Derivation, Expansion, and Motor Neuron Differentiation of Human-Induced Pluripotent Stem Cells with Non-Integrating Episomal Vectors and a Defined Xenogeneic-free Culture System
Derivation, Expansion, and Motor Neuron Differentiation of Human-Induced Pluripotent Stem Cells with Non-Integrating Episomal Vectors and a Defined Xenogeneic-free Culture System
复制标题
DOI：
10.1007/s12035-014-9084-z
复制
发表时间：
2016-04-01
影响因子：
5.1
通讯作者：
Zhang, Suming
中科院分区：
文献类型：
--
作者：
Hu, Wentao;He, Yongpei;Zhang, Suming
从患者源性体细胞产生的诱导多能干细胞（iPSC）为模型开发提供了机会，以便研究具有药物发现潜力的患者特异性疾病状态。然而，慢病毒的使用和iPSC暴露于动物来源的产品限制了它们的治疗效用，并影响iPSC衍生物的谱系分化和随后的下游功能。在本研究的背景下，我们描述了一种简单而实用的方案，使终末分化的成人成纤维细胞有效地重编程为无整合的人iPSC（hiPSC）使用附加型质粒与小分子（SM）的组合。使用这种方法，重编程效率比基于单一质粒载体的方法提高了10倍。我们从1 × 10（5）人成人真皮成纤维细胞（HADF）获得了大约100个iPSC集落，并实现了大约0.1%的重编程效率。同时，我们开发了一个高度有益的文化系统，使用无异种介质和人玻连蛋白。所得到的hiPSC不含DNA整合，完全失去了附加型载体，保持长期自我更新，具有正常核型，表达多能干细胞标志物，并具有在体内分化为所有三个胚层组分的能力。最后，我们证明了无整合的hiPSC可以在无异种培养条件下分化为运动神经元。这种诱导方法将促进患者特异性无整合和无异种iPSC的衍生，并改进运动神经元衍生的策略。我们的方法为人类疾病模型、药物筛选和临床应用提供了有用的工具。
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 x 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.