Induction of osteogenic differentiation of MSCs by GSK3β knockdown through GSK3β siRNAs transfection

The development of effective strategies for treating bone defects can be based on gene therapy methods aimed at regulating the differentiation of osteoprogenitor cells. One of the approaches is to use siRNA molecules in knockdown systems for genes inhibiting osteogenic cell differentiation. In this work, we aimed at developingapproaches to induce osteogenic differentiation of mesenchymal stem cells (MSCs) by siRNAs-mediated knockdown of GSK3β siRNAs in cultures of MSCs derived from human adipose tissue (AD-MSCs). For this purpose, we compared the transfection efficacy of lipoplexes and polyplexes formed with one of four siRNA molecules and five commercial transfection agents most commonly used in laboratory practice. The most effective transfection agent was found to be linear polyethylenimine (PEI) which demonstrated high cytocompatibility both in free form and in polyplexes (even when maximum concentrations were used). Using the polyplexes formed by the newly designed siRNA and PEI, we constructed a highly efficient GSK3β gene knockdown system, which showed effectiveness in AD-MSC cultures. As a result, we demonstrated the osteoinductive properties of GSK3β siRNA molecules in these cultures. These results provide a methodological basis for future siRNA-based strategies targeting GSK3β in osteogenic applications, with in vivo studies needed to establish its translational potential.