Development of a novel human embryonic stem cell (hESC) model of familial amyotrophic lateral sclerosis (ALS)

Funding Type: 
SEED Grant
Grant Number: 
ICOC Funds Committed: 
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
ALS is a devastating disorder that lacks effective treatments. Although animal models of the disease have provided some insight into disease mechanism, therapeutic agents identified in animal studies have failed to be useful in ALS patients. Therefore, new human disease models are necessary that take advantage of recently developed high-throughout research methods to advance basic research and the discovery of new therapies for ALS. This proposal aims to develop and investigate a new and unique model of ALS. Human embryonic stem cell (hESC) lines expressing sod-1 mutants linked to familial ALS will be generated for the first time. The following specific aims will be accomplished: 1. A viral vector, will be used to express human wild type or mutated G93A, A4V or I113T sod-1 DNA and a fluorescent protein as a reporter for sod1 expression. An expression enhancer will ensure that SOD-1 and the fluorescent protein are only expressed in cells differentiating to neurons, thereby allowing an easy identification of SOD-1 expressing nerve cells and motor neurons. Cells are differentiated to motor neuron lineage using established protocols. 2. An initial characterization of the new sod-1 transgenic cell lines will include studies of SOD-1 expression, cell death and cell structure to analyze the effect of the mutants on the differentiating and mature motor neurons. 3. A comprehensive high-throughput screen of thousands of small pharmacological compounds will be performed with the goal to identify new therapies for ALS. This hESC model is extremely versatile because it allows the generation and maturation/aging and subsequent examination of cell types, such as motor neurons and the cells surrounding motor neurons (glia) that are affected by ALS. This proposal addresses the major cell type involved in ALS pathology, motor neurons, and outlines a pharmacological screen. Beyond the scope of this proposal, this hESC model holds the promise of learning much more about cellular pathways involved in ALS. The cell lines can be studied at any stage during cell differentiation. The proposed hESC lines will provide an unprecedented tool not only for examining early cellular changes leading to ALS-related cell loss. They will also present a new tool of studying crucial interactions of motor neuron with the cells surrounding them, which are a key to understanding the complex disease mechanisms of ALS. The overall long-term goal of this project is to provide these cell lines to the ALS research community to advance basic and therapy-oriented research for the benefit of ALS patients. The PI has experience with ALS and frontotemporal dementia research, but is new to hESC research. Two collaborators have joined the team, Dr. Harley Kornblum and Dr. Steven Goldman, to provide expert advise and support the successful completion of the project. A postdoctoral fellow, who has proven his skills in the required cell culture techniques, completes the capable team.
Statement of Benefit to California: 
This proposal aims to advance ALS research and help to bring about desperately needed new therapies for ALS patients in California. To accomplish this goal, a new human embryonic stem cell (hESC) based cell line will be generated and investigated. This first hESC model of ALS will be useful for innumerous studies that explore the mechanisms underlying ALS and search for new therapies. This work will originate in the state of California, thus strongly supporting the notion that the state of California is a driving force in hESC research in the United States. The derived hESC lines will be shared and used by other laboratories in the country, their California origin will always place a spotlight on the research support that the state of California gives to this cutting-edge technology and on the research accomplishments that can be achieved in this state. As the proposed ALS cell lines will be a versatile and useful tool in ALS research, it is expected that subsequently funding mechanisms from federal and private sources will be obtained for future projects involving the lines. In that case, the state of California would have jump-started an innovative and groundbreaking use of human stem cell technology modeling ALS. In addition, the advancement of biotechnology through future expansion of the proposed project will likely lead to increased hiring of laboratory personnel and the potential of developing therapeutic products with subsequent tax revenues. This specific project will significantly enhance basic ALS research in California by supporting cutting-edge stem cell technology in an institution with a dedicated ALS clinic. California has only very few ALS basic research groups, and no group that combines basic research with clinical ALS work. The combination of ALS basic research and clinical work would be unique and have the potential to give California a leading edge in combining basic and clinical ALS research that is currently held by a few East Coast institutions.
Progress Report: 
  • Human embryonic stem cells (hEScs) are derived from embryos early in development before their fate has been determined, and can differentiate into all of the cell types of the body. By exploiting the potential of hESCs to differentiate into multiple lineages medicine stands to benefit enormously. To do so requires a comprehensive understanding of the optimal conditions to grow and differentiate these cells without inducing tumors. What is clear is that the physical, three dimensional (3D) microenvironment in which the hESCs reside, regulates directly or indirectly their tissue-specific differentiation. hESc make physical contact with both an insoluble extracellular scaffold termed the extracellular matrix (ECM) and to other cells through specific proteins (receptors) on their own surface. These interactions are responsible for the structural integrity of the body and are at the center of structural transformations that characterize embryogenesis. While this structural role has long been appreciated it has only recently been shown that these points of contact can be the focus of transmission of mechanical forces originating within and outside the cell and that these forces can be converted into the more familiar signals that influence cell fate decisions. A major goal of our work is to define how these cell derived and externally transmitted forces might regulate hESC behavior. Our hypothesis is that these mechanical forces alter hESC fate by regulating the activity of enzymes called RhoGTPases, that are strongly implicated in ESC behavior. During this past year we have optimized the preparation and culture of hESc on a two dimensional (2D) synthetic matrices of defined composition and stiffness that recapitulate the range of mechanical environments hESC experience during embryogenesis as well as those unnaturally stiff mechanical environments that are currently used for their propagation. For these studies we used inert acrylamide gels cross linked with an ECM molecule (laminin) important in early embryonic development. On 2D surfaces feedback loops originating from cell derived contractile forces sense the mechanical “give” of the surface, and dynamically change their organizational and signaling state both at the single cell level and the multicellular level. Using these 2D gels we have defined the range of mechanical environments in which hESc exhibit mechanosensitivity. We have also shown that this causes changes in their external and internal organizational states across a range of scales from the subcellular (becoming stiffer on stiffer substrates), to the cellular (spreading more on stiffer substrates) to the multicellular (compacting under enhanced cell-cell adhesion on softer substrates). Surprisingly, we have found that the standard (very stiff) substrates that are used routinely for maintenance of a non-differentiated state (pluripotent self renewal) are mechanically suboptimal: hESc have higher rates of cell death and lower rates of growth than on surfaces orders of magnitude softer (operationally termed mid range). At the softest end, although we have found that hESc largely maintain pluripotential self renewal they show signs of either low level differentiation or a move towards a state poised to differentiate. This suggests that precise control of the mechanical environment is an important parameter in the establishment of safe and effective propagation of hESc for regenerative medicine and might be exploited for directed differentiation. To complement these studies we are also developing approaches to imparting external mechanical forces to hESc growing in a 3D context. We have both established novel and robust protocols for the efficient encapsulation of hESc in 3D deformable hyaluronic acid (HA) hydrogels and shown that they support pluripotent self renewal and constructed a bioreactor that will impart oscillatory and static compressive loads to hESCs in these gels. We anticipate that these studies will further illustrate the role(s) by which mechanical forces influence hESC fate and provide additional insight into the underlying molecular mechanisms.

© 2013 California Institute for Regenerative Medicine