Stem Cells and Ischemic Brain Injury

对骨髓细胞的研究表明，将骨髓细胞移植到缺血性脑损伤的动物体内可以恢复神经功能。最近，我们的实验室已经从人骨髓中分离出一种特殊的多能干细胞群，它能够在适当的培养条件下在体外分化为神经元、星形胶质细胞和少突胶质细胞。我们假设这些骨髓来源的干细胞可以为缺血性脑损伤和中风的移植和修复提供自体供体细胞来源。在特异性目的1中，我们将确定骨髓来源的人干细胞移植到自发性高血压大鼠的大脑后产生的神经表型。人类干细胞将被转导表达eGFP来标记供体细胞。骨髓来源的干细胞将被移植到纹状体、海马体或感觉运动皮层。在移植后的不同时期，将研究来自宿主大鼠的脑组织，以确定eGFP标记细胞的存在。eGFP标记的细胞也将被检查，以确定它们是否发育成神经元、胶质细胞和/或少突胶质细胞。位点特异性分化也将被评估，以确定移植神经元是否也共同表达纹状体、海马体和皮层中神经元特有的神经递质标记物。在特异性目标2中，我们将确定移植骨髓干细胞在改善缺血性脑损伤相关神经功能缺陷方面的功效。在分支纹状体血管远端结扎将单侧闭塞大脑中动脉。这种闭塞造成皮层的离散缺血性损伤，并导致损伤侧对侧前肢永久性感觉和运动缺陷。缺血事件发生一周后，将骨髓来源的干细胞移植到损伤区域周围的新皮层区域。在初步研究中，我们发现这些移植能够改善感觉运动缺陷。随着时间的推移，功能恢复的程度将通过一系列行为测试来评估。此外，作为特异性目标2的一部分，我们将确定缺血性脑损伤后干细胞移植的时间窗口，这将导致神经功能缺陷的改善。在这些研究中，缺血大鼠将在缺血损伤后的不同时期接受移植，并使用一组定义的感觉和运动测试来评估随时间推移的功能。在具体目标3中，我们将研究骨髓移植诱导神经功能恢复的可能机制。可能的机制包括对宿主脑神经干细胞的营养作用，诱导宿主脑细胞释放生长因子，以及重组宿主纤维连接。这些研究的结果将提供关于使用特定的骨髓来源干细胞作为缺血性脑损伤移植的自体细胞来源的有效性的信息，以及关于移植物诱导神经可塑性的可能机制的信息。

Studies of bone marrow cells have shown that they can restore neurologic function when transplanted into animals with ischemic brain injury. Recently, our laboratories have isolated a specific multi-potent stem cell population from human bone marrow that is capable of differentiating into neurons, astrocytes, and oligodendroglia in vitro under appropriate culture conditions. We postulate that these bone marrow-derived stem cells can provide an autologous source of donor cells for transplantation and repair in conditions of ischemic brain injury and stroke. In Specific Aim 1 we will determine the neural phenotypes generated from bone marrow-derived human stem cells when transplanted into the brain of spontaneously hypertensive rats. Human stem cells will be transduced to express eGFP to label the donor cells. Bone marrow-derived stem cells will then be transplanted into the striatum, hippocampal formation, or sensorimotor cortex. At various time periods after transplantation, brain tissue from host rats will be studied to identify presence of eGFP labeled cells. eGFP labeled cells will also be examined to determine whether they develop into neurons, glial cells, and/or oligodendroglia. Site-specific differentiation will also be evaluated to determine whether grafted neurons also co-express neurotransmitter markers that are characteristic of neurons that found in the striatum, hippocampus, and cortex. In Specific Aim 2 we will determine the efficacy of transplanted bone-marrow stem cells in ameliorating neurologic deficits associated with ischemic brain injury. The middle cerebral artery will be occluded unilaterally by ligation distal to the branching striatal vessels. This occlusion produces a discrete ischemic lesion of the cortex and results in permanent sensory and motor deficits in the forelimb contralateral to the side of injury. One week after the ischemic event animals will receive transplants of bone marrow-derived stem cells into regions of the neocortex surrounding the area of injury. In preliminary studies we have found that these transplants are capable of ameliorating sensorimotor deficits. The extent of the functional recovery will be assessed over time using a battery of behavioral tests. Also as part of Specific Aim 2 we will determine the time window after ischemic brain injury for stem cell transplantation that will result in an amelioration of neurologic deficits. In these studies ischemic rats will receive transplants at various time periods after ischemic injury and assessed functionally over time using a defined set of sensory and motor tests. In Specific Aim 3 we will investigate possible mechanisms underlying bone marrow-graft induced recovery of neurologic function. Possible mechanisms include trophic effects on neural stem cells of the host brain, the induction of growth factor release by host brain cells, and the reorganization of host fiber connections. The results of these studies will provide information regarding the efficacy of using specific bone marrow-derived stem cells as an autologous source of cells for transplantation in ischemic brain injury, and information regarding possible mechanisms of graft-induced neural plasticity.