Genetic Encoding Novel Amino Acids in Embryonic Stem Cells for Molecular Understanding of Differentiation to Dopamine Neurons

Genetic Encoding Novel Amino Acids in Embryonic Stem Cells for Molecular Understanding of Differentiation to Dopamine Neurons

Funding Type: 
New Faculty I
Grant Number: 
RN1-00577
Award Value: 
$2,587,742
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Status: 
Closed
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 

Year 1

Patients with Parkinson’s disease have malfunctioning or dying dopaminergic (DA) neurons. Human embryonic stem cells can be differentiated into DA neurons for transplantation with the potential to cure this disease, yet the differentiation mechanism is not very clear. A nuclear hormone receptor named Nurr1 is found to regulate the differentiation process. To study the regulation mechanism, we proposed to genetically incorporate nonnatural amino acids into Nurr1 in stem cells, and use the novel properties of these amino acids to identify the interacting protein partners of Nurr1. Once these partners are discovered, effective protocols can be developed to generate high purity DA neurons for therapeutic purposes. In the past year, we made significant progress in genetically inserting nonnatural amino acids in stem cells. We are in the process of making stem cell lines that have this capacity. We also set up functional assays for studying Nurr1 and its mutants containing nonnatural amino acids. These results paved the way for our future identification of Nurr1 interacting networks in stem cells.

Year 2

Patients with Parkinson’s disease have malfunctioning or dying dopaminergic (DA) neurons. Human embryonic stem cells can be differentiated into DA neurons for transplantation with the potential to cure this disease, yet the differentiation mechanism is not very clear. A nuclear hormone receptor named Nurr1 is found to regulate the differentiation process. To study the regulation mechanism, we proposed to genetically incorporate nonnatural amino acids into Nurr1 in stem cells, and use the novel properties of these amino acids to identify the interacting protein partners of Nurr1. Once these partners are discovered, effective protocols can be developed to generate high purity DA neurons for therapeutic purposes. In the past year, we figured out several mechanisms that prevent the efficient incorporation of nonnatural amino acids into proteins in stem cells. We now have developed new strategies to overcome these difficulties. In the meantime, we developed another complementary approach in order to detect unknown proteins that interact with Nurr1 during the differentiation process of stem cells. We are employing these new methods to identify Nurr1 interacting networks in stem cells.

Year 3

Patients with Parkinson’s disease have malfunctioning or dying dopaminergic (DA) neurons. Human embryonic stem cells can be differentiated into DA neurons for transplantation with the potential to cure this disease, yet the differentiation mechanism is not very clear. The differentiation of embryonic stem cells to DA neurons has been found to be regulated by a nuclear hormone receptor Nurr1, but how Nurr1 involves in this complicated process remains unclear - no ligands or protein partners have been uncovered for Nurr1. To understand the regulation mechanism in molecular details, we proposed to incorporate non-natural amino acids into Nurr1 directly in stem cells, and use the novel properties of these amino acids to identify the protein partners of Nurr1. Once these partners are discovered, effective protocols can be developed to generate high purity DA neurons for therapeutic purposes. In the past year, we figured out a right solution for generating stem cell lines capable of incorporating non-natural amino acids. We also created a novel bacterial strain for efficient producing Nurr1 proteins with the non-natural amino acids inserted. With these progresses we are now probing proteins that interact with Nurr1 during the differentiation of stem cells, which should eventually enable us to come up with new strategies for making DA neurons from embryonic stem cells.

Year 4

Patients with Parkinson’s disease have malfunctioning or dying dopaminergic (DA) neurons. Human embryonic stem cells can be differentiated into DA neurons for transplantation with the potential to cure this disease, yet the differentiation mechanism is not very clear. The differentiation of embryonic stem cells to DA neurons has been found to be regulated by a nuclear hormone receptor Nurr1, but how Nurr1 is involved in this complicated process remains unclear - no ligands or protein partners have been uncovered for Nurr1. To understand the regulation mechanism in molecular details, we proposed to incorporate non-natural amino acids into Nurr1 directly in stem cells, and use the novel properties of these amino acids to identify the protein partners of Nurr1. Once these partners are discovered, effective protocols can be developed to generate high purity DA neurons for therapeutic purposes. In the past year, after testing numerous conditions in various cell lines, we discovered that photo-crosslinking is inefficient in capturing proteins interacting with Nurr1, possibly because the affinity between the unknown target protein and Nurr1 is too low. To overcome this challenge, we developed a new strategy of capture interacting proteins based on a novel class of non-natural amino acids, which do not require additional reagents nor external stimuli to function. We demonstrated the ability of these amino acids to crosslink proteins in the process of interacting with other proteins in live cells. We have also generated stable cell lines that are able to incorporate such non-natural amino acids. Using these new methods, we have been probing proteins that interact with Nurr1 during the differentiation of stem cells, which should eventually enable us to come up with new strategies for making DA neurons from embryonic stem cells.

© 2013 California Institute for Regenerative Medicine