Exploring the Potential of Human Pluripotent Stem Cells as a Platform for New ALS Disease Models

Human embryonic stem cells (ESCs) are pluripotent and can serve as a source of human cell types otherwise unobtainable for in vitro study. Recently, reprogramming technology has evolved to allow for the routine derivation of induced pluripotent stem cells (iPSCs) from primary human fibroblasts. By deriving iPSCs from patients with particular conditions, we are able to produce adult cell types in vitro that are genetically identical to the patient and carry any disease causing mutations. The three studies presented in this thesis have sought to understand the utility and potential of these two types of human pluripotent stem cells for disease modeling of amyotrophic lateral sclerosis (ALS). Previous studies in ALS animal models provided evidence that motor neurons, the cell type specifically lost during disease, are compromised through cell autonomous pathology, as well as through toxic interaction with non-neuronal cells of the nervous system. In our first study we demonstrate that human ESCs can serve as a robust source of human motor neurons, which are selectively sensitive to a non-cell autonomous toxicity of primary mouse glia derived from an ALS mouse model. This study confirmed the relevance of previous findings using mouse ESC-derived cultures toward human disease. Using a human motor neuron reporter ESC line that we created for these experiments, we went on to demonstrate how this culture system could be used to identify pathways that modulate glial toxicity. However to study cell autonomous disease processes in human motor neurons, patient-specific ALS iPSC lines would have to be employed. Several iPSC lines from healthy and ALS patients were already available but a comprehensive investigation of their quality and consistency compared to ESCs would be needed to move forward. In our second study we present a cohort of 16 fully characterized human iPSC lines from both healthy patients and from two ALS patients. This study determined that while all pluripotent stem cell lines exhibit reproducible individual behaviors in culture, overall iPSC and ESC lines are similarly capable of generating electrophysiologically active motor neurons for in vitro studies. Additionally we found that among several variables tested for their impact on differentiation ability of iPSC lines only sex and donor of origin significantly correlated with efficiency of motor neuron production. These findings suggest iPSCs can be useful sources of patient-specific motor neurons for ALS disease modeling, as long as possible non-disease related differences are controlled for when multiple cell lines are used for comparative studies. To illustrate this need, we present in a third study, our initial attempts at survival comparisons between motor neurons from healthy and ALS iPSC lines. By the gross measure of change in motor neuron population size over time, we investigate the possible existence of phenotypic differences due to the cell autonomous effects of ALS causing mutations carried by these cells. Although cell line variability complicates analysis of these data, trends suggesting ALS-specific deficiency in motor neuron population growth provide impetus for further study. Taken together, these studies provide a proof of principle for the use of human pluripotent stem cell derived motor neurons for in vitro disease models. They also help us to conclude which aspects of assay design must be optimized to enable complex comparisons of disease processes in multiple forms of ALS using patient-specific motor neurons. These advances provide a framework for mechanistic studies and investigation of therapeutic intervention in human in vitro models of ALS.