Derivation, Expansion, and Motor Neuron Differentiation of Human-Induced Pluripotent Stem Cells with Non-Integrating Episomal Vectors and a Defined Xenogeneic-free Culture System

• DOI: 10.1007/s12035-014-9084-z
• Publication Date: 2015-02-09T03:10:15Z
• Authors: Wentao Hu; Yongpei He; Yongjie Xiong; Hong Lu; Hong Chen; Limin Hou; Zhandong Qiu; Yu Fang; Suming Zhang
• Keywords: Adult; Motor Neurons; 0301 basic medicine; Neurogenesis; Genetic Vectors; Induced Pluripotent Stem Cells; Cell Culture Techniques; Fibroblasts; Culture Media, Serum-Free; Recombinant Proteins; Chromosome Banding; 3. Good health; 03 medical and health sciences; Humans; Vitronectin; Cell Self Renewal; Cells, Cultured; Plasmids

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.