The Aggregate Induced Differentiation of Dopaminergic Neurons in NT2 Cells Mediation by Transcription Factors Pax-2, Pitx3 and Otx2

Date of Award

2008

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Keywords

Dopamine, Stem Cells, Parkinson's Disease, Transcription Factors, Differentiation, Central Nevous System, Neurodegenerative Disorders, Cell Replacement Therapy

Area of Concentration

Biology

Abstract

The Ntera2 (NT2) cell line is a monoclonal, pluripotent line that shows strong similarities to the progenitor cells of the central nervous system (CNS). Aggregation in suspension culture induces differentiation of the NT2 line into neurons expressing a variety of phenotypes, astrocytes, and glial cells. Because of their rapid proliferation in vitro, NT2 cells have been targeted as a cell source for cell replacement treatments for traumatic brain injury, spinal cord injury, and a number of neurodegenerative disorders. Otx2, Pax-2, and Pitx3 are three transcription factors that direct the differentiation and maturation of dopaminergic neurons during in vivo midbrain development. This study uses siRNA gene knockdown, realtime RT-PCR, and immunocytochemistry (ICC) staining to explore the roles of Otx2, Pax-2, and Pitx3 during the in vitro, aggregate-induced differentiation of NT2 cells. siRNA gene knockdown of Pax-2 and Pitx3 during the aggregation stage of induction did not decrease the percent of cells expressing the dopaminergic cell marker tyrosine hydroxylase (TH). However, there was an observed morphological change in TH-positive cells in Pax-2 and Pitx3 siRNA knockdown as compared to control cell cultures. THpositive cells in control cultures grew in large clusters displaying extensive neurite outgrowth, while those within Pax-2 and Pitx3 siRNA cultures typically displayed a spherical morphology and did not grow in clusters. Real-time RT-PCR and ICC staining results showed an up-regulation of all three transcription factors in aggregate-induced, differentiated cell cultures as compared to the pre-differentiated, pluripotent cell cultures.

Rights

This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.

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