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Kathy A. Trzaska
Interdisciplinary Ph.D. Program
B.S., 2003, Rutgers University
Thesis Advisor: Pranela Rameshwar, Ph.D.
Department of Medicine
Friday, April 18, 2008
Dopamine (DA) neurons derived from stem cells are a valuable source for cell replacement therapy in Parkinsonís disease (PD), to study the molecular mechanisms of DA neuron development, and for screening pharmaceutical compounds that target DA disorders. Compared to other stem cells, mesenchymal stem cells (MSCs) derived from the adult human bone marrow (BM) have significant advantages and greater potential for immediate clinical application. MSCs are easily isolated from small volumes of BM aspirates, can be expanded in culture, have unique immunosuppressive properties, and hold immense plasticity. Our laboratory has previously reported the trasdifferentiation of human MSCs to peptidergic neurons using retinoic acid (RA) as an inducing agent. This thesis reports on the identification of in vitro conditions for inducing adult human MSCs into DA neurons. This developed neural induction protocol included a cocktail of sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8) and basic fibroblast growth factor (bFGF). Microarray anaylsis of MSCs induced for 6 days revealed that approximately 30% of the upregulated genes were linked to neuronal or DA differentiation. Further molecular analysis demonstrated ~67% efficiency of induction by 12 days. The analyses were based on tyrosine hydroxylase (TH) expression, morphology, expressions of neural markers, and a post-mitotic neuronal fate with downregulation of cell cycle activator proteins. Other molecular characterization revealed the expression of DA-specific genes: Pitx3, Nurr1, DAT and VMAT2. The induced MSCs also synthesized and secreted DA in a depolarization-independent manner and showed low expression of voltage gated Na+ and Ca2+ channels, suggesting the cells were at an immature stage of development, consistent with progenitors. Subsequent studies in this thesis explored the roles of brain-derived neurotrophic factor (BNDF) and Re-1 Silencing Transcription Factor (REST) as methods to further develop the MSC-derived DA cells to mature functional neurons. At day 9 of induction, the cells highly expressed the BDNF receptor TrkB. BNDF mediated evidence of functional maturation, based on Ca2+ imaging and electrophysiology. Intracellular Ca2+ was increased upon K+-induced depolarization. In addition, the cells responded to neurotransmitters, ACh and GABA. The cells treated with BNDF showed spontaneous postsynaptic currents and increased DA release upon depolarization. REST, which suppresses mature neuronal genes in neuronal progenitors, was not changed during the induction process. The induction of REST knockdown MSCs resulted in upregulation in protein levels of Na+ voltage-gated channel and tyrosine hydroxylase (TH) protein. Electrophysiological analyses showed spontaneous firings and spontaneous postsynaptic currents, similar to native DA neurons. Taken together, these results demonstrate the critical roles of BDNF and REST in the generation of functional DA neurons from adult MSCs.