Derivation and dopaminergic differentiation of human drug addict-specific iPS cel

上瘾可以被定义为尽管有不良后果，但对物质使用失去控制。对…上瘾 合法和非法的物质破坏了成瘾者及其家人的生活，施加了一种 给社会带来巨大的成本和负担。基于分子和细胞的机制有助于 成瘾的发生和发展仍有待阐明。据估计，40%-60%的 上瘾的脆弱性可能归因于基因异常。多个染色体区域已经被 与成瘾有关的物质包括那些含有多巴胺转运体(DAT)和囊泡单胺的物质 转运蛋白(VMAT2)基因。目前正在努力了解这些单胺类化合物的多态 转运蛋白在成瘾的分子机制中的作用受到严重阻碍，因为无法 直接询问患者的神经细胞类型。携带特定基因的患者特定细胞来源 能够稳健且可复制地分化为特定神经谱系的基因变体则不能 是存在的。我们建议开发一种基于细胞的系统，通过该系统，患者的神经细胞可以 功能测试，以询问成瘾的分子机制。为了实现这一目标，我们 开发了一项融合了多个学科的技能和专业知识的尖端提案。在……里面 目的1我们将从成瘾中分离和鉴定患者特异性的诱导多能干细胞(IPS)。 携带单胺转运体基因多态的患者和对照。自中脑多巴胺能系统 在自然和与药物相关的奖励途径中起着突出的作用，并代表着 滥用药物，在目标2中，我们将把患者特有的iPS细胞系分化为多巴胺能神经元并携带 给出这些细胞的详细和功能特征以确定它们的分子特征(即A9， A10，中脑边缘或中皮质多巴胺能神经元)。在目标3中，我们将描述、比较和 对这些来自对照和成瘾的患者特异性iPS细胞来源的多巴胺能神经元进行功能分析 携带hDAT1和hVMAT2基因多态的患者。有很大的潜力可以针对特定的患者 IPS细胞技术通过提供 从基因上看，人类细胞的独特功能来源。通过完成本提案中提出的目标，我们 期望提供患者多巴胺能神经传递功能的详细特征 并提供对这种复杂疾病的病理生理机制的洞察以及对 单胺转运体基因的遗传变异与成瘾有关。我们已经建立了一个跨学科的 在吸毒者的人种学研究、神经分化和多巴胺能方面结合优势的团队 功能分析，以及多能性和iPS细胞，以询问关于细胞和 导致上瘾的分子功能障碍。我们预计我们的研究结果将会有 与理解和治疗这一人类疾病直接相关。

Addiction can be defined as a loss of control of substance use despite adverse consequences. Addiction to legal and illegal substances destroys the lives of both addicted subjects and their families, exerting an enormous cost and burden on society. The molecular- and cellular-based mechanisms that contribute to the initiation and development of addiction remain to be elucidated. Estimates have suggested that 40-60% of the vulnerability to addiction may be attributable to genetic aberrations. Multiple chromosomal regions have been linked to addiction including those containing the dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) genes. Current efforts to understand how polymorphisms in these monoamine transporters contribute to the molecular mechanisms of addiction are severely hindered by the inability to directly interrogate neural cell types from the patients. Patient-specific sources of cells carrying specific genetic variants that are capable of robust and reproducible differentiation into specific neural lineages do not exist. We propose to develop a cell-based system whereby neural cells from afflicted individuals can be functionally assayed to interrogate the molecular mechanisms underlying addiction. To achieve this goal we have developed a cutting-edge proposal that that incorporates the skill and expertise of multiple disciplines. In Aim 1 we will derive and characterize patient-specific, induced pluripotent stem (iPS) cells from addiction patients and controls that carry monoamine transporter polymorphisms. Since midbrain dopaminergic system play a prominent role in natural and drug related reward pathways and represent a common substrate for drugs of abuse, in Aim 2 we will differentiate patient-specific iPS cells line into dopaminergic neurons and carry out a detailed and functional characterization of these cells to identify their molecular characteristics (i.e. A9, A10, mesolimbic or mesocortical dopaminergic neurons). In Aim 3, we will characterize, compare, and functionally assay these patient-specific, iPS cell-derived dopaminergic neurons from control and addiction patients that carry polymorphisms for hDAT1 and hVMAT2 gene. There is great potential for patient-specific iPS cell technology to profoundly impact our understanding of human development and disease by providing genetically distinct, functional sources of human cells. By completing the aims set forth in this proposal we expect to provide a detailed characterization of dopaminergic neurotransmission function in patients afflicted with addiction and provide insight into the pathophysiology of this complex disease as well as the contribution of genetic variants in monoamine transporter genes to addiction. We have established an interdisciplinary team that combines strengths in ethnographic study of drug addicts, neural differentiation and dopaminergic function analysis, as well as pluripotency and iPS cells to interrogate novel questions about the cellular and molecular dysfunction that contributes to addiction. We expect that results from our studies will have immediate relevance to the understanding and treatment of this human disease.