Generating transplantable neurons by in vivo combinatorial screening of transcrip

问题：神经损伤、退行性疾病和疾病，如帕金森病和亨廷顿病、癫痫和中风，仅在美国就影响着数千万人，并且随着人口老龄化，这正成为一个更严重的问题。尽管人们投入了巨大的努力来确定所涉及的分子过程，但现有的化学和物理疗法并不能保证在短期内修复症状之外恢复失去的神经回路。一个潜在的解决方案可能是使用组织移植来恢复神经元功能。在一些人体试验中，尽管存在差异，但移植已经导致帕金森病（PD）和亨廷顿病（HD）的功能恢复。在几项啮齿动物研究中，细胞替代治疗癫痫和中风已经显示出前景，一些实验室正在评估视网膜细胞移植治疗退行性眼病。尽管仍有许多未知因素，但在各种情况下，已经观察到功能的改善是由于移植神经元与现有神经元网络的整合而发生的，而不仅仅是由于移植细胞释放的营养因子。几项研究还表明，当移植物中存在正确的细胞类型时，成人大脑具有显著的能力提供信号线索，指导神经元过程的生长，并诱导具有所需目标的突触的形成。对比啮齿动物大的动物进行的异种移植研究表明，这些线索可以在长距离上起作用。然而，尽管组织移植可能具有巨大的潜力，但仍存在许多科学未知数和本提案中概述的几个基本挑战。处理这些挑战的复杂性目前超出了世界上最大的实验室的能力，并且可能需要部署系统的高通量方法，不仅要解决基本的生物学问题并快速无偏见地测试各种假设，而且要提供有希望的可转化为临床试验的结果。挑战：(1)人类胎儿或ips来源的细胞要么太少，要么会导致肿瘤，无法用于临床；(2)移植细胞的异质性太大；(3)目前不可能在正确和同步的阶段制备移植细胞；(4)不同实验的移植物理部位差异很大；(5)现有的体内移植试验太慢，无法筛选多种不同的假设。创新与方法：在这里，我们提出了一种系统的、无偏见的、在体内的、大规模的、高通量的方法来克服这些挑战，在体外分化和移植神经组织的体内测试。这里提出的方法适用于大多数移植范例。我们将开发的关键技术和策略包括：(A)无基因修饰的RNA介导的核重编程；(B)利用大规模并行技术通过系统的RNA转录因子组合的超高通量筛选对人类细胞系进行重编程；(C)将人类细胞高通量移植到啮齿动物体内（以最小的啮齿动物牺牲），以及对神经元存活和整合的体内分析。

Problem: Neuronal injuries, degenerative diseases, and disorders such as Parkinson¿s and Huntington¿s diseases, epilepsy, and stroke affect tens of millions of individuals in the USA alone, and are becoming a more severe problem with the aging population. Although significant effort is being invested for identification of the molecular processes involved, existing chemical and physical therapies do not promise restoration of lost neuronal circuits beyond the short term remedy of symptoms. A potential solution could be the use of tissue transplantation to restore neuronal function. There have been human trials where transplantations, although variably, have resulted in functional recovery in Parkinson¿s (PD) and Huntington¿s (HD) diseases. Cell replacement for epilepsy and stroke has shown promise in several rodent studies, and the transplantation of retinal cells to treat degenerative eye diseases is under evaluation in several laboratories. Although there are still many unknowns, in various cases, it has been observed that functional improvements occurred owing to the integration of grafted neurons into existing neuronal networks, and was not simply due to trophic factors released by the transplanted cells. Several studies have also shown that the adult brain is remarkably capable of providing signaling cues that guide the growth of neuronal processes and induce formation of synapses with desired targets when correct cell types are present within the grafts. Xenograft studies with animals larger than rodents have shown that these cues can function over long distances. However, while tissue transplantation may have significant potential, there are many scientific unknowns and several fundamental challenges exist as outlined in this proposal. Handling complexity of these challenges is currently beyond the capabilities of the largest laboratories in the world, and will likely require deployment of systematic high-throughput approaches that will not only address fundamental biological questions and rapidly test various hypotheses without bias, but also provide results that are, if promising, translatable to clinical trials. Challenges: (1) Human fetal or iPS-derived cells are either too scarce or tumorogenic for clinical use, (2) Transplanted cells are too heterogeneous, (3) Preparation of transplanted cells in the correct and synchronized stages is currently impossible, (4) Physical site of transplantation significantly varies from experiment to experiment, (5) Existing in vivo transplantation assays are too slow for screening of multitudes of different hypotheses. Innovation & Methodology: Here, we propose a systematic, unbiased, in vivo, large-scale, and high throughput approach for overcoming these challenges to in vitro differentiation and in vivo testing of transplanted neuronal tissues. The proposed methodologies here are applicable to most transplantation paradigms. The key technologies and strategies we will develop include: (A) RNA-mediated nuclear reprogramming without genetic modification, (B) Reprogramming human cell lineages by systematic ultra-high-throughput screening of RNA transcription factor cocktails using a massively parallel technology, (C) High-throughput transplantation of human cells into rodents (with minimal rodent sacrifice) and in vivo analysis of neuronal survival and integration.