Andrzejewska Anna, Grzela Renata, Stankiewicz-Drogon Anna, Rogujski Piotr, Nagaraj Siranjeevi, Darzynkiewicz Edward, Lukomska Barbara, Janowski Miroslaw
NeuroRepair Department, Mossakowski Medical Research Institute, Polish Academy of Sciences, 02-106 Warsaw, Poland.
Center for Advanced Imaging Research, Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland, Baltimore, MD 21201, USA.
Mol Ther Nucleic Acids. 2023 Jul 17;33:454-468. doi: 10.1016/j.omtn.2023.07.006. eCollection 2023 Sep 12.
We previously have shown that mRNA-based engineering may enhance mesenchymal stem cell (MSC) trafficking. However, optimal conditions for mRNA engineering of MSCs are unknown. Here, we investigated several independent variables: (1) transfection factor (Lipofectamine 2000 vs. TransIT), (2) mRNA purification method (spin column vs. high-performance liquid chromatography [HPLC] column), and (3) mRNA capping (ARCA vs. β-S-ARCA D1 and β-S-ARCA D2). Dependent variables included protein production based on mRNA template (measured by the bioluminescence of reporter gene luciferase over hours), MSC metabolic activity corresponding with their wellbeing measured by CCK-8 over days, and endogenous expression of genes by RT-qPCR related to innate intracellular immune response and decapping at two time points: days 2 and 5. We have found that Lipofectamine 2000 outperforms TransIT, and used it throughout the study. Then, we showed that mRNA must be purified by HPLC to be relatively neutral to MSCs in terms of metabolic activity and endogenous protein production. Ultimately, we demonstrated that β-S-ARCA D1 enables higher protein production but at the cost of lower MSC metabolic activity, with no impact on RT-qPCR results. Thus Lipofectamine 2000-based transfection of HPLC-purified and ARCA- or β-S-ARCA D1-capped mRNA is optimal for MSC engineering.
我们之前已经表明，基于mRNA的工程技术可能会增强间充质干细胞（MSC）的迁移能力。然而，MSC的mRNA工程的最佳条件尚不清楚。在此，我们研究了几个独立变量：（1）转染因子（Lipofectamine 2000与TransIT），（2）mRNA纯化方法（旋转柱与高效液相色谱[HPLC]柱），以及（3）mRNA加帽（ARCA与β-S-ARCA D1和β-S-ARCA D2）。因变量包括基于mRNA模板的蛋白质产生（通过报告基因荧光素酶数小时的生物发光来测量）、通过CCK-8在数天内测量的与MSC健康状况相对应的代谢活性，以及通过RT-qPCR在两个时间点（第2天和第5天）测量的与先天性细胞内免疫反应和去帽相关的基因的内源性表达。我们发现Lipofectamine 2000优于TransIT，并在整个研究中使用它。然后，我们表明mRNA必须通过HPLC纯化，以便在代谢活性和内源性蛋白质产生方面对MSC相对中性。最终，我们证明β-S-ARCA D1能够实现更高的蛋白质产生，但代价是MSC代谢活性降低，对RT-qPCR结果没有影响。因此，基于Lipofectamine 2000对HPLC纯化且用ARCA或β-S-ARCA D1加帽的mRNA进行转染是MSC工程的最佳选择。
