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

使用锌指核酸酶技术生成报告基因标记的人类多能干细胞作为优化光感受器移植的工具

基本信息

批准号：
BB/I02333X/1
负责人：
Majlinda Lako
金额：
$ 15.24万
依托单位：
Newcastle University
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2011
资助国家：
英国
起止时间：
2011 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FI02333X%2F1
关键词：
Using zinc finger nuclease technology

项目摘要

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.

多能干细胞是无染色的细胞，可以在实验室中生长并编程以成为所需类型的专门细胞，例如血细胞，肌肉细胞等。人多能干细胞可以从不同的早期胚胎中以不同的方式得出，当它们在体外肥料中以剩余产物的形式可用，或者可以通过更普遍地从造型细胞中获得造型细胞。这种可能性引起了人们对使用干细胞进行治疗应用的极大兴趣，以治疗由于特定类型的细胞损失引起的疾病。例如，原则上可以通过从患者的皮肤细胞样本中处理因视网膜细胞丧失而引起的失明，重新编程，以使皮肤细胞形成无染色的多能干细胞，然后对所得的干细胞进行编程，以使视网膜细胞祖细胞可将视网膜细胞移植到患者的眼睛中，从而引起视网膜细胞。区分人多能干细胞的困难问题之一是跟踪细胞变成专用细胞，然后净化所需的专用细胞。对于小鼠多能干细胞，可以相对容易地完成此操作，通过插入使记者分子用荧光标记的基因使细胞在适当条件下发光的基因，但是该方法在人类多能干细胞中的效率非常低。现在，一种新的尖端技术提供了潜在的解决方案。锌指核酸酶是人为创建的剪刀，可以设计用于仅在一个特定位置的DNA分子的两个链。这些核酸酶在DNA结构中造成了差距，从而激活了细胞对DNA修复的响应。在存在短的DNA拉伸后，该拉伸与包含切除的区域相似，但也含有记者基因，可以将记者基因引入人类多能干细胞感兴趣的基因中。这项技术是最近的，仅在人类多能干细胞中应用了两次。但是，效率比其他报道的方法高得多，因此潜在的应用是巨大的。在此提案中，我们试图实施这项技术来创建标记的人类多能干细胞系线，该线路将用作优化细胞移植到退化视网膜的工具。视网膜具有非常复杂的结构，该结构由几层神经元组成，这些神经元相互关联。对光直接敏感的两种主要细胞类型是棒和锥形感光细胞。我们的小组表明，可以产生具有人类多能干细胞锥和杆的特征的人类细胞。尽管取得了这种进步，我们仍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，使用称为荧光活化细胞分选（FACS）的技术来实现细胞的选择。人体的不同细胞在其表面上具有特定的蛋白质，用彩色或荧光分子标记的抗体可以结合，从而使我们能够使用FACS识别并对其进行分类，但是，很少有这样的标记可用于隔离锥和杆。我们打算将记者引入一个重要的视网膜基因，以标记其与锥体和棒的区分。荧光报告基因的存在将使我们能够使用上面提到的细胞选择策略来净化这些细胞。然后，我们可以提出一个问题，即这些细胞是否使用各种体外干细胞分析和电生理分析表现出与棒和锥相关的特性。如果成功，这种方法将使我们能够前瞻性地分离棒和锥细胞，定义它们的分子表型，并测试它们在视网膜疾病动物模型中恢复视力的能力。