Basic Biology III
$1 355 063
A major goal of stem cell research is to generate various functional human cell types that can be used to better understand how these cells work and to use them directly in therapies. There are currently no effective treatments, let alone a cure, for many neurological conditions. Two particular devastating neurological conditions, spinal cord injury and amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease) share a common element. That is, in both conditions, the corticospinal motor neurons that control skilled voluntary movement are severely damaged, leading to significant loss of motor control. There has been extensive research on spinal cord injury and ALS in recent years. In the field of spinal cord injury, much effort has been devoted to repairing the damaged nerve paths, but this has turned out to be extremely challenging. The work on ALS, on the other hand, has mostly focused on the spinal motor neurons (often referred to as the lower motor neurons in the context of ALS). Our proposed study focuses on the corticospinal motor neurons (or the upper motor neurons) and, more broadly, the subcerebral projection neurons. Taking clues from studies in mice, we aim to understand how the subcerebral projection neurons including the corticospinal motor neurons can be made from human embryonic stem cells. We will focus on the later steps in differentiation that are not well understood, which gave rise to different types of neurons in the cerebral cortex. To aid in this process, we have engineered a fluorescent reporter in human embryonic stem cells, which, when the stem cells are turned into corticospinal motor neurons and related subcerebral projection neurons, will light up – literally. We will probe the molecular control of this process and determine if corticospinal motor neurons made in a culture dish, when introduced back into an organism, can send projections to the spinal cord, as they would normally do during development. Most of our knowledge about the development of corticospinal motor neurons comes from studies with mouse models. As there are likely to be important differences between humans and mice, we will pay special attention to the similarities and differences between mouse and human corticospinal motor neurons. Knowledge gained from this study will pave the way to make better disease-models-in-a-dish for neurological conditions such as ALS and to develop therapies for ALS, spinal cord injury, traumatic brain injury, stroke and other neurological conditions when corticospinal motor neurons are damaged.
Statement of Benefit to California:
Neurological conditions affect millions of Californians each year. Spinal cord injury is one particularly debilitating neurological condition. The disability, loss of earning power, and loss of personal freedom associated with spinal cord injury is devastating for the injured individual, and creates a financial burden of an estimated $400 million annually for the state of California. Research is the only solution as currently there is no cure for spinal cord injury. A major functional deficit for patients of spinal cord injury is the loss of motor control. Corticospinal motor neurons mediate skilled, voluntary movement in humans and damage to these neurons leads to severe disability. Our proposed study focuses on the understanding of how corticospinal motor neurons and, more broadly, subcerebral projection neurons can be made from human embryonic stem cells under culture conditions, and how they can be introduced back to central nervous system. Understanding this process will allow scientists to design ways to use these cells for transplantation therapies not only for spinal cord injury, but also for other neurological conditions such as amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease). Effective treatments promoting functional repair will significantly increase personal independence for people with spinal cord injury and decrease the financial burden for the State of California. More importantly, treatments that enhance functional recovery will improve the quality of life for those who are directly or indirectly affected by spinal cord injury, ALS and other neurological conditions.
Project Synopsis: In this proposal, the Principal Investigator (PI) will generate and characterize corticospinal motor neurons (CSMNs), derived from human embryonic stem cells (hESCs). CSMNs are of interest because the axonal projections of these neurons form the corticospinal tract, which controls skilled voluntary movements in humans, and could be a cell type important for cellular therapies in Amyotrophic Lateral Sclerosis (ALS) and spinal cord trauma patients. To facilitate this study, the PI has engineered a reporter that recapitulates the expression of a specific gene and thus fluoresces as hESCs differentiate into CSMNs and related neurons. In Aim 1, the PI plans to purify and profile reporter-expressing neurons derived from hESCs at different stages of differentiation to assess molecular identity. Then in Aim 2, the PI proposes to conduct a functional analysis of candidate transcription factors that may regulate human CSMN fate specification and screen for small molecules that promote human CSMN fate in vitro. Finally in Aim 3, the PI intends to perform an in vivo evaluation of CSMN and related neuron fate by transplanting the reporter-expressing cells into the mouse brain. Significance and Innovation: - Reviewers considered this project to have high potential impact. Mechanisms controlling specification of CSMNs from hESCs are poorly understood and this project has the potential to substantially further this area. Uncovering these mechanisms is important for both fundamental research and translational approaches to central nervous system repair. - This application is highly innovative in both its design and use of tools. - The application has a logical, sound rationale. Feasibility and Experimental Design: - The application is extremely well written, the experimental plan is clearly articulated, specific aims are logical and achievable, and the proposal is scientifically sound. - The primary and critical weakness is the reliance of the project on the reporter’s specificity toward CSMN fate. If reporter expression is not associated with CSMNs, this will be a major problem. Some reviewers felt the rationale for focusing on the gene chosen for reporter design is experimentally justified and the preliminary data sufficiently mitigates the risk. Other reviewers were more skeptical and were concerned that the reporter protein might also mark earlier stage neural progenitor populations. - Overall, the preliminary data are detailed and sophisticated and clearly demonstrate the PI and research team have the knowledge and arsenal of tools required to perform the experiments described in the proposal. - The transplant studies are critical to understanding the in vivo phenotype of CSMNs and their feasibility is supported by the preliminary data, though additional data showing that purified human cells at relevant stages of differentiation survive transplantation would have been appreciated. Reviewers did suggest that the applicant could enhance the significance of potential findings by including post-natal tissue as a positive control and utilizing more quantitative outcome measures such as cell survival, cell migration, and axonal outgrowth. - The small molecule screen was an innovative addition. However, it was lacking in detail and poorly integrated into the proposal, making it difficult to judge the robustness of the assay or to determine how hits may be useful to the overall goals of the proposal. - The PI presents a good discussion regarding alternate plans. Principal Investigator (PI) and Research Team: - The PI is well trained with appropriate expertise, a productive publication record, and a good funding record. - The research team and collaborators are very strong and have the relevant experience to successfully undertake the project. Responsiveness to the RFA: - The application is entirely responsive to the RFA as it is focused on hESC fate determination during neural differentiation and the regulatory aspects underlying the developmental potential of neural progenitor cells.