Introduction: Human Mesenchymal Stromal Cells (hMSCs)-based tissue engineering is generally regarded as one of the most promising approaches for cartilage regeneration. Nevertheless, suitable protocols for the in vitro manipulation of stem cells for the production of implantable constructs are yet to be found. 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 traditional differentiating agents, such as TGF-β. Recently, miR-221 and Slug transcription factor have emerged as negative chondroregulatory molecules. We investigated if the inhibition of these regulators could be sufficient to address hMSCs from Wharton’s Jelly (WJMSCs) toward a chondrocyte-like phenotype, in the absence of TGF-β. Moreover, in order to confirm the feasibility of this approach for other MSCs sources and for the generation of tissue engineering constructs, the expression of miR-221 and Slug was inhibited in Bone Marrow derived hMSCs (BMSCs) and the effect of cell encapsulation in alginate was assessed. Methods: miR-221 and Slug expression was transiently inhibited in WJMSCs, using specific antagomiR and siRNA molecules, respectively. The effect of miR-221 and Slug silencing on the expression of extracellular matrix proteins and specific transcription factors was evaluated by qRT-PCR and immunocytochemistry. Chromatin immunoprecipitation (ChIP) assay was employed to explore the possibility of a functional correlation between miR-221 and Slug. miR-221 and Slug expression was inhibited in BMSCs, before cell encapsulation in alginate beads; the efficiency of gene silencing was monitored up to 28 days post-encapsulation.

Enhanced chondrogenic potential of Slug and miR-221 depleted human MSCs.

LOLLI, Andrea;LAMBERTINI, Elisabetta;PENOLAZZI, Maria Letizia;ANGELOZZI, MARCO;VEZZALI, Federica;PIVA, Maria Roberta
2014

Abstract

Introduction: Human Mesenchymal Stromal Cells (hMSCs)-based tissue engineering is generally regarded as one of the most promising approaches for cartilage regeneration. Nevertheless, suitable protocols for the in vitro manipulation of stem cells for the production of implantable constructs are yet to be found. 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 traditional differentiating agents, such as TGF-β. Recently, miR-221 and Slug transcription factor have emerged as negative chondroregulatory molecules. We investigated if the inhibition of these regulators could be sufficient to address hMSCs from Wharton’s Jelly (WJMSCs) toward a chondrocyte-like phenotype, in the absence of TGF-β. Moreover, in order to confirm the feasibility of this approach for other MSCs sources and for the generation of tissue engineering constructs, the expression of miR-221 and Slug was inhibited in Bone Marrow derived hMSCs (BMSCs) and the effect of cell encapsulation in alginate was assessed. Methods: miR-221 and Slug expression was transiently inhibited in WJMSCs, using specific antagomiR and siRNA molecules, respectively. The effect of miR-221 and Slug silencing on the expression of extracellular matrix proteins and specific transcription factors was evaluated by qRT-PCR and immunocytochemistry. Chromatin immunoprecipitation (ChIP) assay was employed to explore the possibility of a functional correlation between miR-221 and Slug. miR-221 and Slug expression was inhibited in BMSCs, before cell encapsulation in alginate beads; the efficiency of gene silencing was monitored up to 28 days post-encapsulation.
2014
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2338273
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