EFFECTS OF SMALL MOLECULE LIBRARIES ON DIFFERENTIATION OF EMBRYONIC AND NERAL STEMS CELLS IN DOPAMINERGIC PHENOTYPE
PUBLIC ABSTRACT. Degenerative brain diseases account for a huge portion of the health burden for our society. Most are largely untreatable or, at best, inadequately treated. A number of devastating human neurological diseases are caused by abnormalities of the ways in which signals are transmitted from one brain cell to another and from one part of the brain to another. One of the most important signal systems in the brain and one that is most vulnerable to inherited or acquired damage is the system that is regulated by a special class of nerve cells (neurons) that use the molecule dopamine to transmit signals to other neurons and other cells in the brain. The most common of dopamine-deficiency diseases is the familiar Parkinson’s disease that afflicts millions of people throughout the US and hundred of thousands of Californians. Unfortunately, for many reasons, the causes of the dopamine degeneration in this disease are difficult to study, partly because most cases of this disorder seem to be caused by complex interactions among a number of genes or by mixtures of genetic and environmental factors. Only a few cases are caused by identified simple defects in the genes responsible for producing or maintaining the dopamine neurons. Fortunately for an understanding the genetics of the dopamine signaling system, another disorder of dopamine function, Lesch Nyhan Disease, is caused by defects in one single gene called HPRT. The disorder is associated with severe retardation, abnormal movements and a compulsive and untreatable self-mutilation behavior and is largely untreatable. Because the disease is a direct result of abnormalities in a single, well-understood gene, it is possible to study the ways in which genetic damage can cause defects in the dopamine systems, changes that are directly responsible for the severe neurological consequences. Because the damage to the dopamine pathways produces defects of the dopamine-dependent cells themselves, we propose to study the ways in which stem cells develop into mature normal dopamine nerve cells. There are a variety of cells that have developed along the pathway from stem cells to normal nerve cells but that have not reached the stage of functioning dopamine nerve cells. We wish to determine if we can force these cells as well as the stem cells themselves to become functional dopamine neurons by treating them with a collection of more approximately 50,000 chemicals. If these studies area successful, we may be able to understand the dopamine development process more thoroughly and possibly also to prepare large numbers of normal dopamine-producing cells for eventual transplantation approaches to these devastating diseases.
Statement of Benefit to California:
BENEFIT TO CALIFORNIA Advances in modern molecular genetics are making possible new approaches to understanding the basis of normal and abnormal human biology and to improve treatment of human disease. Studies of human embryonic stem cells promise to become an important part of this new area of biomedicine, but many kinds of studies have been severely hampered in the U.S. by a restrictive national policy and inadequate funding mechanisms. Wisely, the people of California have taken steps to catalyze this field through the formation of the California Institute for regenerative Medicine (CIRM) and through the CIRM programs to support research in this area. Our studies, if successful, will contribute to an understanding of a basic process of brain function and will therefore strengthen the huge basic research effort in neurobiology at the California-based academic institutions. Furthermore, knowledge and techniques derived from this kind of study can be applied to the discovery and development of drugs that specifically affect neuron function and that potentially affect the movement, cognitive, mood, compulsive and aggressive behavioral aspects of brain disorders, all of which represent the central features of Lesch Nyhan Disease. Another potentially useful outcome with commercial implications could involve methods to produce large amounts of dopamine-producing cells for transplantation for treatment of some kinds of brain disorders and the development of similar approaches to other neurotransmitter CNS disorders. Such discoveries will constitute the basis for expanded and new pharmaceutical and biotechnology ventures in California, with the health and economic benefits that such progress carries with it.
SYNOPSIS: The overall aims of the proposal are to produce ES cells with dopaminergic properties and to identify factors that will induce this differentiation - with a future goal of using these cells or knowledge from these experiments to treat Parkinson’s disease and Lesch Nyhan disease (which has mutated HPRT). In Aim I, the PI will screen small molecules on the ability of selected cells (PC12 or neuroblastoma cells) to express EGFP under the control of the human tyrosine hydroxylase (TH) promoter, as an indicator of induced dopaminergic phenotype. Once preliminary identifications are made of inducing molecules in the PC12 cells, the candidates will be used on wild type and HPRT knockout hES cells. Aim II tests the chemically-differentiated cells for dopaminergic and neuronal phenotypes. Aim III makes use of microarrays to determine the effects of the agents identified in Aim 1 on global patterns of gene expression in the differentiated cells in order to identify pertinent transcription. SIGNIFICANCE AND INNOVATION: At present, there are difficulties obtaining a large number of homogeneous dopaminergic neurons that are pure and free of xenobiotic products, such as feeder cells. In addition, the transcription factors and the pathways that are important in the differentiation and maintenance of these cells are poorly defined. Therefore, this proposal is an important one since it may lead to knowledge and the generation of dopaminergic differentiated cells that impacts on the treatment of Parkinson's disease. In addition, although not mentioned by the PI, the small molecules that may be identified in the planned studies of this proposal may have a use in direct treatment of patients since they are likely to cross the blood-brain-barrier, may slow down DA neuronal death or stimulate regeneration, and avoid transplantation into a delicate region of the brain. Lastly, this proposal explores factors that are key to differentiation, and therefore knowledge gleaned from the present proposed studies could lead to advances in the stem cell field more globally. The use of high throughput screens of chemical libraries to identify factors that drive ES cells down a particular differentiation pathway has been rarely performed and therefore should be considered a novel approach. The methods that will be employed by the PI are contemporary ones. There is no question that a better understanding of small molecules that can induce midbrain dopaminergic neuron phenotype from hES cells is important. Many labs around the world are already engaged in the type of drug-screen, HTS technologies on NSCs and hESCs as proposed here, but there is still significance if the PI could discover compounds that do impressively increase the number of dopamine neuron precursors with a midbrain phenotype from hES cells. STRENGTHS: The proposal is directed at important issues related to Parkinson's disease and may help in the disease’s treatment. The proposal is straightforward and depends on a concept (i.e., stem cells exposed to small chemicals can potentially be induced to differentiate into functional dopaminergic cells) that appears to have been successful, as noted in previous publications. The use of an initial screen in TH-GFP transduced PC12 or neuroblastoma cells is a clever first approach prior to screening ES cells. The collaboration with the San Diego Center for Chemical Genomics, which operates as an NIH-funded center of the Multiple Libraries Screening Centers Network, provides assurance that the high throughput screening facility will be of high quality. The assistance of Dr. Theodore Friedman’s lab with respect to their expertise in characterizing the functional properties of dopaminergic differentiated cells provides assurance that the phenotype of the cells will be appropriately analyzed. The PI is experienced with the use of virus vectors for gene delivery. The co-PI has experience with microarray transcription profiling, including studies of FACS-sorted midbrain DA neurons obtained from a transgenic mouse expressing GFP from the TH promoter The PI has recognized and focused on what appears to be the correct transcription factors and signaling molecules involved in induction of DA neuron phenotype. WEAKNESSES: There are some aspects of the proposal that need to be clarified. For example: - What is the rationale for using the HPRT knockout hES cells? - Why will the screening of the neuroblastoma cells allow for testing of a larger library than in the case of PC12 cells? - Will siRNA libraries be used, as suggested on line 2 of p. 10? - Which colleagues will be used for investigations of the neurophysiological properties of the dopaminergic cells? The hypothesis of this proposal, i.e., stem cells exposed to small chemicals can potentially be induced to differentiate into functional dopaminergic cells, is risky. In addition, there may be a group of chemicals that are key to the dopaminergic phenotype, making the identification more complicated. It would have been valuable for the PI to have noted how many positive hits he anticipates and how he will prioritize looking at many hits in Specific Aim II. The third specific aim, involving the microarray profiling, is dependent on success of the first specific aim, i.e., differentiating dopaminergic cells with a small molecule or chemical. There is little discussion of problems that may occur in carrying out the specific aims and what will be done when the problems occur. The PI has had modest productivity and lists no publications related to studies of dopaminergic cells of stem cells. The last publication listed by the co-PI is in 2000; productivity is low. There is really no one on this grant with what appears to be the necessary skills to work with the hESC cells. The attention paid to PC12 and other cells besides hESCs is distracting and not helpful for the cause of studying ES cells. Working with the latter cells is certainly challenging enough without having to perfect the screens and readouts from these other populations of cells. DISCUSSION: The primary reviewer liked the approach of using HTS to effect differentiation of hESC to dopaminergic properties since there is currently no good way to achieve differentiation to dopaminergic cells. The approach and the collaborators were regarded as good but lots of unanswered questions remain after reading the proposal. There was concern about the productivity of the investigator. Also, the rationale was regarded as weak.