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Publication Date

2013-12-13

Availability

UM campus only

Embargo Period

2013-12-12

Degree Name

Master of Science (MS)

Department

Biomedical Engineering (Engineering)

Date of Defense

2013-11-21

First Committee Member

Herman Cheung

Second Committee Member

Chun Yuh Charles Huang

Third Committee Member

Fotios Andreopoulos

Abstract

This research aims to characterize the neural crest (NC) remnant population in Human Exfoliated Deciduous Teeth (SHED). This study applies a recently identified technique that uses connexin43 (Cx43) as a biomarker to select for a homogeneous population of neural crest (NC) stem cells. In addition, this study evaluates the presence of pluripotency-associated transcription factors and neural crest-specific markers and the potential of Cx43+ SHED cells to differentiate into the neural, cardio, and osteo lineages. Cx43+ SHED used as control were cultured in control media. Neuro, cardio and osteo induced Cx43+ SHED were cultured in neurogenic, cardiomyogenic and osteogenic cocktail media. Cells were cultured in differentiation media for 10 days and media was changed every 2-3 days. Following treatment, immunohistochemistry and gene expression analysis through qPCR was evaluated. Immunohistochemistry analysis confirmed the presence of pluripotency-associated factors such as OCT4 and SOX2 and neural crest-specific factors such as SOX 10 and Nestin. In addition, there is a statistically significant increase in the expression of OCT4 (p<0.05) when compared to the non-selected population. Furthermore, results from immunohistochemistry for glial fibrillary acidic protein (GFAP) and beta tubulin III analysis and gene expression for neurofilament medium (NEFM) indicated that neurogenesis was initiated. In addition, the cardiomyogenic phenotype was also expressed by indicating the presence of early cardiomyogenic markers such as myosin enhancer factor-2 (MEF2), NKk-2.5, GATA4 and cardiac troponin. Furthermore, the presence of calcium deposits was confirmed through Alizarin Red S staining. This indicates the initiation of osteogenic differentiation. Moreover the statistically significant increase of the gene RUNX2 (p<0.05) confirmed the pre-commitment into the osteo-lineage. Overall the expression of neurogenic, cardiomyogenic and osteogenic markers either in the immunohistochemistry analysis or the qPCR in the Cx43+ SHED population surpasses that of the non-selected population. The Cx43+ SHED population represents a more homogeneous neural crest stem cell population capable of differentiating into mesodermal and ectodermal germ layers.

Keywords

Neural crest stem cells; Stem Cells Derived from Human Exfoliated Deciduous Teeth; Connexin 43; Pluripotency

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