ES CELL MYELINATION IN INJURED SPINAL CORD

描述：（改编自研究者摘要）： 完整的轴突是导致大多数CNS功能丧失的重要因素 紊乱这些包括脊髓损伤（SCI）和多发性硬化症，其中 天然细胞介导的髓鞘再生的能力是有限的。胚胎干 (ES)细胞为研究髓鞘形成的机制提供了有力的工具。ES 细胞比其他细胞有优势，因为它们可以分裂， 无限期地，提供无限的干细胞来源用于培养， 移植此外，它们在遗传上是正常的、多能的， 只有干细胞可以进行双等位基因基因遗传操作。我们建议 利用ES细胞系统的潜力来评估 髓鞘形成我们计划探索胚胎干细胞的可能性， 维甲酸下调神经胶质细胞分化途径，可产生 在体外和体内能够轴突髓鞘形成的少突胶质细胞（oligos）。 此外，我们建议确定ES细胞是否可以分化为寡核苷酸， 并在移植到脱髓鞘的脊髓中后形成髓鞘。我们 假设增强ES细胞衍生的寡核苷酸（ESoligo）分化和 髓鞘形成将优化SCI后的行为恢复。我们计划建立在 我们之前的工作证明了ES细胞分化为神经细胞， 以及后肢运动功能的适度改善， 移植在中度脊髓挫伤后延迟9天。 在目标1中，我们建议优化ESoligo差异化并开发 丰富的ESoligos来源，用于培养和移植， 实验目标I的一个主要组成部分是开发PLP-lacZ 转基因ES细胞系，其将使得能够鉴定ESoligo衍生的髓鞘 并在体外和体内快速定量。在目标2中，我们将测试几个 优化ESoligo存活和髓鞘形成的策略（在目标1中筛选）， 脊髓脱髓鞘的2个啮齿动物模型。在目标3中，我们将应用 结果，这两个先前的目的，移植在最临床 相关的（但复杂的）SCI模型，重物坠落挫伤。我们预测 髓鞘形成的改善将促进运动能力的恢复。一旦恢复， 优化，ESoligo髓鞘形成的要求，以维持运动 恢复将受到考验。这将通过选择性地诱导 产生髓鞘的ESoligos使用敲入的 bar过表达基因，由髓鞘特异性PLP启动子驱动。的 本项目的长期目标是开发ES细胞系统作为研究 以及旨在了解髓鞘再生机制的治疗工具

DESCRIPTION: (Adapted from the Investigator's Abstract): Demyelination of intact axons is an important factor contributing to lost function in most CNS disorders. These include spinal cord injury (SCI) and multiple sclerosis, where the capacity for native cell-mediated remyelination is limited. Embryonic stem (ES) cells provide a powerful tool to investigate mechanisms of myelination. ES cells provide an advantage over other cells because they can divide indefinitely, affording an unlimited source of stem cells for culture or transplantation. Furthermore, they are genetically normal, pluripotent, and the only stem cell amenable to double allele genetic manipulation. We propose to harness the potential of the ES cell system to evaluate mechanisms of myelination. We plan to explore the possibility that ES cells, induced by retinoic acid down a neuroglial differentiation pathway, can generate oligodendrocytes (oligos) capable of axonal myelination in vitro and in vivo. Additionally, we propose to determine if ES cells can differentiate into oligos and myelinate after transplantation into the demyelinated spinal cord. We hypothesize that enhancing ES cell-derived oligo (ESoligo) differentiation and myelination will optimize behavioral recovery after SCI. We plan to build upon our previous work that demonstrated ES cell differentiation into neural cells and a modest improvement in hindlimb locomotor function even when transplantation was delayed 9 days after moderate spinal cord contusion injury. In Aim 1, we propose to optimize ESoligo differentiation and to develop enriched sources of ESoligos for culture and transplantation in subsequent experiments. One major component of Aim I is development of a PLP-lacZ transgene ES cell line that will enable ESoligo derived myelin to be identified and quantified rapidly both in vitro and in vivo. In Aim 2 we will test several strategies (screened in Aim 1) for optimizing ESoligo survival and myelination, in 2 rodent models of spinal cord demyelination. In Aim 3, we will apply the results of both previous Aims to transplantation in the most clinically relevant (but complex) model of SCI, weight-drop contusion injury. We predict that improved myelination will enhance recovery of locomotion. Once recovery is optimized, the requirement of ESoligo myelination for maintenance of locomotor recovery will be tested. This will be done by selectively inducing myelin-producing ESoligos to undergo apoptotic death using a knocked-in bar-overexpression gene, driven by a myelin-specific PLP promotor. The long-term goal of this project is to develop the ES cell system as a research and therapeutic tool aimed at understanding the mechanisms of remyelination