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。通过在这笔赠款下进行的开创性工作，我们首次确立了肝窦内皮细胞(LSEC)可以成功移植。我们开发了合适的条件来展示移植细胞的植入，包括在这一过程中肝内细胞-细胞相互作用和细胞因子/趋化因子/受体的作用。详细的分析证实，移植的LSEC整合在肝脏的内皮衬里中，并继续表达内皮功能。此外，我们还建立了调节移植的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的永久纠正。随后，我们建立了通过慢病毒载体进行基因修饰的LSEC移植中的治疗性基因转移机制。在其他研究中，我们确定LSEC是否可能起源于肝外来源，例如骨髓。这些研究排除了内皮细胞可能来自供者骨髓来源的细胞，但我们发现了额外的细胞类型，即单个核细胞，包括Kupffer细胞，以及来自骨髓的间充质基质细胞，具有较低水平表达FVIII的能力。最后，我们确定了乙肝和丙型肝炎复制的抗病毒机制，以考虑抗病肝细胞和健康内皮细胞是否可以同时被取代。因此，对LSEC生物学的深入了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，合适的内皮祖细胞可以从健康的供体组织中分离、扩增并在培养条件下进行操作，然后进行移植以取代肝窦内皮细胞，从而促进人类的细胞治疗。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，我们将开展具有良好特性和卓越的细胞移植模型的研究。首先，我们将定义候选人类内皮祖细胞的特性。接下来将对促进移植内皮细胞植入和增殖的机制进行研究。这些研究将允许在血友病A小鼠身上检测人类细胞的细胞治疗潜力。通过这种方式，利用人类内皮细胞进行细胞治疗的创新方法将克服治疗血友病和其他疾病方面的现有障碍。有关人内皮细胞特性和用途的基础知识将促进对肝脏生物学和肝细胞治疗的深入了解，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.