Parkinson's disease is a debilitating chronic and progressive disease characterized by muscle rigidity, tremor, slowing of movement progressing to inability to move, and various difficulties with speech, posture, and cognition. It is generally agreed that the loss of a particular type of brain cell called the dopamine neuron is responsible. Drug therapy is useful in early stages but becomes less effective as the disease progresses. Dopamine cell transplantation has been successful to some degree, but has been limited by the number of cells available for transplant as well as the long term functional viability of such cells. Recently it has been shown that the exposure of human embryonic stem cells to a cocktail of growth factors leads to the production of substantial numbers of dopamine cells. Thus human embryonic stem cells may yield an inexhaustible supply of such neurons. However, the efficacy of such cells in transplant therapy depends on such cells exhibiting the necessary characteristics of dopamine cells, most importantly the ability to produce and release dopamine when transplanted into the brain. The work proposed here will allow a complete characterization of the properties of such cells in terms of their neurochemistry, degree of activity and ability to make and release dopamine. Using a variety of markers we have developed and are developing in live cells, we will be able to isolate a homogeneous population of dopamine cells produced from human embryonic stem cells and optimized for their ability to replace the function of the lost dopamine neurons in Parkinson's disease. We see this as a necessary "quality control" step to assure the maximum therapeutic value of such cells. Eventually we will transplant such functionally verified dopamine cells into animal models of Parkinson's disease as the next step to their eventual therapeutic use in human Parkinsonian patients.
Statement of Benefit to California:
Parkinson's disease is a debilitating chronic and progressive disease characterized by muscle rigidity, tremor, slowing of movement progressing to inability to move, and various difficulties with speech, posture, and cognition. The incidence of newly diagnosed Parkinson's disease in California is estimated at about 13/100,000 citizens per year. Assuming a population of 36 million in California, that yields 468,000 newly diagnosed cases of Parkinson's disease every year in California. Clearly this is one of the most prevalent neurological disorders in California. This translates into a tremendous decrease in productivity of the work force, let alone the immense cost in medical care, and the personal cost in human suffering. The ability to transform human embryonic stem cells into dopamine neurons holds probably the greatest potential for cure of this terrible disease. However, stem cells do not uniformly turn into dopamine neurons when exposed to appropriate growth factors. Thus this proposal is aimed at optimizing the transformation by finding the best conditions for transformation, followed by functional characterization of those cells by a wide variety of neurochemical and physiological assays. The result will be a homogeneous population of functionally optimized dopamine neurons that will be maximally useful for transplantation first into animal models of Parkinson's and ultimately into human patients.
SYNOPSIS: This proposal tests the hypothesis that successful generation of transplantable dopamine (DA) neurons for Parkinson’s disease, from any precursor cell, requires their complete functional equivalency to midbrain substantia nigra DA neurons. Two specific aims are proposed here to examine neurochemical, electrophysiological, and secretion phenotype of DA neurons generated from existing non-ES cell lines and primary SN DA cultures (Aim 1) and hES cells (Aim 2). Appropriate synthesizing enzyme expressions, ion channel and firing patterns, and single cell/vesicle DA exocytosis will be examined using in vitro methods. INNOVATION AND SIGNIFICANCE: It is generally appreciated that dopamine cell replacement for Parkinson’s will most benefit from a stem cell differentiation protocol that promotes precise directed differentiation. The present proposal is not particularly innovative in its choice of DA neuron genes (Nurr 1 and Mash 1) and differentiation protocols, but is in its choice of methodologies used to establish electrophysiological and secretion phenotypes. Understanding how to generate DA neurons from hESCs that facilitate complete midbrain SN DA neuronal phenotype, focusing on appropriate mechanisms of coupling proper electrical activity and secretion is both innovative and significant. STRENGTHS: There is no question that for DA cell replacement approaches for Parkinson’s, a precursor cell will have to be “engineered” to exhibit as much if not all attributes of midbrain nigral functional phenotype as possible in order to generate efficacious transplantable cells for both preclinical studies and eventual human trials. The focus on electrophysiological and secretion phenotypes, particularly the use of the fusion protein SNARE VAMP2 attached to pHluorin for studying dopamine-containing vesicles, is interesting. The PI has a solid ES cell collaborator who has previously worked in the Trounson group. WEAKNESSES: The proposal does not consider the possibility that existing DA neurons generated from ES cells by other investigators such as McKay, Studer, and Zhang may already exhibit appropriate electrophysiological and transmitter synthesis/release phenotype and behavior. It should not be assumed that new cell lines have to be generated rather than testing on existing DA lines. It is not unreasonable to suggest that the hES cells to be studied here might need Nurr 1 and Mash1 transduction to confer DA neuron phenotype, but this is a bit open-ended in design and rational. The use in Aim 1 of primary cultures, four dopamine cell lines, and HEK293 (non dopaminergic) cells seems a bit ambitious, and even generating the primary DA neuron cultures is not without considerable effort. It is appreciated that “baseline” DA phenotype should be established in characterized and easy to manipulate cells before going on to the hES cell studies, but the numbers of markers and physiological approaches to be employed seems to require a commitment that alone will take the entire funding period (without even getting to the hES cell studies). DISCUSSION: There was no further discussion following the reviewers' comments.