Combination therapy in human ES cell transplantation after neonatal stroke

描述（由申请人提供）：缺血性新生儿卒中是一种常见的中枢神经系统疾病，但目前尚无有效的治疗方法。胚胎干细胞移植治疗缺血性脑卒中已被广泛应用于成年动物的脑损伤修复，但目前对胚胎干细胞移植治疗新生儿缺血性脑卒中的研究较少。在拟议的研究中，将在大鼠新生儿缺血模型中测试使用人ES细胞（hESC）的移植治疗，靶向须桶皮质。我们将解决hESC移植中的三个关键问题：1）促进hESC衍生的神经细胞在移植到恶劣的缺血环境中后的存活; 2）促进内源性再生反应中风和hESC移植后（血管生成和神经发生），并了解这些反应中的调节信号; 3）指导和改善hESC移植后须-桶通路中的突触发生、神经血管重塑和功能恢复。基于我们以前对小鼠ES细胞的研究和对hESCs的初步研究，神经分化的hESCs在移植前将经受低氧预处理（HP），以增加其对缺血脑中破坏性环境的耐受性。移植后将评估细胞存活和再生反应的必需调控基因的表达。突触传递代表了神经元的基本活动。我们将在体外（Aim 1）和体内（Aim 2）检验HP可以促进hESC衍生神经元的轴突生长、突触发生和功能活动的假设。作为一种使用依赖性康复策略，将测试由胡须刺激诱导的强化传入信号促进和引导移植的hESC衍生细胞和内源性机制重建胡须-桶皮质和丘脑皮质连接的更好组织和更功能的神经血管结构的能力（目的3）。细胞和分子生物学，免疫组织化学和电生理技术将被应用于获得由HP和丰富的环境促进的人类干细胞治疗的机制和功能益处的全面理解。这些实验的结果可能会提高hESC移植在缺血新生儿中的疗效和效率。使用人ES细胞（hESC）的移植治疗将在大鼠新生儿缺血模型中进行测试，靶向须桶皮质。我们将解决hESC移植中的三个关键问题：1）促进移植后hESC源性神经细胞的存活; 2）促进内源性再生反应; 3）指导和改善hESC移植后须桶通路中的修复过程和功能恢复。

DESCRIPTION (provided by applicant): Ischemic neonatal stroke is a common CNS disorder, yet no effective treatment is currently available. Embryonic stem (ES) cell transplantation after ischemic stroke has been tested extensively as a possible repair therapy in adult animals, however, little investigation has been done to evaluate ES cell transplantation in neonatal ischemic strokes. In the proposed studies, transplantation therapy using human ES cells (hESCs) will be tested in a rat neonatal ischemia model targeting the whisker-barrel cortex. We will address three key issues in hESC transplantation: 1) to promote survival of hESC- derived neural cells after transplantation into the harsh ischemic environment; 2) to promote endogenous regenerative responses (angiogenesis and neurogenesis) after stroke and hESC transplantation, and to understand regulatory signals in these responses; 3) to guide and improve synaptogenesis, neurovascular remodeling and functional recovery in the whisker-barrel pathway after hESC transplantation. Based on our previous investigations on mouse ES cells and preliminary studies on hESCs, neurally differentiated hESCs will be subjected to hypoxic preconditioning (HP) before transplantation in order to increase their tolerance to the destructive environment in the ischemic brain. Expression of the essential regulatory genes for cell survival and regenerative responses will be assessed after transplantation. Synaptic transmission represents a fundamental neuronal activity. We will test the hypothesis that HP can promote neurite outgrowth, synaptogenesis and functional activities of hESC-derived neurons in vitro (Aim 1) and in vivo (Aim 2). As a use-dependent rehabilitation strategy, intensified afferent signals induced by whisker stimulation will be tested for the ability to promote and guide transplanted hESC-derived cells and endogenous mechanisms in rebuilding better organized and more functional neurovascular architecture of the whisker-barrel cortex and thalamocortical connections (Aim 3). Cellular and molecular biological, immunohistochemical and electrophysiological techniques will be applied to acquire a comprehensive understanding of the mechanisms and functional benefits of human stem cell therapy boosted by HP and an enriched environment. Results from these experiments will likely improve the efficacy and efficiency of hESC transplantation in ischemic neonates.Transplantation therapy using human ES cells (hESCs) will be tested in a rat neonatal ischemia model targeting the whisker-barrel cortex. We will address three key issues in hESC transplantation: 1) to promote survival of hESC-derived neural cells after transplantation; 2) to promote endogenous regenerative responses; 3) to guide and improve the repair process and functional recovery in the whisker-barrel pathway after hESC transplantation.