Funding opportunities

Dopaminergic differentiation of hESCs: developing HTS for drug discovery and non-invasive tools for cell therapy strategy.

Funding Type: 
New Faculty II
Grant Number: 
RN2-00951
Funds requested: 
$2 969 718
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Parkinson's disease (PD) is a devastating movement disorder caused by the death of dopaminergic neurons (a type of neurons in the central nervous system) present in the midbrain. These neurons secrete dopamine (a signaling molecule) and are a critical component of the motor circuit that ensures movements are smooth and coordinated. All current treatments attempt to overcome the loss of these neurons by either replacing the lost dopamine, or modulating other parts of the circuit to balance this loss or attempting to halt or delay the loss of dopaminergic neurons. Cell replacement therapy attempts to use cells as small pumps of dopamine that will be secreted locally and in a regulated way and has been evaluated as a potential therapy for PD. Small molecules (e.g., GDNF) have also been tested to determine their efficacy in prolonging the survival or promoting sprouting of dopaminergic neurons in PD patients. Work in these fields has been limited however, partially due to the limited availability of cells for transplantation (e.g. 6-10 fetuses of 6-10 weeks post-conception are required for a single patient) and for high throughput drug screening. We believe that human embryonic stem cells (hESCs) may offer a potentially unlimited source of the right kind of cell required for cell replacement therapy. The fact that hESCs can be expanded and directed to produce specific cell types such as dopaminergic neuron makes it possible to develop cell-based screening models for drug discovery, for example, drugs that are protective to dopaminergic neurons. In this proposal we seek to understand how hESCs differentiate into authentic dopaminergic neurons using a culture method that we have tested. We believe that this differentiation can be broken into several stages and each of which is regulated by growth factors and other molecules. We also believe that these stages can be understood by selecting cells at each stage and comparing their properties using molecular tools and by examining their behavior after transplanting the cells into a rodent model of the disease. We also believe dopaminergic neurons produced from hESCs offer an unprecedented opportunity to develop screening models for assessing small molecule drugs and to clarify the mechanisms of disease. Indeed, we have shown in our preliminary studies that sufficient numbers of cells can be generated from hESCs to run such screening. Finally, we will need to develop a non invasive method of following cells after transplantation and we propose to develop a reporter line carrying a magnetic resonance imaging (MRI) label (and can be detected by MRI) based on previous successful work in mice to monitor dopaminergic neurons in animal models and possibly in future clinical trials. We believe that these experiments are critical to enhancing our understanding of the disease and providing the tools that will be necessary to move cell therapy to the clinic.
Statement of Benefit to California: 
We have proposed three aims in this proposal to identify factors that regulate the process of dopaminergic development and to develop reporter lines that allow us to screen large chemical libraries for drugs that promote dopaminergic differentiation or prolong the survival of dopaminergic neurons. We have also proposed to assess the long-term integration and differentiation of transplanted cells non-invasively in animal models that mimic the human disease using a magnetic resonance imaging (MRI)-based labeling system. We believe these experiments not only provide a proof-of-principle for moving Parkinson’s disease towards the clinic for people suffering with the disorder but also a generalized plan for the development of stem cell therapy and stem cell based drug discovery for multiple degenerative disorders. The tools and reagents that we develop will be made widely available to Californian researchers and we will select California-based companies for commercialization of such therapies. We hope that California-based physicians will be at the forefront of developing this promising avenue of research. We expect that the money expended on this research will benefit the Californian research community and the tools and reagents we develop will help accelerate the research of our colleagues in both California and worldwide.
Review Summary: 
Parkinson’s disease (PD) is a neurodegenerative disorder caused by the selective degeneration of midbrain dopaminergic neurons. The study plan proposed by the applicant outlines a series of aims to better define how stromal cells induce human embryonic stem cell (hESC) differentiation into authentic dopaminergic neurons in culture. In Aim 1, the PI proposes to identify and characterize novel factors that regulate dopaminergic differentiation in hESCs utilizing a defined system of dopaminergic differentiation induced by medium conditioned on stromal cells. In Aim 2, the applicant will develop dopaminergic reporter hESC lines and high throughput screening (HTS) assays for drug screening (e.g., compounds that specifically enhance dopaminergic differentiation or are protective for dopaminergic neurons). In Aim 3 the applicant proposes to develop a non-invasive method for monitoring long-term cell survival after transplantation into PD animals, including transplantation coupled with neuroprotective compounds identified in Aim 2, using a reporter ESC line carrying a magnetic resonance imaging (MRI) label. Overall reviewers found the research plan for this application superficial, overly ambitious and lacking in important details. In Aim 1, the PI does not provide convincing evidence that the cells generated under these conditions are indeed functional dopaminergic neurons. For example, using tyrosine hydroxylase (TH) as a marker is not sufficient as TH is readily induced under various conditions. The applicant cannot rule out that stromal conditioned media simply induces TH expression in hESC derived neural stem cells (NSCs) rather than inducing a midbrain dopamine (DA) neuron patterning response. In Aim 2, reviewers were enthusiastic about the generation of reporter ESC lines, however, the panel also felt that proposing to conduct HTS assays using these lines is premature and overly ambitious for this proposal, as reporter lines are notoriously difficult to establish and validate in hESCs and none of these reporter lines have been generated yet. The in vivo experiments in Aim 3 lacked details on many key parameters and the few details provided were not adequate (for instance, there was little discussion of an immunosuppression regimen or of behavioral testing). One of the main pitfalls in this last aim, however, is the fact that the PI does not provide preliminary data demonstrating adequate DA neuron survival after transplantation. Survival of TH positive cells is an absolute requirement for in vivo tracking. Overall the proposal is interesting theoretically but is likely to run into several problems that the applicant does not address carefully. The principal investigator (PI) has been an assistant professor at the host California institution since 2005. The PI has published a reasonable number of papers in the field and is dedicated to hESC research. However, most of the papers are in low impact journals and rather technical in nature. Moreover, the applicant has not yet obtained any NIH funding during his/her career. Reviewers found the mentoring and career development plans adequate. On the other hand, the review panel found it disappointing that there had not been more input from the mentors based on the considerable problems encountered in the research plan. Reviewers agreed that the applicant’s institution has a good track record for promoting young investigators and providing adequate resources. However, reviewers felt that the institutional commitment to the applicant has not been ideal to date. The PI is leading a core lab facility but has only 400 sq ft available for developing his/her own research. It seemed unclear to reviewers how and/or whether the institution really pushes the applicant’s independence or whether his/her primary role will remain more technical in nature, running the stem cell core facility, which is a considerable responsibility. In summary, the proposal is too ambitious and presents too many weaknesses in the research plan. Moreover, neither the applicant nor the institutional commitment convinced the review panel that the application meets the objectives put forth in the present RFA.
Conflicts: 

© 2013 California Institute for Regenerative Medicine