Elucidating Chemotropic Responses of Human Embryonic Stem Cells to Guidance Cues
Parkinson’s disease is caused by the death of neurons in the substantia nigra. These neurons extend projections, called axons, to another region of the brain called the corpus striatum. Here, they release dopamine, an essential chemical required for smooth and coordinated movement. Loss of 80% of the dopamine producing cells leads to the symptoms of Parkinson’s disease which include muscle rigidity, tremor, and uncoordinated movement.
Current therapy involves supplementing the dwindling dopamine production with drugs, but this therapy loses effectiveness over time. Surgical options also exist, such as neurostimulation from battery-operated implants, but surgical complications occur in addition to problems associated with implanted hardware such as broken or dislocated wires. Successful long-term treatment has been attained for a subset of patients using fetal midbrain tissue grafts that reconstitute the axonal pathway, but general use of this therapy has been limited by requirements for fresh human fetal tissue. Consequently, there are clear advantages to using sources of dopaminergic neurons for transplantation, such as human embryonic stem cells (hESC), that can be perpetually grown in tissue culture
Great strides toward treatment have recently been made in our ability to reliably differentiate hESCs into dopaminergic neurons, but ultimately, therapeutic success will require reconstituting the precise neuronal connections with engrafted hESC-derived dopaminergic neurons. In the nervous system, neurons and their axons make appropriate connections by following cues present in the extracellular environment. Studies on dopaminergic neurons have shown that they are guided by two families of environmental cues called Netrins and Slits.
In this application, we propose to investigate the response of hESC-derived dopaminergic neurons to the Netrin and Slit cues. Neurons respond to cues based on the expression of receptors on their cell surface. Consequently, in Aim I, we propose to determine the profile of receptors expressed on dopaminergic neurons. Studies on mouse embryonic stem cells suggest that at least one receptor for each family of cue will be expressed by hESCs.
In Aim II, we propose to evaluate how hESCs respond to Netrin and Slit cues, using an assay in which aggregates of hESC-derived dopaminergic neurons are placed in a 3-dimensional matrix near a point source of Netrin and Slit. We will determine how axons of these dopaminergic neurons respond to cues by recording the migration behavior of the axons, toward or away from, the point source. If the dopaminergic neurons do not respond, we have previously identified methods to stimulate the neurons to generate a response.
Finally in Aim III, we propose to observe the response of hESC-derived dopaminergic neurons to Netrins and Slits that are present in adult brains by transplanting the neurons into mice and evaluating the response of their axons one and four weeks post-transplantation.
The citizens of California voted to support research on potential stem cell therapies that can be used to treat serious medical conditions that cripple millions of Americans. Parkinson’s disease is one of these conditions. The experiments proposed in this application tackle key issues that must be resolved before neurons derived from human embryonic stem cells can be successfully used to treat this neural disorder. Recently, researchers have developed ways to reliably differentiate human embryonic stem cells into the type of neurons, dopaminergic, that degenerate in patients with Parkinson’s disease. This application addresses the next step required for the development of successful therapeutic strategies. These strategies will require that transplanted dopaminergic neurons respond robustly and appropriately to environmental cues in the patient’s brain so that they supply the crucial chemical, dopamine, to the correct target. Parkinson’s disease is one of the illnesses in which a stem cell-based therapy is within grasp. An early success in using human stem cells to treat a devastating illness will be inspirational to California citizens.