Quantifying Cortical Neuron Production After Transplantation

描述(申请人提供)：大脑皮层基本上通过处理感觉输入来控制哺乳动物的行为，然后通过投射到皮质下结构来协调传出活动。在解剖学上，它由一个复杂的六层层状结构组成。感觉信息处理发生在更浅的层；皮质投射神经元(CPN)出现在深层，并将轴突投射到皮质的其他部分或皮质下区域，因此代表着最重要的皮质输出的细胞调节器。因此，重建CPN复杂的、特定的连接将是任何皮质损伤修复策略的重要目标。最近，通过胚胎干细胞(ES细胞)的植入，用细胞疗法来替代神经元已经引起了人们的极大兴趣。虽然这些细胞很容易被诱导分化为神经元，但移植后不同分化策略和类型的神经元之间是否存在质的差异仍有待确定。我们自己的研究评估了两个体外条件神经元ES细胞池，发现在移植到新生儿皮质后，它们形成CPN的能力出现了意想不到的质的差异。因此，虽然一个细胞池几乎没有产生CPN，但另一个细胞池产生了一个强大的皮质下投射，这在解剖学上是非常合适的。此外，通过对这两个细胞池的连续比较，我们能够确定这种行为是由相对较小的转录因子子集驱动的，这些转录因子在发育过程中发挥作用，产生CPN群体。因此，这些发现提出了一种可能性，即在体外通过诱导(或抑制)有限数量的基因，可以在移植前特异性地调节细胞修复治疗的神经元表型。在这个R21提案中，我们建议通过丰富细胞亚群中与CPN生产相关的标记来优化这个特定神经细胞亚集的生产。此外，我们将通过在非CPN产生的细胞池中诱导它们的活性或在CPN产生的池中沉默它们来评估识别的基因在这一过程中的必要性和充分性。有了这些结果，我们将更好地评估该策略是否具有改善功能的能力，从而使其更接近最终在临床上的应用，主要是作为一种治疗皮质出生损伤的方法。脑损伤是一个重大的公共健康问题，不仅导致生活质量下降，而且代价极其高昂。因此，加强从这些毁灭性问题中恢复具有重大意义。胚胎干细胞可能是脑移植中最有潜力的细胞类型。因此，能够控制这些细胞的神经元分化，使它们能够产生特定的细胞亚群，对于开发更好的治疗策略应该是非常有价值的。

DESCRIPTION (provided by applicant): The cerebral cortex controls mammalian behavior essentially through processing sensory input to then coordinate efferent activity via projection to subcortical structures. Anatomically, it is composed of a complex six-layer laminar structure. Sensory information processing occurs in the more superficial layers; cortical projection neurons (CPNs) arise in the deep layers and project axons either to other parts of the cortex or to subcortical areas, thus representing the most important cellular modulators of cortical output. Reestablishment of a CPN's complex, specific connectivity would therefore be an important goal of any repair strategy for cortical injuries. Recently, there has been much interest in using cellular therapy to replace neurons, usually through implantation of embryonic stem cells (ES cells). Although these cells can be readily induced to differentiate into neurons, it remains to be established whether there are qualitative differences between various differentiating strategies and types of neurons seen after transplantation. Our own studies assessing two pools of in vitro conditioned neuronal ES cells revealed an unexpected qualitative difference in their capacity to form CPN's after transplantation into neonatal cortex. Thus, while one cell pool produced virtually no CPN's, the other produced a robust subcortical projection that was remarkably anatomically appropriate. Furthermore, by serial comparisons of these two pools of cells, we were able to establish that this behavior was driven by a relatively small subset of transcription factors that function during development to produce the CPN population. These findings therefore raise the possibility that one can specifically modulate the neuronal phenotype of a cell repair treatment prior to transplantation through induction (or inhibition) of finite numbers of genes in vitro. In this R21 proposal, we propose to optimize the production of this specific neuronal cell subset through enriching the cell subpopulation for markers associated with CPN production. Additionally, we will assess the necessity and sufficiency of the identified genes in this process through either inducing their activity in the non- CPN producing pool of cells or silencing them in the CPN producing pool. With the results obtained, we will then be in a better position to assess whether this strategy has the capability of effecting functional improvement, thus moving it closer to eventual utility in the clinic, primarily as a treatment of cortical birth injuries. Brain injuries represent a significant public heath problem that is not only quality of life diminishing but also extremely costly. Improving recovery from these devastating problems is therefore of great relevance. Embryonic stem cells represent possibly the best potential cell type for use in brain transplantation. The ability to control the neuronal differentiation of these cells so that they can produce specific cell subsets should therefore be of great value in developing better therapeutic strategies for this problem.