Date on Master's Thesis/Doctoral Dissertation

5-2005

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Anatomical Sciences and Neurobiology

Committee Chair

Roisen, Fred J.

Author's Keywords

Olfactory epithelium; Lineage; Differentiation; Neuroepithelial

Subject

Neurobiology--Research

Abstract

Neurosphere forming cells (NSFCs) have been derived from cultures of adult olfactory neuroepithelium obtained from patients and cadavers in serum rich medium. These neural progenitors remain undifferentiated when maintained in serum rich medium but have the potential to differentiate along glial or neuronal lineages. A totally defined medium (DM) was employed to examine their proliferation, lineage restriction and differentiation. None of the neurotrophic factors evaluated increased NSFCs viability or lineage restriction under the defined experimental conditions. Few of NSFCs ever express mature neuronal or glial markers in DM. To evaluate the potential of NSFCs to form oligodendrocytes, transcription factors, Olig2, Nkx2.2, and Sox10, were introduced into NSFCs to determine if their expression is sufficient for oligodendrocyte differentiation. Simultaneous transfection of Olig2-Nkx2.2, or Nkx2.2-Sox10 cDNA produced characteristic oligodendrocyte morphology and antigenicity including myelin basic protein. Furthermore, a population of Olig2-expressing NSFCs also expressed Sox10. Coculture of NSFCs transfected with Olig2-Nkx2.2, or Nkx2.2-Sox10 with purified sensory neurons demonstrated ensheathment formation between NSFC processes and axons. Retinoic acid, forskolin, and sonic hedgehog (RFS) have been reported to play important roles in neurogenesis in embryonic CNS in vivo. The application of RFS to NSFCs induced a small population to express mature neuronal antigens and to undergo neurite formation. To further increase the neuronal, especially motoneuronal, antigen expression, transcription factors Olig2, Ngn2 and HB9 were introduced into NSFCs to determine if their expression was sufficient for motoneuron differentiation. Simultaneous transfection of Ngn2-HB9 or Olig2-HB9 cDNA, supplemented with RFS produced increased expression of motoneuron antigens. Furthermore, high levels of Olig2 expressing NSFCs also expressed Ngn2. Coculture of NSFCs transfected with Ngn2-HB9, or Olig2-HB9 with purified chicken skeleton muscle cells demonstrated the formation of neuromuscular junctions. Our long-term goal is to develop cell populations for future studies that would be employed to determine the therapeutic utility of these olfactory-derived NSFCs for autologous transplantation into donors with CNS trauma and neurodegenerative diseases as well as for use in diagnostic evaluation.

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