Kozisek Tyler, Hamann Andrew, Nguyen Albert, Miller Michael, Plautz Sarah, Pannier Angela K
1Department of Biological Systems Engineering, University of Nebraska-Lincoln, 231 L.W. Chase Hall, Lincoln, NE USA.
2Department of Biomedical Engineering, Pennsylvania State University, 122 Chemical and Biomedical Engineering Building, University Park, PA USA.
J Biol Eng. 2020 May 19;14:16. doi: 10.1186/s13036-020-00238-1. eCollection 2020.
Human mesenchymal stem cells (hMSCs) are intensely researched for applications in cell therapeutics due to their unique properties, however, intrinsic therapeutic properties of hMSCs could be enhanced by genetic modification. Viral transduction is efficient, but suffers from safety issues. Conversely, nonviral gene delivery, while safer compared to viral, suffers from inefficiency and cytotoxicity, especially in hMSCs. To address the shortcomings of nonviral gene delivery to hMSCs, our lab has previously demonstrated that pharmacological 'priming' of hMSCs with the glucocorticoid dexamethasone can significantly increase transfection in hMSCs by modulating transfection-induced cytotoxicity. This work seeks to establish a library of transfection priming compounds for hMSCs by screening 707 FDA-approved drugs, belonging to diverse drug classes, from the NIH Clinical Collection at four concentrations for their ability to modulate nonviral gene delivery to adipose-derived hMSCs from two human donors.
Microscope images of cells transfected with a fluorescent transgene were analyzed in order to identify compounds that significantly affected hMSC transfection without significant toxicity. Compound classes that increased transfection across both donors included glucocorticoids, antibiotics, and antihypertensives. Notably, clobetasol propionate, a glucocorticoid, increased transgene production 18-fold over unprimed transfection. Furthermore, compound classes that decreased transfection across both donors included flavonoids, antibiotics, and antihypertensives, with the flavonoid epigallocatechin gallate decreasing transgene production - 41-fold compared to unprimed transfection.
Our screen of the NCC is the first high-throughput and drug-repurposing approach to identify nonviral gene delivery priming compounds in two donors of hMSCs. Priming compounds and classes identified in this screen suggest that modulation of proliferation, mitochondrial function, and apoptosis is vital for enhancing nonviral gene delivery to hMSCs.
人间充质干细胞（hMSCs）因其独特性质而在细胞治疗应用方面受到广泛研究，然而，hMSCs的内在治疗特性可通过基因修饰得到增强。病毒转导效率高，但存在安全问题。相反，非病毒基因递送虽比病毒递送更安全，但存在效率低下和细胞毒性问题，尤其是在hMSCs中。为解决hMSCs非病毒基因递送的缺点，我们实验室先前已证明，用糖皮质激素地塞米松对hMSCs进行药理学“预处理”可通过调节转染诱导的细胞毒性显著增加hMSCs中的转染效率。这项工作旨在通过从美国国立卫生研究院临床收藏库中筛选707种经美国食品药品监督管理局（FDA）批准的、属于不同药物类别的药物，以四种浓度测试其调节来自两名人类供体的脂肪来源hMSCs非病毒基因递送的能力，从而建立一个hMSCs转染预处理化合物库。
对用荧光转基因转染的细胞的显微镜图像进行分析，以鉴定能显著影响hMSC转染且无明显毒性的化合物。在两名供体中均能提高转染效率的化合物类别包括糖皮质激素、抗生素和抗高血压药。值得注意的是，糖皮质激素丙酸氯倍他索使转基因产量比未预处理的转染提高了18倍。此外，在两名供体中均能降低转染效率的化合物类别包括类黄酮、抗生素和抗高血压药，与未预处理的转染相比，类黄酮表没食子儿茶素没食子酸酯使转基因产量降低了41倍。
我们对NCC的筛选是首次采用高通量和药物重新利用方法在两名hMSCs供体中鉴定非病毒基因递送预处理化合物。在此筛选中鉴定出的预处理化合物和类别表明，调节增殖、线粒体功能和细胞凋亡对于增强hMSCs的非病毒基因递送至关重要。
