Production of dopamine neurons from human embryonic stem cells

从人类胚胎干细胞产生多巴胺神经元

基本信息

批准号：
7342518
负责人：
KAI CHRISTIAN SONNTAG
金额：
$ 17.61万
依托单位：
MCLEAN HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-01-22 至 2008-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7342518
关键词：
Address Adverse effects Animal Model Attention Automobile Driving Biological Brain Cell Therapy Cells Cellular biology Chemicals Chronic Clinical Cloning Condition Deep Brain Stimulation Development Dopaminergic Cell Drug resistance Dyskinetic syndrome Embryonic Development Engineering Fluorescence Fluorescence-Activated Cell Sorting Functional disorder Gene Transfer Generations Genes Genetic Goals Growth Factor In Vitro Knowledge Lentivirus Vector Mediating Methods Midbrain structure Modeling Modification Motor Mus Neurodegenerative Disorders Neuroglia Neurons Non - mammalian blastula Oxidopamine Parkinson Disease Patients Pharmaceutical Preparations Phenotype Plasmids Play Population Production Property Proteins Protocols documentation Puromycin Rattus Replacement Therapy Reporting Research Personnel Resistance Response Elements Role Safety Source Stem cells Subfamily lentivirinae Substantia nigra structure Symptoms Synapsins System Teratoma Testing Time Tissues Transcription Coactivator Transfection Translating Transplantation Work base blastocyst cell type dopaminergic neuron drug testing embryonic stem cell fetal genetic manipulation human embryonic stem cell human fetus tissue in vivo new technology novel patient safety pressure programs promoter protein expression transcription factor

项目摘要

Parkinson's disease (PD) is a severe neurodegenerative disorder associated with the selective loss of midbrain dopaminergic (DA) neurons. There is proof of principal that DA neuronal replacement therapy can work in patients using fetal-derived tissue. However, this is afflicted with several technical and ethical problems. Muman embryonic stem cells (hESCs) represent a promising new cell source to generate functional DA neurons for cell replacement in PD. Current in vitro differentiation protocols have demonstrated that DA neurons can be produced from hESCs, however, the generation of a homogeneous DA neuronal cell population that is safe, stable and functional after transplantation into the brain has not yet been achieved. This proposal will focus on a novel idea for DA phenotype specification and selection. The transcriptional activator Nurrl plays a major role in DA cell specification during neuronal cell development. To enrich for the appropriate DA cellular phenotype, we will select Nurrl expressing cells by puromycin drug resistance using Lentiviral vectors engineered with a Nurrl-responsive promoter driving the expression of the puromycin resistance gene. Since Nurrl could potentially be expressed in non-neuronal cell populations, we will further select the DA neurons using Lentiviruses expressing the green fluorescence protein (GFP) from the neuronal-specific promoter synapsin by fluorescence activated cell sorting (FACS). The drug-selected and FACS-purified homogeneous DA neuronal cell population will be tested in a rat model of PD to determine its safety, stability and functionality. The use of this new technology in cell replacement paradigms for PD could be a major step towards hESC-based therapies for chronic neurodegenerative disorders.

帕金森病 (PD) 是一种严重的神经退行性疾病，与选择性丧失神经元相关中脑多巴胺能 (DA) 神经元。有证据表明 DA 神经元替代疗法可以使用胎儿来源的组织对患者进行研究。然而，这受到一些技术和道德的困扰问题。 Muman 胚胎干细胞 (hESC) 是一种有前景的新细胞来源用于 PD 细胞替代的功能性 DA 神经元。目前的体外分化方案已证明 DA 神经元可以从 hESC 产生，然而，同质 DA 神经元细胞的产生目前尚未实现移植到大脑后安全、稳定且功能正常的人群。该提案将重点关注 DA 表型规范和选择的新想法。转录的激活剂 Nurrl 在神经元细胞发育过程中的 DA 细胞规范中发挥着重要作用。为丰富适当的DA细胞表型，我们将通过嘌呤霉素耐药性选择Nurrl表达细胞用驱动嘌呤霉素表达的 Nurrl 响应启动子设计的慢病毒载体抗性基因。由于 Nurrl 可能在非神经元细胞群中表达，我们将进一步使用表达绿色荧光蛋白 (GFP) 的慢病毒选择 DA 神经元通过荧光激活细胞分选 (FACS) 检测神经元特异性启动子突触蛋白。药物的选择和 FACS 纯化的均质 DA 神经元细胞群将在 PD 大鼠模型中进行测试，以确定其安全性、稳定性和功能性。在 PD 细胞替代范例中使用这项新技术可以这是基于 hESC 的慢性神经退行性疾病疗法的重要一步。