Graduation Date

Spring 2020

Document Type

Thesis

Program

Master of Science degree with a major in Biology

Committee Chair Name

John W. Steele

Committee Chair Affiliation

HSU Faculty or Staff

Second Committee Member Name

Amy Sprowles

Second Committee Member Affiliation

HSU Faculty or Staff

Third Committee Member Name

Brigitte Blackman

Third Committee Member Affiliation

HSU Faculty or Staff

Fourth Committee Member Name

John Reiss

Fourth Committee Member Affiliation

HSU Faculty or Staff

Subject Categories

Biology

Abstract

Human induced pluripotent stem cells offer a model for human brain development and disease by differentiation into brain organoids; however, current neural culture systems lack the microenvironment, neuronal circuits and connectivity, vascular circulation, and immune system that exist in vivo. After differentiation and development of neuronal and non-neuronal cell types within two formats of cell cultures, we can visualize and recapitulate in vivo protein accumulation, gene expression, and degradative processes such as autophagy. Using RNA extraction, purification methods and reverse transcription I compared traditional monolayer cultures and novel 3-D neural sphere cultures via gene expression analysis. This analysis indicated variable gene expression between formats therefore only monolayer cultures were analyzed for tau protein accumulation with pharmacologic treatments and measured by Western blot. I also report on cell type specific gene expression by transient transfection of plasmid cassette tools and fluorescent microscopy. Here, cell type specific gene targeting is demonstrated in successfully transfected cells. Further development of the tools utilized in this study will significantly expand the field of neurodegenerative research, by giving us the ability to target specific cell types within mixed cultures, and allowing for a more accurate depiction of pathogenesis within diseased cell types.

Citation Style

CBE

Supplemental Gene Table S1.xlsx (27 kB)
Primer sequences and additional information

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