Characterization Of a Human Dorsal Root Ganglion Organoid Patterning Recipe Restricted; Files Only

Abstract

 Human dorsal root ganglion (DRG) organoids derived from human induced pluripotent stem cells (hiPSCs) provide an in vitro model to study sensory neuron development and pain related disorders. This study compared three DRG organoid recipes by varying the timing of 5 fluorodeoxyuridine (FUDR) application to arrest progenitor proliferation at different time points: Regular (d15, d22), Delayed (d29, d36), and No FUDR (No FUDR treatment). Because progenitor cells switch from neurogenesis to gliogenesis during development, arresting proliferation at different timepoints may interrupt one of these 2 processes, resulting in different proportions of glial cells and neurons (such as nociceptors, proprioceptors and mechanoreceptors) in the resultant organoids, which may be of consequence to modelling disease. Quantitative PCR (qPCR) revealed that neural progenitor markers for all recipes, such as SOX10 peaked early, while glial markers such as FABP7 and GFAP increased over time, which was consistent with model of DRG development reflecting the expected gliogenic switch. The Delayed FUDR condition showed significantly higher expression of ISL1 (a neuronal marker) at d63, which might suggest extended neurogenesis. Microelectrode array (MEA) recordings at d120 showed higher baseline firing rates in the Delayed FUDR condition, supporting this claim. Exposure to capsaicin, a nociceptor agonist, showed no significant differences in capsaicin-induced responses across conditions, suggestive of low nociceptor activity. These results highlight how modifying FUDR timing alters cellular composition and activity in DRG organoids – the Delayed FUDR group was found to have increased neuronal activity and expression of neural markers relative to the other group, warranting further investigation into the precise mechanism why and other ways in which the functionality of this recipe may differ relative to the other groups. 

Table of Contents

Contents

1. Introduction ........................................................................................................... 1

Figure 1 ..................................................................................................................... 3

Figure 2 ..................................................................................................................... 6

Figure 3 ..................................................................................................................... 8

2. Experimental Approach & Hypothesis .................................................................... 11

Table 1 .................................................................................................................... 11

3. Methods .............................................................................................................. 12

Table 2 .................................................................................................................... 16

4. Results ................................................................................................................ 18

Figure 4 ................................................................................................................... 18

Figure 5 ................................................................................................................... 20

Figure 6. .................................................................................................................. 25

5. Discussion .......................................................................................................... 26

6. Conclusions & Future Directions ........................................................................... 30

7. Limitations .......................................................................................................... 31

References ................................................................................................................. 33

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Genetics Biology, Neuroscience
Palavra-chave	stem cell dorsal root ganglion organoid patterning
Committee Chair / Thesis Advisor	Jimena Andersen, Emory University
Committee Members	Andreas Fritz, Emory University Fikri Birey, Emory University

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