A new strategy to express the granulocyte macrophage colony-stimulating factor in mesenchymal stromal cells by in vitro transcribed mRNA.

CANTERO M; GONZALEZ LLAMAZARES L; FIORE E; BAYO J; DOMINGUEZ L; ATORRASAGASTI C; MALVICCINI M; MAZZOLINI G; GARCIA M

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencia (SAIC SAFE SAB SAP); 2019

In the last years, mesenchymal stromal cells (MSCs) have been used for therapeutic purposes, and particularly in cancer, to deliver anti-tumoral agents due to their capacity to home to tumors. Most of the strategies to engineer MSCs are based on virus-mediated gene transfer. However, the use of in vitro transcribed mRNA (IVT mRNA) is gaining attention as a promising tool to induce the expression of therapeutic agents by MSCs. It has also been demonstrated that the granulocyte macrophage colony-stimulating factor (GM-CSF) reduced tumoral growth by improving the antitumor immune response. The aim of this work was to express GM-CSF in MSCs by IVT mRNA with the final purpose to treat liver tumors. To this end, mRNAs (of GM-CSF or DsRed as control) were in vitro transcribed, modified to be more stable within the cell, and then transfected into human umbilical cord derived MSCs. GM-CSF production by MSCs was assayed by ELISA and DsRed expression by flow cytometry. Different quantities of mRNA were used and we observed that low amount of mRNA (0.2 µg mRNA/40.000 cells) was efficient to express DsRed (57.1 ± 3.9%) and GM-CSF (2,48 ± 0,23 µg /ml/1x106 cells) in MSCs. Since the use of MSCs for therapeutic purposes relies on their migration capacity and their low immunogenicity, we evaluated their migration capacity (by a modified Boyden chamber) and their surface markers (CD90, CD44, CD34, CMHII, CD80 and CD86) by flow cytometry. We observed that MSCs expressing IVT mRNA of GM-CSF have the same migratory capacity than unmodified MSCs and their surface markers remained unchanged. Moreover, we evaluated GM-CSF functionality analyzing the ability of conditioned media produced by MSCs expressing GM-CSF to mature dendritic cells in vitro. We observed that the GM-CSF produced by MSC generated matured dendritic cells (68,21 ± 0,24 % CD11c+, CD86+, CMHII+). We conclude that the expression of GM-CSF in MSCs by IVT mRNA is a good and useful strategy for therapeutic purposes.