Using human IPS cells to study fate, function and neurodegenerative disease

使用人类 IPS 细胞研究命运、功能和神经退行性疾病

• 批准号: 9444793
• 负责人: LORRAINE IACOVITTI
• 金额: $ 37.88万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9444793
• 关键词: 1-Methyl-4-phenylpyridinium; Affect; Aftercare; Amyotrophic Lateral Sclerosis; Animal Model; Autologous; Behavior; Binding; Cell Line; Cell surface; Cells; Chromatin; Chromatin Structure; Clinic; Cues; DNA; DNA biosynthesis; Data; Development; Disease; Disease model; Dopamine; Epigenetic Process; Genetic study; Goals; Grant; Harvest; Heterogeneity; High Pressure Liquid Chromatography; Histones; Human; Hydroxydopamines; Implant; In Vitro; Investigational Therapies; LRRK2 gene; Midbrain structure; Mind; Modeling; Molecular Conformation; Motor; Motor Neuron Disease; Motor Neurons; Muscle; Names; Neurodegenerative Disorders; Neurons; Parkinson Disease; Pathogenicity; Pathway interactions; Patients; Phenotype; Physiological; Population; Positron-Emission Tomography; Process; Rattus; Recruitment Activity; Replacement Therapy; Specific qualifier value; Stem cells; Technology; Testing; Therapeutic; Therapeutic Uses; Time; Toxic Environmental Substances; Translations; Transplantation; Undifferentiated; Work; alpha synuclein; cell type; dopaminergic neuron; drug discovery; human embryonic stem cell; in vivo; induced pluripotent stem cell; insight; nerve stem cell; progenitor; stem cell differentiation; synaptic function; tool; transcription factor

Human induced pluripotent stem cells (iPSCs), with their potential to generate autologous patient-derived cells, hold great promise for the study and treatment of a host of devastating diseases, including Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS), to name a few. One of the major obstacles slowing the translation of this powerful technology to the clinic is the heterogeneity of both desired and unwanted cell types generated in grafts of iPSCs, even after cells have been directed down specific differentiation pathways. Similarly in culture, a multitude of cell types are generated after treatment of iPSCs with lineage-specifying cocktails. These realities, combined with the current lack of suitable cell surface markers for the selection of specific desired cell types, has significantly impacted the field, hampering our ability to develop cell replacement therapies or to accurately model diseases in the dish. One plausible explanation for the observed cell heterogeneity is that presumptive undifferentiated pluripotent cells sometimes spontaneously initiate the process of differentiation after encountering lineage- specifying cues in culture, thereby precluding their subsequent directed differentiation by exogenously added differentiation cocktails. Currently, there are no assured ways to know if iPSCs have begun to spontaneously differentiate. However, an exciting new discovery made during our previous grant cycle suggests that the epigenetic state of chromatin shortly after DNA replication serves as a reliable and very early indicator of the state of differentiation of a stem cell. Our results suggest that it may be possible to uniformly direct the differentiation of all iPSCs toward a specific cell fate if chromatin can be kept closed until incubation with exogenous fate-specifying differentiation factors. If these insights are indicative of a more generalized principle, then it should be possible to generate pure populations of neural progenitors (NPs) of different subtypes which can give rise to homogeneous populations of various neurons for studies in culture and in animal models of multiple diseases. With these goals in mind, our Specific Aims for this proposal are: 1) to assess chromatin status during commitment to a motor neuron phenotype; 2) to generate pure populations of midbrain dopamine (mDA) and motor NPs and neurons which will be characterized for phenotype and synaptic function in culture and 3) to further determine whether homogeneous mDA-committed NPs/neurons can accurately model PD in the dish and be used in transplants to therapeutically treat PD rat models.

人类诱导多能干细胞 (iPSC)，具有产生自体患者来源的潜力 细胞为研究和治疗许多破坏性疾病带来了巨大希望，包括 帕金森病 (PD) 和肌萎缩侧索硬化症 (ALS) 等。主要之一 阻碍这一强大技术向临床转化的障碍是两者的异质性 即使在细胞被向下定向后，iPSC 移植物中也会产生所需和不需要的细胞类型 特定的分化途径。同样，在培养物中，处理后会产生多种细胞类型 iPSC 与谱系指定鸡尾酒。这些现实，加上目前缺乏合适的电池 用于选择特定所需细胞类型的表面标记，对该领域产生了重大影响， 阻碍了我们开发细胞替代疗法或在培养皿中准确模拟疾病的能力。 对观察到的细胞异质性的一种合理解释是，假定未分化细胞 多能细胞有时会在遇到谱系后自发启动分化过程 指定培养物中的线索，从而排除它们随后通过外源性定向分化 添加差异化鸡尾酒。目前，没有确定的方法可以知道 iPSC 是否已经开始 自发分化。然而，我们在上一个资助周期中取得了一个令人兴奋的新发现 表明 DNA 复制后不久染色质的表观遗传状态是可靠且非常有效的 干细胞分化状态的早期指标。我们的结果表明，有可能 如果染色质可以保持关闭，则统一引导所有 iPSC 向特定细胞命运分化 直到与外源命运指定分化因子一起孵育。如果这些见解表明 更普遍的原则，那么应该有可能产生纯神经祖细胞群 不同亚型的（NP）可以产生用于研究的各种神经元的同质群体 在多种疾病的培养物和动物模型中。考虑到这些目标，我们为此制定了具体目标 建议是：1）评估运动神经元表型形成过程中的染色质状态； 2）到 产生纯中脑多巴胺 (mDA) 和运动 NP 和神经元群， 表征培养物中的表型和突触功能，3) 进一步确定是否 同质 mDA 承诺的 NP/神经元可以准确地模拟培养皿中的 PD，并可用于 移植治疗帕金森病大鼠模型。