Optimization of guidance response in human embryonic stem cell derived midbrain dopaminergic neurons in development and disease
A promising approach to alleviating the symptoms of Parkinson’s disease is to transplant healthy dopaminergic neurons into the brains of these patients. Due to the large number of transplant neurons required for each patient and the difficulty in obtaining these neurons from human tissue, the most viable transplantation strategy will utilize not fetal dopaminergic neurons but dopaminergic neurons derived from human stem cell lines. While transplantation has been promising, it has had limited success, in part due to the ability of the new neurons to find their correct targets in the brain. This incorrect targeting may be due to the lack of appropriate growth and guidance cues as well as to inflammation in the brain that occurs in response to transplantation, or to a combination of the two. Cytokines released upon inflammation can affect the ability of the new neurons to connect, and thus ultimately will affect their biological function. In out laboratory we have had ongoing efforts to determine the which guidance molecules are required for proper targeting of dopaminergic neurons during normal development and we have identified necessary cues. We now plan to extend these studies to determine how these critical guidance cues affect human stem cell derived dopaminergic neurons, the cells that will be used in transplantation. In addition, we will examine how these guidance cues affect both normal and stem cell derived dopaminergic neurons under conditions that are similar to the diseased and transplanted brain, specifically when the brain is inflamed. Ultimately, an understanding of how the environment of the transplanted brain influences the ability of the healthy new neurons to connect to their correct targets will lead to genetic, and/or drug-based strategies for optimizing transplantation therapy.
Statement of Benefit to California:
The goal of our work is to further optimize our ability to turn undifferentiated human stem cells into differentiated neurons that the brain can use as replacement for neurons damaged by disease. We focus onParkinson’s disease, a neurodegenerative disease that afflicts 4-6 million people worldwide in all geographical locations, but which is more common in rural farm communities compared to urban areas (Van Den Eeden et al., 2003), a criteria important for California’s large farming population. In Parkinson’s patients, a small, well-defined subset of neurons, the midbrain dopaminergic neurons have died, and one therapeutic strategy is to transplant healthy replacement neurons to the patient. Our work will further our understanding of the biology of these neurons in normal animals. This will allow us to refine the process of turning human ES cells onto biologically active dopaminergic neurons that can be used in transplantation therapy. Our work will be of benefit to all Parkinson’s patients including afflicted Californians. In addition to the direct benefit in improving PD therapies, discoveries from this work are also likely to generate substantial intellectual property and further boost clinical and biotechnical development efforts in California.
SYNOPSIS: In order to facilitate the survival, integration, and axonal innervation of grafted dopamine neurons in the Parkinson's disease (PD) brain, it is important to understand the molecular milieu of the degenerating host brain before evaluating which cell is best to restore the dopaminergic nigrostriatal axis. This proposal focuses on two aspects of this molecular milieu: axonal guidance molecules and neuroinflammatory molecules. hES-derived DA neurons are looked at, and compared with ventral mesencephalon dopamine neurons, for the role of differentiation state in vitro and response to axon guidance (e.g. ephrins) and inflammatory (interlukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interferon-γ (Infγ) molecules. Three aims are proposed that 1. Confirm the roles, on hES-derived dopamine neurons, of certain guidance molecules established in previous funded studies on mouse cells; 2. Determine the influences of inflammation on DA neuron axon guidance; chemokines and immune effector. SIGNIFICANCE AND INNOVATION: This is a very innovative grant that will address the roles of axon guidance and neuroinflammation molecules in getting cells to respond to guidance cues. A co-culture method previously used by the PI, will now be used with hESC aggregates, and COS cells secreting guidance molecules. The idea is innovative and should yield novel and important findings on factors that direct guidance of DA axons. STRENGTHS: This proposal combines expertise from two well established and respected groups to study axon guidance and inflammatory molecules in a co-culture bioassay with hESC derived DA neuron precursors. A key strength is the inclusion of Mary Hynes, a senior researcher in the PI's lab, who is a world expert in dopaminergic neurons. She will be directing the proposed studies. The goal of this work is highly meritorious - to understand the potential neurodegenerative disease inflammatory environment of the PD brain, and shed light on how this environment influences guidance and connectivity for future studies that exploit genetic and drug-based approaches for maximizing stem cell based neuron replacement strategies. WEAKNESSES: A potential weakness is which dopaminergic neuron will one get from hESC differentiation. There are many different sub-types of dopaminergic neurons. Only a specific sub-type is lost in PD. Therefore it is critical to focus on ensuring that the differentiation protocol specifically targets this sub-type, and not just any neuron that makes tyrosine hydroxylase. This kind of differentiation has not yet been perfected and the applicant knows this and recognizes the challenge. Additionally, some of the previous studies showed effects of these molecules on midbrain DA neuron topography, so it is not certain how axons will be the only elements affected by these molecules. Also, there is little discussed with regard to diverse outcomes from studying “immune effector cells” on a heterogeneous population of hES-derived cells in these cultures. DISCUSSION: This proposal aims to investigate the guidance response in hESC-derived midbrain dopaminergic neurons in development and disease. The PI proposes to look at axon guidance molecules and the inflammatory response. Two issues were raised regarding the potential effects of these molecules, first with respect to axons being the only affected elements, and second with respect to the diverse outcome that may arise from treating a heterogeneous cell population. The PI should be able to overcome the latter. One suggestion was that the protocol of Ericson, Perlman and Studer should be used rather than the older protocols cited.