Uhlin Elias, Marin Navarro Ana, Rönnholm Harriet, Day Kelly, Kele Malin, Falk Anna
Department of Neuroscience, Karolinska Institutet.
Department of Neuroscience, Karolinska Institutet; Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet.
J Vis Exp. 2017 Jul 7(125):56146. doi: 10.3791/56146.
Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.
无动物源且成分完全确定的条件是稳健且可重复地生成同质人诱导多能干细胞（hiPS细胞）的关键参数。将hiPS细胞维持在饲养细胞或成分未明的基质上易受批次差异、病原体污染及免疫原性风险的影响。利用成分确定的重组人层粘连蛋白521（LN-521）基质并结合无动物源且成分确定的培养基配方可减少变异性，并能持续生成hiPS细胞。仙台病毒（SeV）载体是一种基于RNA的非整合系统，因此避免了与整合型载体可能对基因组完整性产生的潜在破坏作用相关的担忧。此外，这些载体在重编程真皮成纤维细胞方面已显示出相对较高的效率。此外，通过酶促单细胞传代培养细胞有助于hiPS细胞的同质维持，而无需干细胞培养方面的大量前期经验。在此，我们描述了一个经过广泛测试和开发的方案，该方案侧重于可重复性和易用性，提供了一种从成纤维细胞生成成分确定且无动物源的人hiPS细胞的稳健且实用的方法。
