Identification and characterization of human ES-derived DA neuronal subtypes

Identification and characterization of human ES-derived DA neuronal subtypes

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01358
Award Value: 
$1,405,345
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Status: 
Closed
Public Abstract: 
Parkinson’s disease (PD) is a neurodegenerative movement disorder that affects 1 in 100 people over the age of 60, one million people in the US and six million worldwide. Patients show a resting tremor, slowness of movement (bradykinesia), postural instability and rigidity. Parkinson's disease results primarily from the loss of neurons deep in the middle part of the brain (the midbrain), in particular neurons that produce dopamine (referred to as “dopaminergic”). There are actually two groups of midbrain dopaminergic (DA) neurons, and only one, those in the substantia nigra (SN) are highly susceptible to degeneration in Parkinson’s patients. There is a relative sparing of the second group and these are called ventral tegmental area (VTA) dopaminergic neurons. These two groups of neurons reside in different regions of the adult ventral midbrain and importantly, they deliver dopamine to their downstream neuronal targets in different ways. SN neurons deliver dopamine in small rapid squirts, like a sprinkler, whereas VTA neurons have a tap that provides a continuous stream of dopamine. A major therapeutic strategy for Parkinsons’ patients is to produce DA neurons from human embryonic stem cells for use in transplantation therapy. However early human trials were disappointing, since a number of patients with grafts of human fetal neurons developed additional, highly undesirable motor dyskinesias. Why this occurred is not known, but one possibility is that the transplant mixture, which contained both SN and VTA DA neurons, provided too much or unregulated amounts of DA (from the VTA neurons), overloading or confusing the target region in the brain that usually receives dopamine from SN neurons in small, regular quantities. Future human trials will likely utilize DA neurons that have been made from human embryonic stem cells (hES). Since stem cells have the potential to develop into any type of cell in the body, these considerations suggest that we should devise a way to specifically produce SN neurons and not VTA neurons from stem cells for use in transplantation. However, although we can produce dopaminergic neurons from hES cells, to date the scientific community cannot distinguish SN from VTA neurons outside of their normal brain environment and therefore has no ability to produce one selectively and not the other. We do know, however, that these two populations of neurons normally form connections with different regions in the brain, and we propose to use this fact to identify molecular markers that distinguish SN from VTA neurons and to determine optimal conditions for the differentiation of hES to SN DA neurons, at the expense of VTA DA neurons. Our studies have the potential to significantly impact transplantation therapy by enabling the production of SN over VTA neurons from hES cells, and to generate hypotheses about molecules that might be useful for coaxing SN DA neurons to form appropriate connections within the transplanted brain.
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 on Parkinson’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, 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 embryonic stem 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. Further, this project will utilize California goods and services whenever possible.
Progress Report: 

Year 1

Parkinson's disease results primarily from the loss of neurons deep in the middle part of the brain (the midbrain), in particular neurons that produce dopamine (referred to as “dopaminergic”). In this region of the midbrain there are actually two different groups of dopaminergic (DA) neurons, and only one of them, the neurons of the substantia nigra (SN) are highly susceptible to degeneration in patients with PD. There is a relative sparing of the second group of midbrain dopaminergic neurons, called the ventral tegmental area (VTA) dopaminergic neurons. These two groups of neurons reside close to each other in the brain and both make dopamine. They are virtually indistinguishable except for one major functional difference—they release dopamine, the transmitter that is lost in Parkinson’s patients, to their downstream neuronal targets in different ways. SN neurons deliver dopamine in small rapid squirts, like a sprinkler, whereas VTA neurons have a tap that provides a continuous stream of dopamine. A major therapeutic strategy for patients with PD is to make new DA neurons from human embryonic stem cells (hES). As stem cells have the potential to develop into any type of cell in the body, these considerations suggest that we should devise a way to produce SN neurons in the absence of VTA neurons from stem cells for use in transplantation. At present although we can produce dopaminergic neurons from hES cells, the scientific community cannot distinguish SN from VTA neurons in vitro due to lack of molecular markers or a bioassay, and we are therefore unable to identify culture conditions that favor the production of one over the other, In addition to releasing dopamine differently, SN and VTA neurons have axons that project to different regions of the striatum. It has been shown over the last decade that specific classes of guidance cues guide axons to their particular targets. One approach we have taken has been to investigate whether differences in axon guidance receptor expression and or responses to guidance cues in vitro might provide both markers and a bioassay that will distinguish SN from VTA neurons. Over the last year we have shown that VTA and SN neurons respond differentially to Netrin-1 and express different markers associated with the guidance cue family. We now have a bioassay and markers that distinguish these two populations of neurons in vitro and in the coming year we plan to utilize this information to identify cultures conditions that favor the production of SN over VTA neurons, from hES cells.

Year 2

Parkinson’s disease results primarily from the loss of neurons deep in the middle part of the brain (the midbrain), in particular neurons that produce dopamine (referred to as “dopaminergic”). In this region of the midbrain there are actually two different groups of dopaminergic (DA) neurons, and only one of them, the neurons of the substantia nigra (SN) are highly susceptible to degeneration in patients with PD. There is a relative sparing of the second group of midbrain dopaminergic neurons, called the ventral tegmental area (VTA) dopaminergic neurons. These two groups of neurons reside close to each other in the brain and both make dopamine. They are virtually indistinguishable except for one major functional difference—they release dopamine, the transmitter that is lost in Parkinson’s patients, to their downstream neuronal targets in different ways. SN neurons deliver dopamine in small rapid squirts, like a sprinkler, whereas VTA neurons have a tap that provides a continuous stream of dopamine. 
A major therapeutic strategy for patients with PD is to make new DA neurons from human embryonic stem cells (hES). As stem cells have the potential to develop into any type of cell in the body, these considerations suggest that we should devise a way to produce SN neurons in the absence of VTA neurons from stem cells for use in transplantation. At present although we can produce dopaminergic neurons from hES cells, the scientific community cannot distinguish SN from VTA neurons in vitro due to lack of molecular markers or a bioassay, and we are therefore unable to identify culture conditions that favor the production of one over the other, 
In addition to releasing dopamine differently, SN and VTA neurons have axons that project to different regions of the striatum. It has been shown over the last decade that specific classes of guidance cues guide axons to their particular targets. One approach we have taken has been to investigate whether differences in axon guidance receptor expression and or responses to guidance cues in vitro might provide both markers and a bioassay that will distinguish SN from VTA neurons. We showed previously that VTA and SN neurons respond differentially to Netrin-1 and express different markers associated with the guidance cue family. Also, in this year using backlabeling, laser capture and microarray analysis of SN vs VTA neurons, we have identified a number of genes expressed in on or the other population. We now have a bioassay and markers that distinguish these two populations of neurons in vitro and in the coming year we plan to utilize this information to identify cultures conditions that favor the production of SN over VTA neurons, from hES cells.

Year 3

Parkinson's disease (PD) is a neurodegenerative movement disorder that affects more than six million people worldwide. The main symptoms of the disease result from the loss of neurons from the midbrain that produce dopamine (referred to as "dopaminergic" or DA neurons).Human embryonic stem cells (hESC) offer an exciting opportunity to treat Parkinson’s disease by transplanting hESC-derived DA neurons to replace those that have died. There are actually two groups of midbrain DA neurons in the human brain. Those from the substantia nigra (SN) are highly susceptible to degeneration in Parkinson's patients while those from the ventral tegmental area (VTA) are not. These two types of neurons have similar features but have different functions and it is important to ensure that DA neurons from hESC are the correct SN type before they are used in therapy. The primary goal of this research was to study these two neuronal types in animals and determine if the distinguishing features discovered in mice or rats can be used to more easily recognize and purify SN-type DA neurons made from hESC. One of the discoveries made in this research is that SN and VTA neurons show differences in how they make connections within the brain. We have been able to identify some of the molecules that guide each neuron to connect to it appropriate target and have found that SN and VTA neurons placed in the petri dish can be distinguished from each other by their response to guidance molecules. Work in the final period of this grant has focused on testing guidance response in hESC-derived DA neurons and we have found that many of the neurons produced from hESC do show SN-like responses to guidance molecules. This discovery is being further developed as a screening tool to help guide our ongoing efforts to make increasingly pure populations of DA neurons from hESC. Future human trials will likely utilize such DA neurons but since embryonic stem cells have the potential to develop into any type of cell in the body, it is important to ensure that the production methods used to make a therapeutic product for Parkinson’s disease do indeed specifically produce SN neurons. Prior to the research supported under this CIRM grant, the scientific community was not able to distinguish SN from VTA neurons outside of their normal brain environment and therefore had no ability to confirm whether a method produced one type selectively and not the other. Further refinements of the assay tools developed in our research may provide a practical means of quantifying the purity of a DA neuron preparation. This would have a significant impact transplantation therapy as well as provide useful insights into the molecular mechanisms that underlie proper connectivity and function of SN and VTA DA neurons in humans.

© 2013 California Institute for Regenerative Medicine