Biology Ph.D. Dissertations

Stem-like cells and glial progenitors in the adult mouse suprachiasmatic nucleus

Date of Award

2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Michael Geusz (Advisor)

Second Advisor

George Bullerjahn (Committee Member)

Third Advisor

Howard Cromwell (Committee Member)

Fourth Advisor

Paul Morris (Committee Member)

Fifth Advisor

Pascal Bizarro (Other)

Abstract

Reports have described cells with stem-like protein expression in the hypothalamic suprachiasmatic nucleus (SCN), which contains the principal circadian pacemaker of the body. Additionally, there are oligodendrocyte progenitor cells (OPCs) scattered throughout the SCN and other brain areas with reported abilities to differentiate into neurons and glia. The SCN is a particularly good structure for studying adult neurogenesis because its cellular manipulation has known quantifiable effects on specific parameters of circadian rhythms. The objectives of this study were to characterize stem and progenitor cells in the SCN and to study neurogenesis from SCN OPCs in vitro. We first performed a meta-analysis to identify the expression of stem cell-related genes in the SCN and then used defined serum-free media for inducing stem and progenitor cell proliferation in SCN explant cultures, identified by immunocytochemistry and confocal microscopy. In the meta-analysis, we analyzed 25 genes associated with stem cell maintenance and increased motility, out of which over 90% were expressed at higher levels in the SCN than in other brain areas. In explant cultures maintained in stem and progenitor cell medium (SPM), cells expressed stem cell proteins: SOX2, nestin, MSI2 and OCT4. Explant cultures had ongoing mitotic activity and extensive cell loss. Despite neuronal loss, tissue remained viable for over 7 weeks in culture, as shown by bioluminescence imaging. The circadian rhythm in SCN gene expression persisted in brain slice cultures in SPM. SCN explants maintained in NeuralX medium supporting OPC proliferation, formed a cell monolayer and a suspended cell culture that included 87% OPCs. These cells were then induced to differentiate into neurons, which were identified by immunocytochemistry and electrical impulses recorded with microelectrode arrays. In differentiating cultures, a subset of OPCs formed oligodendrocytes that myelinated nascent neurons. These results provide evidence that the mature SCN has cells with regenerative properties providing plasticity that may enable circadian rhythms to adjust to changing environmental timing cues, seasonal behavioral cycles or aging. These immature cells can be used to prepare an SCN cell line that may provide a consistent source of rhythmic cells that would enable simpler genetic manipulation of key mammalian clock genes.

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