Title: Human Stem Cells for Modeling Amyotrophic Lateral Sclerosis Disease Mechanisms and Modifiers
Description: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor system. Although ALS has been extensively studied in post-mortem patient samples and animal models, there are currently no very effective treatments and there is no cure. One reason for the lack of treatment options in ALS may stem from the inaccessibility of living human motor neurons for use in disease research and subsequent therapeutic target validation. Recent developments in the field of stem cell biology can potentially provide access to living human motor neurons from individual ALS patients. It is now possible to derive induced pluripotent stem cells (iPS cells) from the somatic tissues of ALS patients and then to differentiate these iPS cells into motor neurons with the precise genetic makeup of the donor patient (iPS-MNs). Before iPS-MNs can be put to productive use, however, the iPS system as a whole must be validated as a reliable source of motor neurons with characteristics that closely resemble their endogenous or hES-derived counterparts.

This thesis will first address a series of issues relating to the validation of iPS cells as a reliable source of motor neurons a then move on to expression profiling studies aimed at identifying a transcriptional signature of ALS in iPS-MNs. I will first describe a collaborative study aimed at determining whether or not iPS cells are as useful as ES cells for the production of motor neurons. By comparing motor neuron differentiation efficiency across a panel of 6 ES lines and 16 iPS lines, we demonstrated that iPS cells are equally capable of producing electrophysiologically active motor neurons as ES cells. Moreover, both ALS and control iPS lines produce motor neurons with equal efficiency, suggesting that iPS cells will be useful in the production of ALS iPS-MNs for disease research. In addition, our results identify some of the variables that contribute to differentiation efficiency, including donor identity and individual iPS/ES line identity. The following section will serve to provide a deeper molecular and electrophysiological understanding of human stem cell-derived motor neurons.

I first generated expression profiles from purified hES-MNs to identify potential motor neuron-specific surface markers as well as maturational changes occurring in motor neurons in vitro. Using calcium imaging techniques, I then demonstrated that iPS-MNs behave functionally similarly to ES-MNs and described culture-wide rhythmic depolarizations that are likely influencing multiple properties of iPS-MNs. After characterizing the iPS-MN culture system, I made a first attempt at defining the transcriptional phenotypes of ALS in iPS-MNs. This work relied on the use of a motor neuron-specific lentiviral reporter that I developed to isolate and transcriptionally profile iPS-MNs from two control iPS lines and four ALS iPS lines. I show evidence of significant transcriptional differences between motor neurons isolated from ALS lines and those from control patients. These differences may in the future help to define ALS-specific phenotypes. Lastly, I conducted a meta-analysis comparing transcriptional changes in ALS iPS-MNs to those in existing models of ALS and identified some common stress-related features of ALS in iPS-MNs.

In order to form new hypotheses about what sorts of individual patient-specific phenotypes may be present in iPS-MNs, I will then utilize published expression profiles from post-mortem ALS patient motor neurons to identify a previously-overlooked class of genes that exhibit expression levels highly correlated with individual age at ALS onset. This group of 43 onset-correlated genes contains many members with known or hypothesized relationships to neurodegenerative disease. I discuss how onset-correlated genes may function as disease-modifiers or biomarkers and design experiments to investigate these possibilities. Taken together, the work in this thesis will lay the foundations for developing a human iPS-based model of ALS and point toward numerous avenues of future investigation.