Division of Physiology, Livestock Research Institute, Council of Agriculture, Tainan, Taiwan.
Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan.
Sci Rep. 2019 Oct 31;9(1):15745. doi: 10.1038/s41598-019-52282-7.
The objective of this study was to establish a versatile cell line for replication-incompetent virus production and inactivation with formaldehyde to generate a model of cell-based vaccine manufacturing process. To achieve this goal, we took advantage of the easily accessed chick embryonic fibroblasts. Nine-day old chick embryonic fibroblasts were obtained and subjected to be transduced with a set of lentivirus to develop a chick induced pluripotent stem (ciPS) cell line. Morphological features, positive periodic acid-Schiff staining as well as strong immunocytofluorescence of alkaline phosphatase, intestinal (ALPI) and POU class 5 homeobox 1 (POU5F1) proteins suggested that these chick embryonic fibroblasts have been transformed into ciPS cells. Further differentiation and immunocytofluorescence assays confirmed that this ciPS cell line possesses capacities and potentials to form embryoid bodies, differentiate into all three embryonic layers: ectoderm, mesoderm and endoderm with evidence of strongly positive and specific molecular markers. Immunoblot analysis next demonstrated that through recombinant DNA technology and the 2 generation lentiviral transfer system, the goose hemagglutinin gene (H5) gene was packaged into the replication-incompetent virus and highly expressed in a bladder cancer-derived cell line, T24, after transduction. The titer of ciPS-generated replication-incompetent virus is comparable to that from the Phoenix-AMPHO cell line, which is a commercial and high productive retrovirus producer. Our study successfully established a ciPS cell line which is able to produce replication-incompetent virus, providing a new strategy for cell-based vaccine production after virus inactivation.
本研究的目的是建立一种多功能细胞系，用于复制缺陷型病毒的生产和甲醛灭活，以生成基于细胞的疫苗制造工艺模型。为了实现这一目标，我们利用了易于获得的鸡胚成纤维细胞。获得 9 日龄鸡胚成纤维细胞，并进行一组慢病毒转导，以开发鸡诱导多能干细胞（ciPS）细胞系。形态特征、过碘酸希夫（PAS）阳性染色以及碱性磷酸酶、肠（ALPI）和 POU 类 5 同源框 1（POU5F1）蛋白的强免疫细胞荧光提示这些鸡胚成纤维细胞已转化为 ciPS 细胞。进一步的分化和免疫细胞荧光分析证实，该 ciPS 细胞系具有形成胚状体的能力和潜力，并分化为外胚层、中胚层和内胚层这三个胚胎层，具有强烈阳性和特异性分子标志物的证据。免疫印迹分析进一步表明，通过重组 DNA 技术和第二代慢病毒转移系统，将鹅血凝素基因（H5）基因包装到复制缺陷型病毒中，并在转导后在膀胱癌衍生细胞系 T24 中高表达。ciPS 产生的复制缺陷型病毒的滴度可与 Phoenix-AMPHO 细胞系相媲美，后者是一种商业上高产的逆转录病毒生产细胞系。我们的研究成功建立了一种能够产生复制缺陷型病毒的 ciPS 细胞系，为病毒灭活后的基于细胞的疫苗生产提供了一种新策略。
