Induced pluripotent stem cells (iPSCs) generated from patient-derived somatic cells provides the opportunity for model development in order to study patient-specific disease states with the potential for drug discovery. However, use of lentivirus and exposure of iPSCs to animal-derived products limit their therapeutic utility and affect lineage differentiation and subsequent downstream functionality of iPSC derivatives. Within the context of this study, we describe a simple and practical protocol enabling the efficient reprogramming of terminally differentiated adult fibroblasts into integration-free human iPSCs (hiPSCs) using a combination of episomal plasmids with small molecules (SMs). Using this approach, there was a 10-fold increase in reprogramming efficiency over single plasmid vector-based methods. We obtained approximately 100 iPSCs colonies from 1 × 10(5) human adult dermal fibroblasts (HADFs) and achieved approximately 0.1% reprogramming efficiencies. Concurrently, we developed a highly conducive culture system using xeno-free media and human vitronectin. The resulting hiPSCs were free of DNA integration and had completely lost episomal vectors, maintained long-term self-renewal, featured a normal karyotype, expressed pluripotent stem cell markers, and possessed the capability of differentiating into components of all three germ layers in vivo. Finally, we demonstrate that the integration-free hiPSCs could be differentiated into motor neurons under xeno-free culture conditions. This induction method will promote the derivation of patient-specific integration-free and xeno-free iPSCs and improve the strategy for motor neuron derivation. Our approach provides a useful tool for human disease models, drug screen, and clinical applications.
Keywords: Human-induced pluripotent stem cells; Integration-free; Motor neurons; Vitronectin; Xeno-free.