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bioRxiv · 10.1101/093419

Quantitative Analysis of Dopamine Neuron Subtypes Generated from Mouse Embryonic Stem Cells

Dingle, Y.-T. L.·Xiong, K.·Machan, J. T.·Seymour, K. A.·Ellisor, D.·Hoffman-Kim, D.·Zervas, M.
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Abstract

Dopamine (DA) neuron subtypes modulate specific physiological functions and are involved in distinct neurological disorders. Embryonic stem cell (ESC) derived DA neurons have the potential to aid in the study of disease mechanisms, drug discovery, and possibly cell replacement therapies. DA neurons can be generated from ESCs in vitro, but the subtypes of ESC-derived DA neurons have not been investigated in detail despite the diversity of DA neurons observed in vivo. Due to cell culture heterogeneity, sampling methods applied to ESC-derived cultures can be ambiguous and potentially biased. Therefore, we developed a quantification method to capture the depth of DA neuron production in vitro by estimating the error associated with systematic random sampling. Using this method, we quantified calbindin+ and calretinin+ subtypes of DA neurons generated from mouse ESCs. We found a higher production of the calbindin+ subtype (11-27%) compared to the calretinin+ subtype (2-13%) of DA neuron; in addition, DA neurons expressing neither subtype marker were also generated. We then examined whether exogenous sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) affected subtype generation. Our results demonstrate that exogenous SHH and FGF8 did not alter DA neuron subtype generation in vitro. These findings suggest that a deeper understanding DA neuron derivation inclusive of mechanisms that govern the in vitro subtype specification of ESC-derived DA neurons is required.\n\nNoteAll research was planned and conducted while members were at Brown University\n\nResearch fundingNIH/NCRR/NIGMS RI Hospital COBRE Center for Stem Cell Biology (8P20GM103468-04) (MZ) Brown Institute for Brain Science Pilot Grant (4-63662) (MZ/DHK)

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Dingle, Y.-T. L., Xiong, K., Machan, J. T., Seymour, K. A., Ellisor, D., Hoffman-Kim, D., Zervas, M.. 2016-12-13. Quantitative Analysis of Dopamine Neuron Subtypes Generated from Mouse Embryonic Stem Cells. https://doi.org/10.1101/093419

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