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

多能干细胞是一种未特化的细胞，可以在实验室中培养并被编程为所需类型的特化细胞，如血细胞、肌肉细胞等。人类多能干细胞可以通过不同的方式获得，可以从体外受精过程中作为多余产物的非常早期的胚胎获得，或者更广泛地说，通过重新编程来自个体的容易获得的细胞，例如来自皮肤细胞的样本。这种可能性导致了人们对使用干细胞治疗因特定类型细胞丢失引起的疾病的极大兴趣。例如，由于视网膜细胞丧失而导致的失明，原则上可以通过从患者身上提取皮肤细胞样本，对皮肤细胞进行重新编程以生成未特化的多能干细胞，然后对生成的干细胞进行编程以提供视网膜细胞前体细胞，这些细胞可以移植到患者的眼睛中形成视网膜细胞。分化人类多能干细胞的难题之一是跟踪细胞转化为特化细胞的过程，然后提纯所需的特化细胞。对于小鼠多能干细胞来说，这可以相对容易地完成，方法是插入一些基因，使报告分子标记上荧光，使细胞在合适的条件下发光，但这种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。锌指核酸酶是一种人工制造的剪刀，可以被设计成只在一个特定的位置专门切割DNA分子的两条链。这些核酸酶在DNA结构上产生一个缺口，从而激活细胞对DNA修复的反应。当存在一小段DNA片段，显示出与包含切除的区域相似，但也包含报告基因时，就有可能将报告基因引入到人类多能干细胞的感兴趣基因中。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，这种方法的效率远远高于其他已报道的方法，因此潜在的应用是巨大的。在这项提案中，我们试图实施这项技术来创建标记的人类多能干细胞系，作为优化细胞移植到退化视网膜的工具。视网膜有一个非常复杂的结构，由相互连接的几层神经元组成。对光直接敏感的两种主要细胞类型是视杆细胞和视锥细胞。我们的团队已经证明，从人类多能干细胞中制造出具有锥体和杆状特征的人类细胞是可能的。尽管取得了这一进展，但我们不能从分化过程中产生的其他细胞类型中选择这些细胞。正常情况下，细胞选择是使用一种称为荧光激活细胞分选(FACS)的技术来实现的。人体不同的细胞在其表面有特定的蛋白质，标记了有色或荧光分子的抗体可以结合到这些蛋白质上，使我们能够使用FAC来识别和分类它们，然而，很少有这样的标记可以用于分离视锥和视杆。我们打算向记者介绍一个重要的视网膜基因，它标志着它们向视锥和视杆分化。荧光报告的存在将使我们能够使用上面提到的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分析和电生理分析来询问这些细胞是否表现出与杆和锥体相关的特性。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.