Transplantation of Endothelial Cells

描述（由申请人提供）：肝细胞移植对各种遗传和获得性疾病具有广泛的潜力。通过在这项资助下进行的开创性工作，我们首次确定肝窦内皮细胞（LSEC）可以成功移植。我们开发了合适的条件来证明移植细胞的植入，包括肝内细胞-细胞相互作用和细胞因子/趋化因子/受体在这一过程中的作用。详细分析表明，移植后的LSEC与肝脏内皮内层融合，并继续表达内皮功能。此外，我们还建立了调节移植LSEC在肝脏中增殖的机制。移植的LSEC合成和释放FVIII的能力使我们能够在小鼠模型中实现血友病A的永久矫正。随后，我们在移植的LSEC中建立了治疗性基因转移的机制，LSEC已被慢病毒载体进行了遗传修饰。在其他研究中，我们确定了LSEC是否可以起源于肝外来源，例如骨髓。这些研究排除了内皮细胞可能来源于供体骨髓来源的细胞，但我们发现了其他细胞类型，即单核细胞，包括Kupffer细胞，以及骨髓来源的间充质间质细胞，它们具有较低水平表达FVIII的能力。最后，我们确定了乙型肝炎和丙型肝炎复制的抗病毒机制，以考虑是否可以同时替换抗病肝细胞和健康内皮细胞。因此，LSEC生物学的见解将为肝脏相关疾病的细胞/基因治疗提供合适的框架。为了推进细胞/基因治疗，我们现在建议对人类LSEC祖细胞进行研究。我们的假设是，可以从健康的供体组织中分离出合适的内皮祖细胞群，在培养条件下进行扩增和操作，然后移植以取代肝窦内皮，从而推进人类细胞治疗。由于适当的策略对于诱导移植细胞的植入和增殖至关重要，我们将在具有良好特征和卓越的细胞移植模型中开展研究。首先，我们将定义候选人内皮祖细胞的特性。这将随后检查机制，以改善移植内皮细胞的植入和增殖。这些研究将允许检查人类细胞在血友病A小鼠中的细胞治疗潜力。通过这种方式，利用人内皮细胞进行细胞治疗的创新方法将克服血友病和其他疾病的现有障碍。关于人类内皮细胞的特性和用途的基本知识将推进肝脏生物学和肝细胞治疗的见解，这将对改善人类健康具有巨大的价值。

DESCRIPTION (provided by applicant): Liver cell transplantation has extensive potential for a variety of genetic and acquired conditions. Through pioneering work performed under this grant, we established for the first time that liver sinusoidal endothelial cells (LSEC) can be successfull transplanted. We developed suitable conditions to demonstrate engraftment of transplanted cells, including roles in this process of intrahepatic cell-cell interactions and cytokines/chemokines/receptors. Detailed analysis established that transplanted LSEC integrated in the endothelial lining of the liver and continued to express endothelial functions. Also, we established mechanisms regulating proliferation of transplanted LSEC in the liver. The ability of transplanted LSEC to synthesize and release FVIII permitted us to achieve permanent correction of hemophilia A in a mouse model. Subsequently, we established mechanisms in therapeutic gene transfer in transplanted LSEC that had been genetically-modified by lentiviral vectors. In other studies, we determined whether LSEC could originate from extrahepatic sources, e.g., bone marrow. These studies excluded that endothelial cells could originate from donor bone marrow-derived cells but we identified additional cell types, i.e., mononuclear cells, including Kupffer cells, as well as mesenchymal stromal cells derived from bone marrow with ability to express FVIII in lower levels. Finally, we identified antiviral mechanisms for hepatitisB and C replication, to consider whether disease-resistant hepatocytes and healthy endothelial cells could be simultaneously replaced. Therefore, insights in the biology of LSEC will offer suitable frameworks for cell/gene therapy in liver-related conditions. To advance cell/gene therapy, we now propose studies with human LSEC progenitor cells. Our hypothesis is that suitable endothelial progenitor cell populations can be isolated, expanded and manipulated in culture conditions from healthy donor tissues, followed by transplantation to replace liver sinusoidal endothelium, such that cell therapy in people will be advanced. As appropriate strategies are critical for inducing engraftment and proliferation in transplanted cells, we will develop studies in well-characterized and superb cell transplantation models. First, we will define properties of candidate human endothelial progenitor cells. This will be followed by examination of mechanisms to improve engraftment and proliferation in transplanted endothelial cells. These studies will permit examination of the cell therapy potential of human cells in hemophilia A mice. In this way, innovative approaches for cell therapy with human endothelial cells will overcome existing barriers in curing hemophilia and other disorders. Fundamental knowledge in respect to the properties and uses of human endothelial cells will advance insights in liver biology and liver cell therapy, which will be of enormous value for improving human health.