Efficient germ-line transmission obtained with transgene-free induced pluripotent stem cells

Significance Using a single, nonintegrating episome, containing an optimized assembly of reprogramming factors and positive/negative selection markers, we generated germ-line–competent induced pluripotent stem (iPS) cells. To ensure that the iPS cells were transgene-free (i.e., were independent of exogenous reprogramming factors to achieve and maintain their pluripotent ground state) required the inclusion on the episome more that the classical four (POU5F1/OCT4, KLF4, SOX2, and cMYC) reprogramming factors. Also critical for the transgene-free iPS cells exhibiting competency for germ-line transmission was the requirement for growth in 2i medium. Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine. To overcome potential problems associated with transgene insertions, efforts have been directed over the past several years to generate transgene-free iPS cells by using non-viral-vector approaches. To date, however, cells generated through such procedures have had problems producing reproductively competent animals, suggesting that their quality needed further improvement. Here we report the use of optimized assemblies of reprogramming factors and selection markers incorporated into single plasmids as nonintegrating episomes to generate germ-line–competent iPS cells. In particular, the pMaster12 episome can produce transgene-free iPS cells that, when grown in 2i medium, recapitulate good mouse ES cells, in terms of their competency for generating germ-line chimeras.