Protein-induced human iPS cells for personalized cell therapy of PD

蛋白质诱导的人类 iPS 细胞用于 PD 个性化细胞治疗

基本信息

批准号：
8481598
负责人：
Kwang-Soo Kim
金额：
$ 32.69万
依托单位：
MCLEAN HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-06-01 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8481598
关键词：
Animal Model Behavioral Biological Biomedical Research Calculi Cell Line Cell Therapy Cell Transplantation Cells Clinical Data Clinical Research Collaborations Controlled Clinical Trials Copy Number Polymorphism Corpus striatum structure Custom DNA Data Defect Development Disease Disease model Double-Blind Method Embryo Epigenetic Process Exhibits Family Fibroblasts Functional disorder Gene Expression Generations Genes Genetic Genomics Goals Human In Vitro Infant Karyotype Laboratories Lead Lesion Letters Location Methods Midbrain structure Molecular Morphology Motor Neurodegenerative Disorders Neurons Newborn Infant Oligonucleotides Outcome Oxidopamine Parkinson Disease Pathology Patients Peptides Physiological Placebo Control Pluripotent Stem Cells Property Proteins Publishing Rattus Recovery Regimen Resolution Retroviridae Rodent Model Somatic Cell Source Southern Blotting Staging Stem Cell Research Stem cells Substantia nigra structure Techniques Testing Therapeutic Tissues Transgenes Transplantation Validation Viral Virus Work aphakia mice base c-myc Genes cell type clinical application disease mechanisms study dopaminergic neuron embryonic stem cell fetal human disease human embryonic stem cell human embryonic stem cell line improved in vivo induced pluripotent stem cell insight male nerve stem cell neurotransmission novel open label preclinical study public health relevance research study retroviral transduction stem cell technology tumor vector viral DNA

项目摘要

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is the second most common neurodegenerative disorder in which the main pathology is selective degeneration of midbrain dopaminergic (mDA) neurons in the substantia nigra. Because of this specific cell loss, PD is considered to be a prime target disease for cell-based therapy. Indeed, numerous clinical and preclinical studies demonstrated the proof-of-principle that cell transplantation is a viable therapeutic regimen for PD treatment once an ideal and unlimited cell source can be established (reviewed in (Redmond, 2002; Li et al., 2008a; Lindvall and Kokaia, 2009). In 2006, Shinya Yamanaka and his colleagues published their groundbreaking work showing that pluripotent stem cells, so called "induced pluripotent stem cells (iPSCs)", can be generated from somatic cells by retroviral transduction of four reprogramming factors (i.e., Oct4, Sox2, Klf4 and c-Myc)(Takahashi and Yamanaka, 2006). Subsequent successful generation of human iPSCs by similar methods (Takahashi et al., 2007; Yu et al., 2007; Park et al., 2008a) offered the possibility to generate disease- or patient-specific stem cells without destruction of embryos. Indeed, these iPSCs offer unprecedented potentials for biomedical research, disease mechanism study, and personalized cell-based therapies. However, current iPSCs suffer from major drawbacks including multiple viral integrations and remaining transgenes at various chromosomal locations, any of which may cause unpredictable genetic dysfunction and/or tumor formation, making these cells unsuitable for clinical applications (Yamanaka, 2009a). With the long-term goal of developing a personalized cell-based therapy of PD using iPSCs, we propose the following three specific aims. First, based on our preliminary results (Kim et al., 2009a), we will establish iPSC lines from healthy and sporadic PD subjects by a novel, DNA-free reprogramming method (i.e., direct delivery of reprogramming proteins). We will extensively evaluate morphological, gene expression, epigenetic, and in vitro and in vivo differentiation properties to establish iPSC lines exhibiting properties similar to human embryonic stem cells (hESCs). We will compare their properties with those of hESC lines and iPSCs generated by conventional retroviral methods. In addtion, we will investigate their chromosomal integrity by state of the art copy number variation analysis. Second, once authentic iPSC lines are established, we will fully characterize and compare their in vitro differentiation properties into neural progenitors and midbrain DA neurons. We will optimize their differentiation into A9 DA neurons and evaluate the molecular, cellular, and electrophysiological characters of DA neurons from these iPS cells. Third, we will initiate to evaluate the potential functional benefits of these iPSC-derived DA neurons in two rodent models of PD; aphakia mice and 6-OHDA lesioned rats, both of which are well established in our laboratory. Biological and behavioral outcomes of these transplantation studies will be systematically investigated. Overall, our proposed experiments will provide invaluable insights and stepping stones, leading to the development of safe, realistic, and ideal cell source for personalized cell-based therapy of PD.

描述（由申请人提供）：帕金森病（PD）是第二常见的神经退行性疾病，其主要病理学是黑质中脑多巴胺能（mDA）神经元的选择性变性。由于这种特定的细胞损失，帕金森病被认为是细胞治疗的主要目标疾病。事实上，大量临床和临床前研究证明，一旦建立了理想且无限的细胞来源，细胞移植就是一种可行的 PD 治疗方案（综述见（Redmond，2002 年；Li 等人，2008a；2008 年）。 Lindvall 和 Kokaia，2006 年）Shinya Yamanaka 和他的同事发表了他们的开创性工作，表明多能干细胞，即所谓的“诱导”。多能干细胞（iPSC）”，可以通过逆转录病毒转导四种重编程因子（即 Oct4、Sox2、Klf4 和 c-Myc）从体细胞产生（Takahashi 和 Yamanaka，2006）。随后通过类似方法成功产生人类 iPSC方法（Takahashi 等人，2007 年；Yu 等人，2007 年；Park 等人， 2008a）提供了在不破坏胚胎的情况下生成疾病或患者特异性干细胞的可能性。事实上，这些 iPSC 为生物医学研究、疾病机制研究和个性化细胞治疗提供了前所未有的潜力。包括多个病毒整合和不同染色体位置的剩余转基因，其中任何一个都可能导致不可预测的遗传功能障碍和/或肿瘤形成，使这些细胞不适合临床应用（Yamanaka， 2009a）。长期目标是利用 iPSC 开发个性化的基于细胞的 PD 疗法，我们提出以下三个具体目标。首先，根据我们的初步结果（Kim 等人，2009a），我们将通过一种新颖的、无 DNA 重编程方法（即直接递送重编程蛋白）从健康和散发性 PD 受试者中建立 iPSC 系。我们将广泛评估形态学、基因表达、表观遗传以及体外和体内分化特性，以建立表现出与人类胚胎干细胞 (hESC) 相似特性的 iPSC 系。我们将它们的特性与传统逆转录病毒方法产生的 hESC 系和 iPSC 的特性进行比较。此外，我们将通过最先进的拷贝数变异分析来研究它们的染色体完整性。其次，一旦建立了真正的 iPSC 系，我们将充分表征并比较它们向神经祖细胞和中脑 DA 神经元的体外分化特性。我们将优化它们向 A9 DA 神经元的分化，并评估来自这些 iPS 细胞的 DA 神经元的分子、细胞和电生理特征。第三，我们将开始评估这些 iPSC 衍生的 DA 神经元在两种 PD 啮齿动物模型中的潜在功能益处；无晶状体小鼠和 6-OHDA 损伤大鼠，这两种模型均已在我们的实验室中得到了很好的应用。这些移植研究的生物学和行为结果将得到系统研究。总体而言，我们提出的实验将提供宝贵的见解和垫脚石，从而为个性化细胞治疗 PD 开发安全、现实和理想的细胞来源。