Using zinc finger nuclease technology to generate reporter-labelled human pluripotent stem cells as a tool to optimize photoreceptor transplantation

多能干细胞是可以在实验室中生长并被编程为所需类型的特化细胞的非特化细胞，例如血细胞、肌肉细胞等。人类多能干细胞可以以不同的方式衍生，当它们在体外受精期间作为剩余产物时来自非常早期的胚胎，或者更一般地通过重新编程来自个体的容易获得的细胞，例如来自皮肤细胞样品。这种可能性已经引起了对使用干细胞用于治疗应用以治疗由特定类型的细胞损失引起的病症的极大兴趣。例如，由于视网膜细胞丢失而导致的失明原则上可以通过从患者身上提取皮肤细胞样本来治疗，重新编程皮肤细胞以产生非特异性多能干细胞，然后编程所得干细胞以产生视网膜细胞祖细胞，这些视网膜细胞祖细胞可以移植到患者的眼睛中以产生视网膜细胞。分化人类多能干细胞的困难问题之一是在细胞转变为特化细胞时对其进行跟踪，然后纯化所需的特化细胞。对于小鼠多能干细胞，这可以相对容易地通过插入基因来完成，这些基因使报告分子标记有荧光，使细胞在合适的条件下发光，但该方法在人类多能干细胞中效率非常低。一项新的尖端技术现在提供了一个潜在的解决方案。锌指核酸酶是人工制造的剪刀，可以被设计成在一个特定位置特异性地切割DNA分子的两条链。这些核酸酶在DNA结构中产生缺口，激活细胞对DNA修复的反应。当存在显示与含有切除的区域相似但也包含报道基因的短DNA片段时，可以将报道基因引入人多能干细胞中的目的基因中。这项技术是非常新的，只在人类多能干细胞中应用过两次;然而，效率比其他报道的方法高得多，因此潜在的应用是巨大的。在这项提案中，我们寻求实施这项技术来创建标记的人类多能干细胞系，这些干细胞系将用作优化细胞移植到退化视网膜中的工具。视网膜有一个非常复杂的结构，由几层相互连接的神经元组成。直接对光敏感的两种主要细胞类型是视杆细胞和视锥细胞。我们的研究小组已经证明，从人类多能干细胞中产生具有视锥细胞和视杆细胞特征的人类细胞是可能的。尽管取得了这一进展，但我们无法在分化过程中出现的其他细胞类型中选择这些细胞。通常，细胞选择使用称为荧光激活细胞分选（FACS）的技术来实现。身体的不同细胞在其表面上具有特定的蛋白质，标记有彩色或荧光分子的抗体可以结合，使我们能够使用FACS对其进行识别和分类，然而，很少有这样的标记物可用于分离视锥细胞和视杆细胞。我们打算将一个报告基因引入一个重要的视网膜基因，标志着他们的分化锥和杆。荧光报告基因的存在将允许我们使用上述细胞选择策略来纯化这些细胞。然后，我们可以使用各种体外干细胞测定和电生理学分析来询问这些细胞是否表现出与视杆细胞和视锥细胞相关的特性。如果成功，这种方法将使我们能够前瞻性地分离视杆细胞和视锥细胞，确定它们的分子表型，并在视网膜疾病的动物模型中测试它们恢复视力的能力。

Pluripotent stem cells are unspecialized cells that can be grown in the laboratory and programmed to become specialized cells of a desired type, such as blood cells, muscle cells etc. Human pluripotent stem cells can be derived in different ways, from very early embryos when they become available as surplus products during in vitro fertilization, or more generally by re-programming easily accessible cells from individuals, such as from a sample of skin cells. This possibility has led to great interest in using stem cells for therapeutic applications to treat disorders caused by loss of cells of a particular type. For example, blindness due to loss of retinal cells could in principle be treated by taking a sample of skin cells from the patient, re-programme the skin cells to make unspecialized pluripotent stem cells and then programme the resulting stem cells to give retinal cell progenitors that can be grafted into the patient's eye to give rise to retinal cells. One of the difficult problems in differentiating human pluripotent stem cells is to track the cells as they change into specialized cells and then to purify the desired specialized cells. This can be done relatively easily for mouse pluripotent stem cells by inserting genes that make reporter molecules tagged with a fluorescent that makes the cells glow under suitable conditions, but the method is very inefficient in human pluripotent stem cells. A new cutting edge technology now offers a potential solution. Zinc finger nucleases are artificially created scissors that can be designed to specifically cut both strands of DNA molecules at just one specific location. These nucleases create a gap in the DNA structure which activates the cell's response for DNA repair. Upon presence of a short DNA stretch which shows similarity to the region containing the excision but also harbouring the reporter gene, it is possible to introduce the reporter gene into the gene of interest in human pluripotent stem cells. This technology is very recent and has only been applied twice in human pluripotent stem cells; however the efficiency has been much higher than other reported methods and as such the potential applications are immense. In this proposal we seek to implement this technology to create labelled human pluripotent stem cells lines that will be used as tools to optimise cell transplantation into the degenerate retina. The retina has a very complex structure consisting of several layers of neurons that are interconnected with each other. The two main cell types that are directly sensitive to light are the rod and cone photoreceptors cells. Our group has shown that it is possible to produce human cells that have the characteristics of cones and rods from human pluripotent stem cells. Despite this progress, we are not able to select these cells amongst other cell types that arise during differentiation process. Normally cell selection is achieved using a technique called fluorescence activated cell sorting (FACS). The different cells of the body have specific proteins on their surface to which antibodies tagged with coloured or fluorescent molecules can bind, allowing us to identify and sort them using FACS, however, there are few such markers that can be used for isolating cones and rods. We intend to introduce a reporter into an important retinal gene that marks their differentiation to cone and rods. The presence of the fluorescent reporter will allow us to use the cell selection strategy mentioned above to purify these cells. We can then ask the question of whether these cells exhibit the properties associated with rods and cones using a variety of in vitro stem cell assays and electrophysiological analysis. If successful, this approach will allow us to prospectively isolate rod and cone cells, define their molecular phenotype and test their ability to restore vision in animal models of retinal disease.