Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate

Kaimin Wu,^1,* Jie Xu,^2,* Mengyuan Liu,¹ Wen Song,¹ Jun Yan,¹ Shan Gao,³ Lingzhou Zhao,² Yumei Zhang<sup>1

1</sup>Department of Prosthetic Dentistry, ²Department of Periodontology and Oral Medicine, School of Stomatology, The Fourth Military Medical University, Xi’an, People’s Republic of China; ³The Interdisciplinary Nanoscience Center and Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark; School of Stomatology, Tianjin Medical University, Tianjin, People’s Republic of China

^*Both authors contributed equally to this work

Abstract: MicroRNA (miRNA) regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes on a tissue culture plate. The lipoplexes can be immobilized on a tissue culture plate with an intact pseudospherical structure and lyophilization without any lyoprotectant. In this study, reverse transfection resulted in highly efficient cellular uptake of miRNA and enabled significant manipulation of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 µL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection formulations did not deteriorate during 90 days of storage at 4°C and -20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing osteogenic differentiation, as indicated by enhanced osteogenesis-related gene expression, amount of alkaline phosphatase present, production of collagen, and matrix mineralization. Overall, the miRNA reverse transfection formulation developed in this study is a promising approach for miRNA transfection which can control stem cell fate and is suitable for loading miRNAs onto various biomaterials.

Keywords: microRNAs, lyophilization, tissue culture plate, mesenchymal stem cells, osteogenic differentiation