Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Uhlin E., Marin Navarro A., Rönnholm H., Day K., Kele M., Falk A.

J Vis Exp. 2017

In this video publication, the authors describe a consistent, highly reproducible and easy-to-use protocol, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts. It also offers a user-friendly culture system for the maintenance of established hiPS cells. Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. 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. This protocol has been extensively tested and used to derive more than 300 hiPS cell lines in the Swedish national human iPS Core facility at Karolinska Institutet of which some lines have previously been described.