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

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• 作者：Wentao Hu ; Yongpei He ; Yongjie Xiong ; Hong Lu ; Hong Chen…
• 关键词：Human ; induced pluripotent stem cells ; Xeno ; free ; Integration ; free ; Vitronectin ; Motor neurons
• 刊名：Molecular Neurobiology
• 出版年：2016
• 出版时间：April 2016
• 年：2016
• 卷：53
• 期：3
• 页码：1589-1600
• 全文大小：7,595 KB
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• 作者单位：Wentao Hu (1) (2)
  Yongpei He (1)
  Yongjie Xiong (1)
  Hong Lu (2)
  Hong Chen (3)
  Limin Hou (3)
  Zhandong Qiu (1)
  Yu Fang (1)
  Suming Zhang (1)
  
  1. Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China
  2. Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450000, Henan, China
  3. Department of Rehabilitation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China
  
• 刊物主题：Neurosciences; Neurobiology; Cell Biology; Neurology;
• 出版者：Springer US
• ISSN：1559-1182

文摘

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 × 105 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.