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Show 121 school of medicine and health sciences INTRODUCTION: Parkinson's Disease (PD), the 2nd most common neurological disease, is a progressive neurodegenera-tive disorder resulting from decreased levels of dopamine caused by the death of dopaminergic (DA) neurons from the substantia nigra subcompacta. Lewy bodies are commonly found in the nucleus of surviving DA neurons in the brains of patients with familial and genetic forms of PD. Lewy bodies are made of aggregates of alpha-synuclein and other proteins such as ubiquitin. The gene encoding alpha-synuclein is named SNCA.Duplication and triplication of the wild type SNCA gene have also been discovered in families with PD, resulting in increased levels of alpha-synuclein in neurons of PD patients. Supported by data from SNCA animal models, it is suggested that increased levels of the wild type form of alpha-synclein can also cause neuronal death in PD patients. Furthermore, increased alpha-synuclein levels have also been found in patients with GBA mutations, in which GBA gene codes for a lysosomal protein (acid β-glucosidase). Homozygous mutations of the GBA gene inactivate acid β-glucosidase and cause the lysosmal storage disease called Gaucher's disease. Heterozygous mutations of GBA partially inactivate acid β-glucosidase and, therefore causing manifest symptoms in PD patients. OBJECTIVES: The study has 3 aims: 1) generation of induced pluripotent stem cells (iPSC) from skin fibroblasts of a control (IMR90) and a patient with a GBAAsn370Ser (GM10915) and without PD symptoms; 2) confirmation of iPS colonies by antibody markers specifically for iPS cells were confirmed; and 3) conversions of iPS cells to tyrosine hydroxylase positive cells. METHODS: 1) Reprogramming fibroblasts to iPS cells. Day 1: 1 x 10⁶ fibroblasts of a GBAN370S patient were placed into a 10 cm² plate. Day 2-3: Fibroblasts were transduced for 48 hours with the GFP-marker Ad-GFP-SOKcM & adenoviruses which produce 4 reprogramming factors: SOX2, OCT3/4, KLF4, c-MYC. Day 4: Viruses were removed and fresh iPS cell culture me-dium (DMEM-F2 + 20% Replacement medium containing 20 ng/ml FGF2 were added. Small colonies with the GFP (Green fluorescent protein) were formed. Day 5-7: Small stem-cell-like colonies formed and grew into bigger colonies. Media was continuously changed every 2 days. Cells were transferred to irradiate/ mouse embryonic fibroblasts (MEF). 2) Expansion and direct differentiation of iPS cells into DA neurons: Day 8-30: iPS colonies were scraped off, dispersed and transferred to new 10 cm2 dish pre-coated with irradiated MEF feeder cells .The process was continuously repeated every 6 days to expand the population of iPS cells. 3) Confirmation of iPS cells: iPS colonies were immunostained with alkaline phosphatase, SSEA-1, SSEA-3 TRA1-60, and TRA1-81. Western blots of protein extracts from transduced iPS cells and untransduced fibroblasts were performed to reas-sert the presence of reprogramming factors. 4) Differentiation of iPS cells to dopamine neurons. iPS media with 4% FGF2 was added, as cells with neurites appearance was observed. Most of these cells were morphologically similar to neurons. DA neurons were confirmed by immunoflu-roscent staining with a rabbit antibody against a DA marker, tyrosine hydroxylase (TH) and a mouse antibody to alpha synuclein. RESULTS AND DISCUSSION: Skin fibroblasts from an unaffected normal control and a patient with a mutation of the GBA gene that replaced Asn at position 370 with Ser (GBAAsn370Ser) have been successfully converted into induced pluripotent stem (iPS) cells. Almost all transduced cells were converted to iPS cells. The high successful rate of fibroblast-iPSC conver-sion was achieved because of equal expression of all 4 deprogramming factors OCT3/4, SOX2, KLF4, c-MYC in all infected cells. These iPS cells have been confirmed by iPS morphology of the colonies as visualized by nuclear staining with DAPI, immunopositive with 4 of the iPS specific markers TRA1-60, SSEA4, KFL4, and SSEA3. All colonies were positive with iPS markers and alkaline phosphatase. Alkaline phosphatase is a functional enzymatic marker for iPS cells that have been widely used to confirm iPS colonies. After terminal neuronal differentiation, specific dopamine producing (DA) neurons have been identified using the TH antibody specific for DA neurons. Only 1-2% of cells with morphology similar to neurons were TH positive. The levels of alpha synuclein in TH positive cells were low in both the IMR90 normal control and the GM10915 cells. This observation was expected since the GM10915 fibroblasts were derived from a patient with a GBA mutation but exhibited only symptoms of Gaucher's disease and no PD symptoms. However, since the level of fluorescent is weak, it is possible that the the fluorescent observed may represent background fluorescent. GENERATION AND CHARACTERIZATION OF INDUCED PLURITOTENT STEMCELLS FROM FIBROBLASTS Linh P. Huynh (Duong P. Huynh) Department of Neurology University of Utah UNDERGRADUATE RESEARCH ABSTRACTS Linh P. Huynh Duong P. Huynh |
