Date Approved

5-1-2024

Graduate Degree Type

Thesis

Degree Name

Biomedical Sciences (M.H.S.)

Degree Program

Biomedical Sciences

First Advisor

Dr. Merritt DeLano-Taylor

Second Advisor

Dr. Dawn Hart

Third Advisor

Dr. Ruijie Liu

Academic Year

2023/2024

Abstract

Dopaminergic neuron generation by human embryonic stem cells is a difficult, lengthy, and expensive process yielding a small percentage of neurons that express dopaminergic characteristics. NATO3 is endogenously expressed in the floor plate of the developing nervous system during dopaminergic neurogenesis. PM-NATO3 is a novel, phosphorylating mimicking version of NATO3. Data from our lab shows PM-NATO3 can enhance multiple factors important for dopaminergic neurogenesis when it is expressed in the developing nervous system. This thesis studies how PM-NATO3 affects dopaminergic neuron generation by human embryonic stem cells in two different culture conditions. In one condition (called “enriched”) PM-NATO3 is expressed with pro-dopaminergic supplements using a commercially available kit. The other culture condition uses minimal media conditions (“minimal”) with an established neuron-generating factor (NEUROG2) and PM-NATO3 expressed by a TET-on inducible promoter. We measured key dopaminergic maturation markers through mRNA expression including NURR1, FOXA2, EN1, and TH. We also looked at protein expression through immunocytochemistry staining for nuclei, β3 tubulin, and TH in one of the conditions. We hypothesized that in both the enriched and minimal condition PM-NATO3 would be able to accelerate maturation of dopaminergic neurons and increase the yield of dopaminergic neurons. The results from this thesis do not show a significant increase in a broad spectrum of key dopamine neuron maturation markers in the PM-NATO3 containing conditions. However, some markers show near significant changes that may inform future investigation. Overall, these findings aim to provide a connection between dopaminergic neuron generation and the action of PM-NATO3. More experiments must be conducted in order to further elucidate the action of PM-NATO3 on human embryonic stem cells. Looking forward, this is an important area of 4 research when considering the potential implications in developing treatments for Parkinson's disease and potentially cell replacement therapy for patients.

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