TRANSLATING HUMAN EMBRYONIC STEM CELL-DERIVED ENDOTHELIAL CELL THERAPY TO THE CLINIC

人胚胎干细胞（hES）和诱导多能干细胞（iPS）在再生医学中具有广泛的潜力。这些细胞可以在刺激下产生所有细胞类型，并沿着定义的细胞承诺途径分化。我们感兴趣的是控制这些细胞进入血管内皮细胞的刺激及其随后在再生医学中的应用的机制。这些细胞排列在所有血管上，注射入体内后具有诱导血管生长的能力。我们最近发表了一项研究，显示了来自人类胚胎干细胞的人类内皮细胞的潜力（Kane et al.， ATVB, 2010）。我们发现内皮细胞在最初的hES细胞分化开始后仅10天内就能非常有效地产生。这种快速的系统诱导了强大的变化，即非常有效地减少多能性标记和诱导内皮选择性标记。我们还通过在细胞培养和动物模型中展示诱导新血管生长导致受损肌肉更好的血液流动，定义了这些细胞的“治疗”潜力。这种表型的诱导表明，这些细胞和这种分化系统可能在临床上治疗心血管疾病患者的缺血性疾病中有用。在最近结束的之前的资助中，我们能够从hES细胞中生产出临床级的内皮细胞，从而证明了这些细胞可以在潜在临床应用所需的质量和数量上生产出来。这是一种强大的方法，但尚未在人体中进行过试验。这就是我们本次拨款申请的目的。在本提案中，我们将解决一个非常重要的问题，该问题现在需要进一步开发该产品以首次应用于人类。我们需要这笔拨款来完成细胞的开发，使其达到临床使用可接受的水平。我们将进一步完善生产方案，在细胞培养和详细分析中充分测试产生的细胞。然后，我们将在小鼠和兔子两种重要的动物模型上测试这些细胞的安全性以及它们在诱导血管生长方面的效果。最后，我们将通过观察细胞在组织中的滞留，向其他组织和器官的扩散以及它们在那里停留的时间来评估注射后细胞的去向。这些都是重要的问题，如果拨款成功，我们将获得监管部门的批准。

Human embryonic stem (hES) cells and induced pluripotent stem cells (iPS) hold broad potential in regenerative medicine. Such cells can generate all cell types upon stimulation to differentiate along defined cell commitment pathways. We are interested in the mechanisms that govern stimulation of these cells into vascular endothelial cells and their subsequent application to regenerative medicine. These cells line all blood vessels and have the capacity to induce blood vessel growth upon injection into the body. We have recently published a study that shows the potential of human endothelial cells derived from human embryonic stem cells (Kane et al., ATVB, 2010). We showed a very efficient production of endothelial cells in just 10 days following initiation of differentiation from the original hES cells. This rapid system induced robust changes, i.e. very effective reduction in pluripotency markers and induction of endothelial selective markers. We also defined the "therapeutic" potential of these cells in cell cultures and in animal models by demonstrating the induction of new blood vessel growth leading to better blood flow in the muscle that was damaged. The induction of this phenotype suggests that these cells and this differentiation system may be useful clinically in the treatment of ischemic conditions in patients with cardiovascular disease. In previous funding that ended recently, we were able to produce endothelial cells from hES cells at clinical grade thus showing proof of concept that these cells can be produced at the quality and quantity required for potential clinical use. This is a powerful approach that has not been tested in man yet. This is our aim for the present grant application.In this proposal we will address a very important issue that now is required for further development of this product towards a first application in man. We require this grant funding to complete development of the cells at a grade acceptable for clinical use. We will further refine the protocol for production, fully test the cells produced in cell culture and by detailed analysis. We will then test the safety the cells and how good they are at inducing blood vessel growth in two important animal models, in mouse and rabbit. Finally, we will assess where the cells go after injection looking at retention in the tissue, dissemination to other tissues and organs and how long they stay there. These are important issues that will allow us to gain regulatory approval if the grant is successful.