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.

多能干细胞是非特化细胞，可以在实验室中生长并编程为所需类型的特化细胞，例如血细胞、肌肉细胞等。人类多能干细胞可以通过不同的方式衍生，从非常早期的胚胎中获得在体外受精过程中，它们可以作为剩余产品获得，或者更一般地说，可以通过对来自个体的容易获得的细胞（例如皮肤细胞样本）进行重新编程来获得。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，以治疗由特定类型细胞丢失引起的疾病。例如，原则上可以通过从患者身上采集皮肤细胞样本，重新编程皮肤细胞以产生非特化的多能干细胞，然后对所得干细胞进行编程以产生视网膜细胞祖细胞来治疗因视网膜细胞丢失而导致的失明。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难题之一是追踪细胞转变为特化细胞的过程，然后纯化所需的特化细胞。对于小鼠多能干细胞来说，这可以相对容易地完成，通过插入基因，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。锌指核酸酶是一种人工制造的剪刀，可专门在一个特定位置特异性切割 DNA 分子的两条链。这些核酸酶在 DNA 结构中产生间隙，从而激活细胞对 DNA 修复的反应。当存在与含有切除区域相似但也含有报告基因的短DNA片段时，可以将报告基因引入人多能干细胞中的目的基因中。这项技术是最近才出现的，仅在人类多能干细胞中应用过两次；然而，其效率远高于其他报道的方法，因此潜在的应用是巨大的。在本提案中，我们寻求实施这项技术来创建标记的人类多能干细胞系，该细胞系将用作优化细胞移植到退化视网膜中的工具。视网膜具有非常复杂的结构，由彼此互连的多层神经元组成。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，可以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。尽管取得了这些进展，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，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。身体的不同细胞表面有特定的蛋白质，带有彩色或荧光分子标记的抗体可以结合这些蛋白质，使我们能够使用 FACS 对它们进行识别和分类，但是，很少有这样的标记可用于分离视锥细胞和视杆细胞。我们打算向记者介绍一个重要的视网膜基因，该基因标志着视网膜向视锥细胞和视杆细胞的分化。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，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。