Modelling eye specification in human embryonic stem cells

In the human body, there are cells that can still grow, like skin or blood cells, and other that cannot, like brain cells. When cells die in the body, naturally or as a consequence of accident or disease, they can be substituted by new ones only if those cells that died belong to the kind of cells that can still grow. This is the reason why some diseases cannot be cured to date. One type of disease that cannot be treated is blindness due to the loss of eye cells responsible for the detection of the light and the transmission of that information to the brain. If those cells could be substituted by new ones, we could potentially cure some forms of blindness. The investigation that we propose here has the ultimate goal of producing cells able to take the place of cells lost in the retina, the part of the eye that receives the visual information and transmits it to the brain. To do so, we propose to use human embryonic cells with the ability of transforming into any type of cell of the body, which are called embryonic stem cells and which are already available for experimentation. The key to transform embryonic stem cells into the kind of cell you need is to treat them with the right molecular messengers that tell the cells what to do. In our previous research, we have characterised in animal model systems many of the messengers that are required to inform a cell that it has to become a retinal cell. The next step is to use this knowledge to direct the fate of human embryonic stem cells to produce the retinal cells that could be used to replace those lost because of disease.

Over the last decade, our work on the genetic control of eye specification in Xenopus has been very successful. We have identified many key genes involved in this process, and shed light on their interactions. Similar work performed in other model organisms (zebrafish, medakafish, chicks, mice) has identified several homologous molecules that play a crucial role in eye specification in these organisms and has led to a better understanding of a conserved programme of eye specification in vertebrates. Here, we propose to refine our knowledge of the signalling systems controlling the specification of neural progenitors towards eye fates, by using human embryonic stem (hES) cells as a model system. hES cell lines have been derived from pre-implantation embryos generated for the purpose of reproductive in vitro fertilization. hES cells are extremely pluripotent and genetically stable. They retain these features, together with their ability to proliferate and differentiate, even after several passages in culture. The proposed study can be performed with hES cell lines already available, and does not need the use of embryos. hES cells can be neuralized using the same signals inducing neural specification of ectodermal cells in the vertebrate embryos, namely BMP and Nodal inhibition in the presence of active FGF signalling. We will expose neuralized hES cells to the molecular signals that are the best candidates to specify eye fates on the basis of studies performed in a variety of vertebrate embryos. These signals include BMP, Wnt, IGF, Notch, Tiarin, TSK. Gain- and/or loss-of function studies show that manipulation of these signalling pathways affects eye formation in the embryo. hES cells represent an excellent in vitro system to test for a direct action of these factors on the specification of neuralized cells. We will expose neuralized hES cells to signalling molecules or their antagonists in the form of purified soluble reagents, most of which are commercially available. In the few cases when soluble factors will not be available, we will also transfect hES cells with DNA constructs coding for inducible agonists or antagonists of signalling pathways. This study will be done following time-course and dose-response curves of signalling activation (or inhibition) in an extensive fashion, as, in addition to the molecular identity of the inducing agents, the timing and the levels of signalling activation are expected to be crucial factors in eye specification. The use of hES cells as a model system will provide two additional advantages. First, it will allow us to study the mechanisms of eye specification directly on human neural progenitors, so that a comparison with the mechanisms acting in other vertebrate species will be possible. Second, the results of this study could pave the way for clinical approaches using stem cells theraputically for certain retinal diseases. After identifying the molecular signals specifying eye fates in hES cells with maximal efficiency, we will use the same signals on stable hES cell lines expressing GFP under the control of retina-specific promoters, in order to purify retinal progenitors by FACS sorting. We will analyze the three-dimensional neuronal patterning of hES cells that differentiate into retinal tissue to test whether such hES derived tissues have the ability to generate the normal cellular arrangements of the human retina. We aim to devise a well-defined and reproducible procedure to generate homogeneous cultures of retinal progenitors from human stem cells in vitro, as a first step towards their use in medical applications.