Molecular mechanisms of direct neuronal programming

直接神经元编程的分子机制

• 批准号: 9094285
• 负责人: Esteban Orlando Mazzoni
• 金额: $ 30.66万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9094285
• 关键词: Address; Axon; Binding; Bypass; Cell Therapy; Cells; Cephalic; Cervical; ChIP-seq; Chest; Chromatin; Clinic; Clinical; Communication; Complement; Consensus; Data; Development; Disease; Environment; Epigenetic Process; Fibroblast Growth Factor; Future; Gene Expression; Generic Drugs; Genes; Genetic; Genomics; Goals; Health; Hour; Implant; Knowledge; Lumbar spinal cord structure; Maps; Medical Research; Molecular; Motor Neurons; Mus; Muscle; Neuraxis; Neurons; Nucleic Acid Regulatory Sequences; Organ; Patients; Pattern; Population; Protocols documentation; Safety; Signal Transduction; Speed; Spinal; Spinal Cord; Spinal cord injury; Staging; Technology; Testing; Tissues; Translating; Transplantation; Uncertainty; base; cell type; clinical application; clinically relevant; design; diagnostic panel; embryonic stem cell; extracellular; histone modification; implantation; improved; in vivo; innovation; insight; knock-down; nerve supply; new technology; novel strategies; progenitor; programs; stem cell differentiation; stem cell technology; stem cell therapy; success; transcription factor; transcriptome sequencing

DESCRIPTION (provided by applicant): Embryonic stem cells (ESC) will revolutionize medical research and patient treatment. However, two major hurdles do not allow the rapid transition of ESC therapies to clinical settings: 1) the lack of protocols precluding homogenous cell populations that exactly reproduce those in vivo; 2) the uncertainty about the fate stability of ESC-derived cells after in vivo grafting. Overcoming these limitations will ameliorate safety concerns associated with ESC-derived cell therapies. Our long term goal is to efficiently generate ESC-derived cells that functionally integrate into organs. We have recently developed efficient protocols to derive terminal cell fates from ESC. When expressed in differentiating ESC, Ngn2- Isl1-Lhx3 (the NIL transcription factors) and Ngn2- Isl1-Phox2a (the NIP transcription factors) are sufficient to program spinal or cranial motor neuron identity respectively. This also happens extremely rapidly: Within 48 hours more than 97% of the cells acquire all the features of terminally differentiated motor neurons. Moreover, programmed neurons correctly integrate into the developing spinal cord projecting axons mirroring the endogenous neurons. Although, NIL and NIP factors do not provide the motor neuron subtype identity required for precise muscle innervation, our preliminary data suggests that it can be acquired by the activity of developmentally relevant signals. We hypothesize that NIL and NIP factors program "generic" motor neuron fate through a rapid transcriptional sequence, and that subtype identity can be independently imposed either by genetic factors or by the host environment after grafting. Here we propose 3 aims to test these ideas: Aim 1- To understand the molecular mechanisms of direct cell programming, we will map the NIL and NIP genetic and epigenetic requirements for efficient programming. This knowledge will facilitate the future design of programming strategies for different clinically relevant cell types. Aim 2- To increase the cellular precision of direct programming, we will impose subtype identity to NIL- programmed neurons by the activity of developmentally relevant signals or transcription factors. This novel strategy will generate neurons at a level of efficiency and precision compatible with clinical applications. Aim 3- To dissect the influence of the host tissue on ESC-derived neurons, we will test the cellular stability of NIL-programmed neurons after implantation into the spinal cord. These results will investigate if ESC-derived neurons change fate after interacting with the host tissue. Completing this proposal will impact not only future therapies for spinal cord injurie, but will also produce general principles to differentiate disease relevant cells at high efficiency These are necessary steps to accelerate the transition of ESC to clinical applications.

描述（由申请人提供）：胚胎干细胞（ESC）将彻底改变医学研究和患者治疗。但是，两个主要障碍不允许ESC疗法快速过渡到临床环境：1）缺乏固定细胞群体的方案，这些方案完全可以在体内重现这些细胞； 2）体内移植后ESC衍生细胞的命运稳定性的不确定性。克服这些局限性将减轻与ESC衍生的细胞疗法有关的安全问题。我们的长期目标是有效地生成在功能上集成到器官的ESC衍生细胞。我们最近开发了有效的方案，以从ESC得出末端细胞命运。当在分化ESC中表达时，NGN2-ISL1-LHX3（NIL转录因子）和NGN2- ISL1-PHOX2A（NIP转录因子）足以分别编程脊柱或颅运动神经元身份。这也发生得非常迅速：在48小时内，超过97％的细胞获得了末端分化的运动神经元的所有特征。此外，编程的神经元正确整合到反映内源神经元的发育中的脊髓投射轴突中。尽管NIL和NIP因子无法提供精确肌肉神经支配所需的运动神经元亚型身份，但我们的初步数据表明可以通过发育相关信号的活性来获取它。我们假设NIL和NIP因子通过快速的转录序列“通用”运动神经元的命运“通用”运动神经元命运，并且可以通过遗传因素或托管后的宿主环境独立强加的亚型身份。在这里，我们提出3个旨在测试这些想法的目的：目的1-了解直接细胞编程的分子机制，我们将绘制零和nip遗传和表观遗传要求，以进行有效的编程。这些知识将促进针对不同临床相关细胞类型的编程策略的未来设计。目标2-为了提高直接编程的细胞精度，我们将通过发育相关的信号或转录因子的活性对NIL编程的神经元施加亚型身份。这种新颖的策略将以效率水平和与临床应用兼容的精度产生神经元。目标3-为了剖析宿主组织对ESC衍生神经元的影响，我们将在植入脊髓后测试NIL程序化神经元的细胞稳定性。这些结果将研究ESC衍生的神经元与宿主组织相互作用后是否改变了命运。完成该建议不仅会影响脊髓损伤的将来的疗法，而且还将产生一般原则，以高效地区分疾病相关细胞，这是加速ESC向临床应用过渡的必要步骤。