Efficient Targeting of Therapeutic Cells in Stroke and EAE

描述（申请人提供）：干细胞用于治疗目的的前景是近年来科学和医学最有前途的领域之一。在这一领域取得了重大进展，包括更好地了解干细胞生物学，确定干细胞的新来源，并在各种疾病中取得令人鼓舞的治疗结果。神经系统疾病仍然是医学上最大的挑战之一，几乎没有有效的治疗方法。使用干细胞的临床前研究一直令人鼓舞，并导致了帕金森病，多发性硬化症和中风的一些临床试验的开始。不幸的是，这些试验都没有表现出令人满意的治疗结果。失败的原因有很多，其中一个主要原因是干细胞的生物分布和靶向效率低下。动脉内递送可能会绕过这一限制，并且已经进行了一些尝试来使用这种方法直接靶向脑病变。限制这种方法的主要障碍是缺乏能够使细胞与内皮有效结合的技术，以及由于过度细胞结合而导致微栓塞的风险。我们的初步结果表明，对接受体VLA-4的过表达大大提高了人类神经胶质祖细胞对炎症区域的靶向效率。使用微流体体外粘附测定，与非VLA-4对照相比，细胞与活化的脑内皮细胞的结合大大增加（分别为71.5 <$11.7 vs. 36.4 <$3.3个细胞/FOV，p=0.045）。在LPS诱导的大鼠全脑炎症模型中，含有VLA-4转基因的细胞在动脉内注射后表现出更强的体内归巢。磁标记细胞的实时、定量系列全脑MR成像显示，VLA-4+细胞仅停靠在同侧颈动脉的血管床内，表明首过粘附机制。逐像素分析显示，在LPS处理的动物中注射VLA-4+细胞导致3，979 <$705个低信号像素，而在VLA-4-LPS处理的对照中为868 <$317个（p=0.014）。有了这些令人鼓舞的结果，本提案的总体目标是诱导多能干细胞衍生的神经胶质前体过表达粘附分子VLA-4和LFA-1以及趋化因子受体CXCR-4和CCR 2。结合颈动脉内给药，我们假设在炎性脑损伤内将发生高效和特异性植入。细胞与内皮细胞结合并外渗到脑实质中的能力最初将被破坏。 使用微流体模型血脑屏障进行体外测试。然后在中风和自身免疫性脑脊髓炎的大鼠模型中进行体内实验。为了确保这种方法的安全性，我们将使用MRI实时监测细胞输送，脑血流和氧合。在我们的研究成功完成后，这种新的靶向方法可以显着提高基于细胞的治疗的疗效，并在许多医学领域中应用。

DESCRIPTION (provided by applicant): The prospect of using stem cells for therapeutic purposes has been one of the most promising fields of science and medicine in recent years. Progress in this area has been substantial, including a better understanding of stem cell biology, the identification of new sources of stem cells, and encouraging therapeutic results in a variety of diseases. Neurological disorders remain one of the greatest challenges in medicine, with little or no effective treatments available. Preclinical studies using stem cells have been encouraging and have led to the initiation of a few clinical trials for Parkinson's disease, multiple sclerosis and stroke. Unfortunately, none of these trials demonstrated a satisfactory therapeutic outcome. There are many suggested reasons for that failure, with one of the primary reasons being an inefficient biodistribution and targeting of stem cells. Intraarterial delivery could potentially bypass this limitation, and a few attempts have been made to use this approach for direct targeting of brain lesions. The major obstacle limiting this approach is the lack of techniques tha enable efficient binding of cells to endothelium, as well as the risk of microembolism as a result of excessive cell binding. Our preliminary results indicate that overexpression of the docking receptor VLA-4 greatly improves the targeting efficiency of human, glial progenitors towards areas of inflammation. Using a microfluidics in vitro adhesion assay, cell binding to activated brain endothelial cells greatly increased as compared to non-VLA-4 controls (71.5¿11.7 vs. 36.4¿3.3 cells/FOV, respectively, p=0.045). In a LPS-induced rat global inflammatory brain model, cells containing the VLA-4 transgene demonstrated much enhanced homing in vivo following intraarterial injection. Real-time, quantitative serial whole brain MR imaging of magnetically labeled cells revealed that, VLA-4+ cells docked exclusively within the vascular bed of the ipsilateral carotid artery indicating a first pass adhesion mechanism. Pixel-by-pixel analysis revealed that injection of VLA-4+ cells in LPS-treated animals resulted in 3,979¿705 hypointense pixels as compared to 868¿317 in VLA-4- LPS-treated controls (p=0.014). With these encouraging results, the overall aim of this proposal is to induce pluripotent stem cells-derived glial precursors to overexpress the adhesion molecules VLA-4 and LFA-1 and the chemokine receptors CXCR-4 and CCR2. Combined with intracarotid delivery, we hypothesize that a highly efficient and specific engraftment within inflammatory brain lesions will occur. The ability of cells to bind to endothelium and extravasate into the brain parenchyma will be initially tested in vitro using a microfluidics model blood brain barrier. Experiments will then be performed in vivo in rat models of stroke and autoimmune encephalomyelitis. To ensure the safety of this approach, we will monitor cell delivery, cerebral blood flow, and oxygenation in real-time using MRI. Upon successful completion of our studies, this new targeting approach could significantly improve the efficacy of cell-based therapy with applications in many areas of medicine.