In diseases of the nervous system such as Parkinson’s disease (PD) and Lou Gehrig’s disease very specific groups of nerve cells die. At least in the case of PD, the surgical methods exist for the implantation of new cells into the area of the brain where the nerve cells are dying. However, since fetal brain cells are almost impossible to obtain, a viable and untested potential source of brain neurons are human embryonic stem (ES) cells. In order to get these cells to function in the brain, it is mandatory that the ES cells be converted to nerve cells before they can be surgically implanted. In our past work with rat and mouse stem cells we have been able to identify and purify factors that are made by nerve precursor cells that cause stem cells to become neurons. In addition, we have a very large potential source of these types of factors that is unique to our laboratory. Finally, we have identified a new family of drugs that may help keep the ES cell-derived neurons alive when they are transplanted into the brain, a situation in which most of them normally die. Therefore, the goal of this proposal is to identify new factors that convert human ES cells to specific types of neurons and to test the new family of compounds to determine if they promote the survival of ES cell-derived neurons in the brain.
Statement of Benefit to California:
Our work will benefit the State in a number of ways. 1) There could be a tremendous health benefit for individuals with diseases of the brain such as Parkinson’s and Lou Gehrig’s diseases as well as damage due to stroke or trauma. 2) Support for this work will provide current employment within the State and help educate scientists in the stem cell field. 3) The advancement of work on novel growth factors and drugs will require the collaboration with commercial (for profit) companies. Most of early stage preclinical development is done in small biotech companies, many of which are within the State. Therefore, there is an economic benefit to the State as well as a health benefit to the State and the world if some of the most debilitating human diseases could be cured.
SYNOPSIS: The PI proposes a broad screen of peptides produced by 150 stored cell lines from the early 1970s as to their effects on human embryonic stem cells (hESCs); then, identification of the optimistically predicted "positive hits". SIGNIFICANCE AND INNOVATION: This application proposes to screen secreted factors from stem cell-like cells derived from the rat central nervous system (CNS) and to examine their effects on the differentiation of hESCs to neurons. Active secreted proteins will be isolated, characterized and assayed for other effects, such as neuronal survival. The proposal is innovative in the use of the Principal Investigator's (PI's) own library of 150 rat CNS cell lines. If successful, novel active compounds that enhance hESC neuronal differentiation and neuronal survival will be identified. This may possibly lead to an advance in the treatment of neurodegenerative disorders. This is a high risk project with a potentially modest to high payoff. It could be a total waste, or could result in finding something important. STRENGTHS: The PI has a library of 150 rat CNS stem cell-like cell lines, generated many, many years ago. These cell lines could make something that is as yet not identified. Potentially, these cell lines secrete factors into the medium which may affect neuronal differentiation of hESC lines and neuronal survival. The PI and his team are experienced with this type of research; they are proficient in the in vitro and in vivo screening methodologies. The Salk Institute is an excellent facility for this work and has the necessary infrastructure and support. The PI recognizes that this proposal is somewhat of a "fishing expedition." However, there are times when screens are appropriate and can yield valuable results. This may be one of those. WEAKNESSES: The proposal is very unfocused and written rather simplistically. There is a lack of detail in the experimental design - e.g. there are no details as to how the hESC lines will be grown, how often they will be passaged, how the analyses will be performed. The team has no hESC expertise and does not collaborate with an hESC researcher. There may be difficulties in growing and manipulating the cell lines. In addition to concerns about hESCs, the proposal is over ambitious but has a very poorly planned process for discovery. If this line of work is really promising, why has it not at least "limped along" over all these years. If there were no additional "hits" (beyond activin and F-spondin) after 30+ years, is it likely to blossom now? One reviewer recommended a more focused approach to see if the first steps will yield any biological motivations for further identification of culture "hits". Teaming up with one of the local high-throughput/high-content screening groups at the cell culture level may be a good move. DISCUSSION: This proposal aims to find secreted peptides that will influence hESC differentiation. The goal is to take 150 immortalized rat CNS cell lines, generated many, many years ago, make conditioned media and then hunt/screen the media for factors that works on hESCs. Both reviewers acknowledged that the Principal Investigator (PI) had a likely unique resource and that "something cool" could come from proposed studies. In general the PI is considered a very experienced investigator in microsequencing cloning, proteomics, mass spectrocopy, etc but with no experience in developmental neurobiology. Both also noted that the application was badly put together. One reviewer was torn between wanting to recommend a poor score due to the fact that the project is highly likely to fail, and a high score because the PI may have a treasure trove of cell lines that produce as yet unknown factors. By NIH standards, this research is highly likely to fail, but there is a small chance that something exciting might come out where no other methods would produce the same result. The environment is strong as there is great proteomics and mass spectrometry at the Salk Institute. The PI has no hESC experience, and no collaborations with high throughput labs in the area. It's not clear that the library of 150 rat CNS lines is viable after 30 years in the freezer. The analysis plan is "somewhat sketchy", such as the plan for growth from 1 to 14 days. How does the PI plan to do this?