Combination therapy in human ES cell transplantation after neonatal stroke

描述（由申请人提供）：缺血性新生儿中风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。胚胎干细胞移植在缺血性脑卒中后作为一种可能的修复疗法已经在成年动物中进行了广泛的测试，然而，很少有研究评估胚胎干细胞移植在新生儿缺血性脑卒中中的作用。在拟议的研究中，使用人类胚胎干细胞（hESCs）的移植治疗将在针对须桶皮层的大鼠新生儿缺血模型中进行测试。我们将解决hESC移植的三个关键问题：1)促进hESC来源的神经细胞在移植后进入恶劣的缺血环境后的存活；2)促进脑卒中和hESC移植后的内源性再生反应（血管生成和神经发生），并了解这些反应中的调控信号；3)引导和促进hESC移植后须-桶通路的突触发生、神经血管重构和功能恢复。根据我们之前对小鼠胚胎干细胞的研究和hESCs的初步研究，神经分化hESCs在移植前需要进行缺氧预处理（hypoxia preconditioning， HP），以增加其对缺血脑内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要调控基因的表达。突触传递是一种基本的神经元活动。我们将在体外（目的1）和体内（目的2）验证HP可以促进hesc来源神经元的神经突生长、突触发生和功能活动的假设。作为一种使用依赖的康复策略，须刺激诱导的传入信号增强将被测试促进和引导移植hesc来源细胞的能力，以及重建更有组织和更功能的须桶皮层和丘脑皮质连接的内源性机制（Aim 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全面了解HP和富集环境促进的人类干细胞治疗的机制和功能益处。这些实验结果有望提高缺血新生儿hESC移植的疗效和效率。人类胚胎干细胞（hESCs）的移植治疗将在针对须桶皮层的大鼠新生儿缺血模型中进行测试。我们将解决hESC移植的三个关键问题：1)促进hESC来源的神经细胞移植后的存活；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.