Graduation Date

Fall 2024

Document Type

Thesis

Program

Master of Science degree with a major in Biology

Committee Chair Name

Dr. Amy Sprowles

Committee Chair Affiliation

HSU Faculty or Staff

Second Committee Member Name

Dr. Kyle Fink

Second Committee Member Affiliation

Community Member or Outside Professional

Third Committee Member Name

Brigitte Blackman

Third Committee Member Affiliation

HSU Faculty or Staff

Fourth Committee Member Name

Dr. Jenny Cappuccio

Fourth Committee Member Affiliation

HSU Faculty or Staff

Keywords

Organoid, CDKL5, Stem Cells, Cell Modeling, Immunohistochemistry, Electrophysiology, RT-qPCR, Neuroscience, Neurodevelopmental Disorder

Subject Categories

Biology

Abstract

CDKL5 deficiency disorder (CDD) is an X-linked disorder that leads to severe cognitive impairments as well as infantile epilepsy. The only FDA approved therapy helps to reduce seizure frequency, however there are currently no cures approved. While murine models have proven useful in characterizing CDD, there are limitations due to differences in brain structure and development across species that may lead to the reported lack of spontaneous seizures in the mouse, a hallmark in humans affected by this disorder. By the utilization of patient-specific iPSCs generated into cerebral organoids, a more comprehensive and in-depth analysis into rare neurological disorders, such as CDD may be better understood. This project focused on phenotypic characterization of the c.1412delA patient-derived iPSC line that was differentiated into neuronal stem cells and cerebral organoids. This cell line was sorted for the X-allele expressing a CDKL5 mutation and the X-allele expressing the healthy allele (isogenic control). CDKL5 deficient organoids were labeled as CDD, and the healthy isogenic control was labeled as ISO. The cellular composition of the organoids was assessed with immunohistochemistry and gene expression analysis was evaluated with RT-qPCR. CDD cerebral organoids tended to express more markers associated with early stem cell/neural progenitor fate, while healthy organoids (ISO) tended to express mature markers at higher rates. To functionally characterize CDD disease phenotype using cerebral organoids, electrophysiological data was collected using the MaxTwo high-density microelectrode arrays (HD-MEA) system, which quantifies and analyzes electrical activity of cells. MaxTwo recordings resulted in ISO organoids with a significantly higher active area percentage, a higher number of bursts, burst peak, and burst duration when compared to CDD.

Citation Style

APA

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