Efficient Targeting of Therapeutic Cells in Stroke and EAE

描述（由申请人提供）：近年来，干细胞用于治疗目的的前景已成为科学和医学中最有前途的领域之一。这一领域取得了重大进展，包括对干细胞生物学有了更好的了解，发现了干细胞的新来源，并在各种疾病中取得了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，很少或没有有效的治疗方法。使用干细胞的临床前研究令人鼓舞，并导致了一些帕金森病、多发性硬化症和中风的临床试验的启动。不幸的是，这些试验都没有显示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。动脉内给药可以潜在地绕过这一限制，并且已经进行了一些尝试，将这种方法用于直接靶向脑病变。限制这种方法的主要障碍是缺乏使细胞与内皮有效结合的技术，以及由于细胞过度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的过表达极大地提高了人类胶质祖细胞对炎症区域的靶向效率。使用微流体体外粘附试验，与非vla -4对照相比，细胞与激活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注射后在体内表现出明显增强的归巢。磁标记细胞的实时、定量序列全脑磁共振成像显示，VLA-4+细胞完全停靠在同侧颈动脉血管床内，表明首过黏附机制。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个低密度像素，而VLA-4- lps处理的对照组为868¿317个（p=0.014）。鉴于这些令人鼓舞的结果，本研究的总体目标是诱导多能干细胞衍生的胶质前体过度表达粘附分子VLA-4和LFA-1以及趋化因子受体CXCR-4和CCR2。结合颈动脉内输送，我们假设在炎症性脑病变内将发生高效和特异性的植入。细胞与内皮细胞结合并向脑实质外渗的能力将被初步削弱

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.