Generation of Nonviral Integration-Free Induced Pluripotent Stem Cells from Plucked Human Hair Follicles

Human induced pluripotent stem cells (hiPSCs) provide a unique experimental reagent for dissecting the complex transcriptional, regulatory, and epigenetic mechanisms of pluripotency, as well as for studying normal and diseased human development. However, the utility of current iPSC lines may be limited by the utilization of integrating viral vectors for transgenic ectopic expression of oncogenic reprogramming factors (e.g., SOX2 , OCT4, KLF4 , MYC, NANOG , LIN28 , and SV40 T antigen ). Leaky expression of integrated pluripotency factor transgenes may inhibit completion of the somatic cell reprogramming process, pose great potential for subsequent malignant transformation, and ultimately limit differentiation strategies and their future clinical application. hiPSCs generated with transgene and vector-free approaches may more faithfully resemble human embryonic stem cells (hESCs) and obviate some or all of these important caveats. In this chapter, we describe a simple and reproducible methodology for generating clinically safe nonviral, integration-free hiPSCs from keratinocytes noninvasively obtained and expanded from a donor’s single plucked hair follicle. Nonintegrated hiPSCs free of viral and transgene sequences should provide a potent tool for studies of pluripotency, and ultimately be more clinically useful in regenerative medicine.