Date on Master's Thesis/Doctoral Dissertation

12-2011

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Anatomical Sciences and Neurobiology

Committee Chair

Roisen, Fred J.

Author's Keywords

Olfactory epithelium; Parkinson's disease; Adult stem cells; Dopamine

Subject

Epithelium; Parkinson's disease; Stem cells

Abstract

Human adult olfactory epithelium contains neural progenitors (hONPs) which replace damaged cellular components throughout life. Methods to isolate and expand the hONPs have been developed in our laboratory. In response to morphogens, the hONPs differentiate along several neural lineages. This study optimized conditions for the differentiation of hONPs towards dopaminergic neurons. The hONPs were treated with Sonic Hedgehog, in the presence or absence of Retinoic acid and/or forskolin. Transcription factors (Nurrl, Pitx3 and Lmxla) that promote embryonic mouse or chicken dopaminergic development were employed to determine if they would modulate lineage restriction of these adult human progenitors. Transcription factor expression and tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, were detected in the transfected cells after 4-month selection with G418, indicating transfected hONPs were stably restricted towards a dopaminergic lineage. Furthermore, enzyme immunoassay was employed to detect the synthesis and release of dopamine. The most efficient dopamine transfection paradigm was determined. Equivalent levels of several neurotrophic factors were detected in both the pre- and post-transfected hONPs which have potential roles in the maintenance, survival and proliferation of dopaminergic neurons. This study engrafted cells modified by the most efficient transfection paradigm for dopamine formation into a unilateral neurotoxin, 6-hydroxydopamine (6-OHDA)-induced Parkinsonian rat model. Thirty-five percent of the animals engrafted with hONPs had improved behavioral recovery as demonstrated by the amphetamine induced rotation test as well as a comer preference and cylinder paw preference, over a period of more than 24 weeks. No difference was observed between the pre- and post-transfected groups indicating that the host environment facilitated dopaminergic differentiation in situ. Human fibroblasts did not diminish the Parkinsonian rotational deficits at any point during the study. The engrafted hONP population remained intact and TH positive for a minimum of six months in vivo. Higher dopamine leveis were detected in the striatum of behaviorally recovered animals than in equivalent regions of their non-recovered counterparts. Throughout these experiments, no evidence of tumorigenicity was observed. These studies support our hypothesis that human adult olfactory epithelial-derived progenitors represent a unique autologous cell type for a cell-based strategy for the treatment of Parkinson's disease.

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