Date of Completion

11-7-2013

Embargo Period

11-1-2015

Keywords

Genetics, neuroscience, neural differentiation, stem cells

Major Advisor

Xue-Jun Li

Associate Advisor

James Li

Associate Advisor

Nada Zecevic

Associate Advisor

Ren-He Xu

Associate Advisor

Alexander Lichtler

Field of Study

Genetics and Genomics

Open Access

Campus Access

Abstract

The specification of various neuronal subtypes from pluripotent stem cells is critical for better understanding neuron subtypes, disease modeling, and for screening therapeutics. The experiments described in this thesis were designed in order to determine how to specify both deep layer cortical and nociceptive neurons from stem cells using knowledge gained through genetic and developmental studies. Both of these neuron types play critical roles in the lives of humans, are not well characterized, and have been linked to diseases with genetic origins. The importance of particular genes (Fezf2, Neurog1) which were previously implicated to play a role in the specification of these neuron subtypes have also been and continue to be evaluated using this system.

In humans, cortical spinal motor neurons (CSMNs) enable voluntary movement, are harmed during spinal cord injuries, and are degenerated in diseases such as ALS. We sought to generate deep layer cortical neurons from mESCs. To do this, we evaluated the role of Fezf2 and found that when it was overexpressed at early time points, it significantly increased the generation of rostral forebrain progenitors. This was partially achieved by the regulation of Wnt signaling. The opposite effect was seen in the Fezf2 knockdown cells. It was also found that Fezf2 promotes the generation of dorsal telencephalic progenitors and deep-layer cortical neurons at later stages.

Nociceptive neurons play an essential role in the sensation of pain as they are responsible for transmitting painful stimuli to the nervous system. However, a major limitation in studies to determine mechanisms of action as well as potential therapeutics in the human system has been a lack of nociceptive neurons to evaluate. However, generating them from hESCs would yield an unlimited supply to study and screen drugs on. When morphogens (BMP4, RA) were added at specific doses and time points, it yielded a high population of neural crest progenitor cells. When Neurog1 was then over-expressed at a particular time point in these cells, it further promoted the specification of the nociceptive lineage which resulted in a population of mature nociceptive neurons as demonstrated by immunostaining and their responsiveness to capsaicin.

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