a novel Slug/miR-221 circuit is crucial for the regulation of the chondrogenic program of hMSCs. We showed that the combination of the engineered hMSCs with 3D-culture conditions and biocompatible scaffolds preserves the efficiency of silencing, particularly for miR-221, demonstrating the feasibility of our approach for the generation of tissue engineering constructs. On-going experiments are aimed at 1. evaluating the ability of miR-221 depleted hMSCs to synthesize cartilage repair tissue in vivo, by using a specific model of osteochondral defect, and 2. determining specific miR-221 targets during the chondrogenic process.
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|Titolo:||DEPLETION OF THE ANTI-CHONDROGENIC REGULATORS MIR-221 AND SLUG: A NOVEL APPROACH TO INDUCE CHONDROGENESIS|
|Data di pubblicazione:||2016|
|Appare nelle tipologie:||03.5 Abstract su rivista|