title: Derivation, Expansion, and Motor Neuron Differentiation of Human-Induced Pluripotent Stem Cells with Non-Integrating Episomal Vectors and a Defined Xenogeneic-free Culture System.
abstract: 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.
authors: Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China.|Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450000, Henan, China.
authors: Wentao
authors: W
authors: Hu
authors: Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China.
authors: Yongpei
authors: Y
authors: He
authors: Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China.
authors: Yongjie
authors: Y
authors: Xiong
authors: Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450000, Henan, China.
authors: Hong
authors: H
authors: Lu
authors: Department of Rehabilitation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China.
authors: Hong
authors: H
authors: Chen
authors: Department of Rehabilitation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China.
authors: Limin
authors: L
authors: Hou
authors: Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China.
authors: Zhandong
authors: Z
authors: Qiu
authors: Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China.
authors: Yu
authors: Y
authors: Fang
authors: Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China. summing_zhang@yeah.net.
authors: Suming
authors: S
authors: Zhang
mesh_terms: D000328:Adult; D018929:Cell Culture Techniques; D000066673:Cell Self Renewal; D002478:Cells, Cultured; D002871:Chromosome Banding; D016895:Culture Media, Serum-Free; D005347:Fibroblasts; D005822:Genetic Vectors; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D009046:Motor Neurons / Q000166:cytology*; D055495:Neurogenesis; D010957:Plasmids; D011994:Recombinant Proteins / Q000494:pharmacology; D019096:Vitronectin / Q000494:pharmacology
chemical_list: D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D019096:Vitronectin
keywords: Human-induced pluripotent stem cells; Integration-free; Motor neurons; Vitronectin; Xeno-free