Molecular mechanisms of direct neuronal programming

胚胎干细胞（ESC）将彻底改变医学研究和患者治疗。然而，两个主要障碍不允许ESC疗法快速过渡到临床环境：1）缺乏排除在体内精确复制那些的同质细胞群体的方案; 2）关于体内移植后ESC衍生细胞的命运稳定性的不确定性。克服这些限制将改善与ESC衍生细胞疗法相关的安全性问题。我们的长期目标是有效地产生功能性整合到器官中的ESC衍生细胞。我们最近开发了有效的协议，从ESC获得终末细胞的命运。当在分化的ESC中表达时，Ngn 2-Isl 1-Lhx 3（NIL转录因子）和Ngn 2-Isl 1-Phox 2a（NIP转录因子）足以分别编程脊髓或颅运动神经元身份。这也发生得非常快：在48小时内，超过97%的细胞获得了终末分化运动神经元的所有特征。此外，编程神经元正确地整合到发育中的脊髓中，投射轴突反映内源性神经元。虽然，NIL和NIP因子不提供精确的肌肉神经支配所需的运动神经元亚型身份，我们的初步数据表明，它可以通过发育相关信号的活动获得。我们假设，NIL和NIP因子程序“通用”运动神经元的命运，通过快速转录序列，亚型身份可以独立施加的遗传因素或嫁接后的宿主环境。在这里，我们提出了3个目的来测试这些想法：目的1-为了了解直接细胞编程的分子机制，我们将映射NIL和NIP的遗传和表观遗传要求，以实现有效的编程。这些知识将有助于未来设计不同临床相关细胞类型的编程策略。目的2-为了提高直接编程的细胞精度，我们将通过发育相关信号或转录因子的活性对NIL编程的神经元施加亚型身份。这种新策略将以与临床应用兼容的效率和精度水平生成神经元。目的3-为了分析宿主组织对ESC衍生神经元的影响，我们将测试植入脊髓后的NIL编程神经元的细胞稳定性。这些结果将研究ESC衍生的神经元在与宿主组织相互作用后是否改变命运。完成这一提案不仅将影响未来脊髓损伤的治疗，而且还将产生高效分化疾病相关细胞的一般原则，这是加速ESC向临床应用过渡的必要步骤。

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.