Funding opportunities

In vitro differentiation of hESCs into corticospinal motor neurons

Funding Type: 
SEED Grant
Grant Number: 
RS1-00170
Principle Investigator: 
Funds requested: 
$500 000
Funding Recommendations: 
Recommended if funds allow
Grant approved: 
Yes
Public Abstract: 
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurological disease that leads to the degeneration of motor neurons in the brain and in the spinal cord. There are currently 20,000 ALS patients in the United States, and 5,000 new patients are diagnosed every year. Unfortunately no cure has been found for ALS. The only medication approved by the FDA to treat ALS can only slow the disease’s progression and prolong life by a few months in some patients. Thus it is critical to explore other therapeutic strategies for the treatment of ALS such as cell replacement strategy. Because of the ability to generate many different cell types, human embryonic stem cells (hESCs) may potentially serve as a renewable source of cells for replacing the damaged cells in diseases. However, transplanting ESCs directly may cause tumor growth in patients. To support cell transplants, it is important to develop methods to differentiate hESCs into the specific cell types affected by the disease. In this application, we propose to develop an effective method to differentiate hESCs into corticospinal motor neurons (CSMNs), the neurons in the cerebral cortex that degenerate in ALS. We will test whether these CSMNs generated from hESCs in culture conditions can form proper connections to the spinal cord when transplanted into mouse brains. To direct hESCs to become the CSMNs, it is critical to establish a reliable method to identify human CSMNs. Recent progress in developmental neuroscience have identified genes that are specifically expressed in the CSMNs in mice. However no information is available for identifying human CSMNs. We hypothesize that CSMN genes in mice will be reliable markers for human CSMNs. To test this hypothesis we will investigate whether mouse CSMN markers are specifically expressed in the human CSMNs. The therapeutic application of hESCs to replace damaged CSMNs in ALS depends on the ability to direct hESCs to develop into CSMNs. Currently a reliable condition to direct hESCs to differentiate into CSMNs has not been established. We will attempt to differentiate hESCs into CSMNs based on the knowledge gained from studying the development of nervous system. We will achieve this goal in two steps: first we will culture hESCs in a condition to make them become progenitors cells of the most anterior region of the brain; then we will culture these progenitors to become neurons of the cerebral cortex, particularly the CSMNs. We will study the identities of these neurons using the CSMN markers that we have proposed to identify. To apply the cell replacement strategy to treat ALS, it will be critical to test if human CSMNs generated from cultured hESCs can form proper connections in an animal model. We will transplant the CSMNs developed from hESCs into the brains of mice and test whether they can form connections to the spinal cord. When carried out, the proposed research will directly benefit cell replacement therapy for ALS.
Statement of Benefit to California: 
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurological disease that leads to the degeneration of motor neurons in the brain and in the spinal cord. There are currently 20,000 ALS patients in the United States, and 5,000 new patients are diagnosed every year. Unfortunately no cure has been found for ALS. The only medication approved by the FDA to treat ALS can only slow the disease’s progression and prolong life by a few months in some patients. Thus it is critical to explore other therapeutic strategies for the treatment of ALS such as cell replacement strategy. Because of the ability to generate many different types of cells, human embryonic stem cells (hESCs) may potentially serve as a renewable source of cells for replacing the damaged cells in diseases. However, transplanting ESCs directly may cause tumor growth in patients. To support cell transplants, it is important to develop methods to differentiate hESCs into the specific cell types affected by the disease. In this application, we propose to develop an effective method to differentiate hESCs into corticospinal motor neurons (CSMNs), the neurons in the cerebral cortex that degenerate in ALS. We will test whether these CSMNs generated from hESCs in culture conditions can form proper connections to the spinal cord when transplanted into mouse brains. Everyday, 15 people die from ALS. For patients diagnozied with ALS, time is running out very fast. It is critical to explore novel therapeutic strategies for this rapidly progressive and fatal disease. The research proposed in this application may provide the basis for a novel cell replacement therapy for ALS, thus it will greatly benefit the State of California and everyone in the State.
Review Summary: 
SYNOPSIS: In this proposal an attempt will be made in-vitro to guide hESC towards corticospinal motor neuron (CSMN) differentiation. A connection between the mouse work from a few labs to human CSMN biology will also be tested. Three questions will be addressed in this proposal: 1) Is Fez-like biology functional in humans, 2) Are focused markers of differentiation pathways turned on when hESC are differentiated into CSMN, and 3) Can new neurons be formed in Fez-like knockout mice that themselves do not form them? SIGNIFICANCE AND INNOVATION: Relatively little attention has been given to characterizing the differentiation and development of CSMNs compared to spinal motor neurons, perhaps at least partly because of the absence of markers for these cells. A greater knowledge concerning these cells and ways to derive them from hESCs may increase our knowledge of the motor system as well as our understanding of the pathogenesis and treatment of various motor neuron disease, such as amyotrophic lateral sclerosis. For these reasons, the topic of the proposal is a significant one. The focus on methods of deriving CSMNs from hESCs and the planned transplantation of these cells into Fezl-/- mice to look at their functional capacities are novel directions and approaches. This is important yet risky work, that will provide important foundations if successful, and will likely give useful negative data even if not fully achieved. STRENGTHS: This proposal is based on strong biology, and attempts to be at the forefront of the field of directed differentiation of ES cells. The efficient differentiation of hESCs into CSMNs is an important goal, and the PI, a newly appointed Assistant Professor in the Department of Molecular, Cellular, and Developmental Biology at UCSC, has experience in transplantation procedures into rodents as well as issues related to CSMNs. The plan of the experiments is logical, progressing from the identification of markers to differentiation to transplantation. The PI also addresses some anticipated problems/pitfalls related to the planned experiments. The preliminary data of the PI with respect to Fezl and the generation of the Fezl-/- mouse are interesting and lend support to the planned studies. WEAKNESSES: This is a risky proposal, and the PI would have benefited from collaborations with others who are expert in the differentiation of ESCs. A failure to identify appropriate markers for CSMNs will make Specific Aim II difficult to achieve, and a failure to differentiate CSMNs will make Specific Aim III impossible to achieve. If the three markers listed by the PI under Specific Aim I are not relevant to human CSMNs, what will the PI do? The description of the roles of Fezl and others are a bit oversimplified from what is known about the need for combinatorial controls. Further details related to the transplantation and other experimental details would have been valuable. For example, how were the times of sacrifice (3 weeks to 3 months) determined? Also, in Specific Aim III it would be valuable to stain the transplanted knockout animals for Fezl. DISCUSSION: ALS is an important disease focus, and focusing on one of the two neuron types that die is important. A key drawback to this proposal is the lack of collaborators with a track record in the embryonic stem cell field and culture. The PI is well trained, and the primary author on a very relevant publication in PNAS, but has only produced one paper since receiving a PhD in 2000. The approach is somewhat naïve in that the description of differentiation into CSMN does not include a discussion of possible combinatorial approaches (Fezl and other genes). Also, there is human homology for Fez-like, but whether it will be a marker is unclear. Aim 3 would produce a very nice knockout mouse model, but it will take a lot to get there. One reviewer commented that the applicant should go slower and more stepwise, and stick to the fundamental biology. PROGRAMMATIC REVIEW: In programmatic review, it was noted that this is a young investigator working in the area of coticospinal motor neurons which degenerate in ALS. It was also noted that based on the scientific score, this investigator was near the top of the 'Recommended if funds available' pool. It was agreed that this investigator be moved to a 'Special funding pool' that would have priority for funding if additional monies became available.
Conflicts: 

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