Central nervous system (CNS) disorders such as those that affect the brain and eye are particularly debilitating because repair is hampered by the fact that neurons don’t divide to replace damaged areas and adult neural stem cells that can divide and potentially replace neurons don’t do so in the adult (for reasons we don’t understand). Therefore, it is intriguing to think that disorders such as stroke and macular degeneration of the eye may benefit from stem cell therapy. Treatment with stem cells derived from human embryonic stem cells (hESCs) requires pre-formation of a particular cell type, since transplantation of hESCs themselves leads to tumor formation. However, current methods for making CNS neurons are inadequate since the neurons are usually immature and unable to connect with the other neurons in the damaged area. Interestingly, treatment of animal models of CNS disorders with stem cells has shown some limited benefits without generation of mature neurons, possibly due to factors that the cells produce. However, transplantation of brain- or eye-specific cells derived from hESCs may lead to greater functional recovery (e.g. treatment of the damaged brain with a brain-specific neuron able to make functional connections with the host tissue). Certain regions of the brain (cortical) and eye (retinal) arise from the same area during development and share expression of a variety of markers, including regulatory proteins called transcription factors. Thus, it should be possible to make brain and eye cells from hESCs using similar conditions. Our goal is to use transcription factors to preferentially form brain and eye cells from hESCs. In one set of experiments, we will analyze transcription factors made by the hESCs in response to exposure to different conditions. In our second set of experiments, we will put transcription factors into hESCs during differentiation to test whether a particular transcription factor can direct formation of brain or eye neurons. This approach was recently shown to improve generation of the particular type of neurons damaged in Parkinson’s disease from mouse ESCs. We hope to use the cells we make for transplantation and also for drug testing, since there is currently no large-scale source of human versions of brain and eye neurons for these purposes.
Statement of Benefit to California:
The goal of this project is to make brain- and eye-specific cells from hESCs that can be used for transplantation for disorders that affect specific brain regions (such as stroke) or the eye (retinal disorders such as macular degeneration). We also expect that the cells derived in this project could be used as a source of human brain or eye neurons for drug testing. Currently, there is no large-scale supply of such neurons. As such, if the goals of this project are realized, the benefits to the State of California and its citizens would include a greater range of therapeutic options for central nervous system disorders as well as an important source of cells for pharmaceutical and biotechnology companies.
SYNOPSIS: The goal of this proposal is to use expression of developmentally regulated transcription factors to preferentially differentiate hESCs along cortical and retinal lineages. In one aim, the applicant will analyze expression of endogenous transcription factors stimulated by differentiation conditions in which matrix and soluble modulators will be varied to mimic those that the cells would experience during development. In the second aim, the applicant will express exogenous transcription factors in hESCs during differentiation. These experiments will assess whether a particular transcription factor can direct development of an in vitro phenotype that more closely matches that of in vivo cells. This approach was recently shown to improve generation of “bona fide” midbrain dopaminergic cells from mouse ESCs. The ultimate aim is to use the predifferentiated cells for transplantation and as in vitro sources of human cortical and retinal neurons for drug testing. Three different hESC lines will be used that vary in their time in culture and derivation methods. Two of these lines were generated after the federal deadline and would not be eligible for federal funds. SIGNIFICANCE AND INNOVATION: The proposed study intends to differentiate human ESCs to neural progenitors that are potentially fated to cortical neurons and retinal cells using both non-genetic and genetic alterations (over-expression of Lhx2). There are only limited reports on differentiation of neural progenitors from ESCs that possess forebrain phenotypes. Detail studies on cortical and retinal neuron differentiation are still largely lacking possibly because of our limited knowledge about molecular regulation of forebrain neuronal specification and because of technical difficulty in maintaining the forebrain identity of the ESC differentiation cultures. The proposal is novel in this regard. Besides, the ability to direct human ESCs to specific cortical or retinal neurons may also have implication in treating a wide range of neurological diseases that affect cortical neurons. STRENGTHS: The strength of the proposal is its important question and the potential implication of the study to generate forebrain cells including retinal cells from human ESCs. The PI proposed parallel non-genetic approach and a genetic means to over-express Lhx2, a transcription factor mainly expressed by forebrain progenitors. The PI and her associates have begun to use human ESCs and provided quite convincing data that they were able to differentiate human ESCs to neuroepithelial cells, many of which expressed Lhx2. Therefore, the likelihood of success of the proposed study is high. WEAKNESSES: The PI provided a list of growth factors and extracellular matrix proteins for inducing forebrain neural progenitors but did not give a clear plan of how the experiments would be done. How will the various factors be prioritized and how will they be appled applied (single, combination, sequence, etc). The same is true of the marker experiments, which just give a list of markers. In this sense, the proposal lacks a strong research plan with a clear rationale of which factors (or markers) are likely to be important and why they will be chosen. A related weakness is that although the applicant has expertise in microfluidics and optimization of culture conditions, the proposal is in some respects biologically naive and not conversant with the relevant neural development literature. The applicant appears not to understand, for example, that there is more than one cortical lineage in the forebrain. DISCUSSION: There was some discussion and disagreement on the significance of the preliminary data, and whether the investigator has the requisite biological expertise to carry out the project. In spite of a number of weaknesses, the importance of the project and the fact that this was a beginning investigator strengthened enthusiasm for the proposal.