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

Yu-Ting L. Dingle, Katherine B. Xiong, Jason T. Machan, Kimberly A. Seymour, Debra Ellisor, Diane Hoffman-Kim, Mark Zervas
doi: https://doi.org/10.1101/093419
Yu-Ting L. Dingle
1Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, Rhode Island, USA
2Center for Biomedical Engineering, Brown University, Providence, Rhode Island, USA
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Katherine B. Xiong
3Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island, USA
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Jason T. Machan
4Departments of Orthopedics and Surgery at Rhode Island Hospital and The Warren Alpert Medical School at Brown University, Providence, Rhode Island, USA
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Kimberly A. Seymour
3Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island, USA
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Debra Ellisor
3Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island, USA
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Diane Hoffman-Kim
1Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, Rhode Island, USA
2Center for Biomedical Engineering, Brown University, Providence, Rhode Island, USA
5Brown Institute for Brain Science, Providence, Rhode Island, USA
6School of Engineering, Brown University, Providence, Rhode Island, USA
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  • For correspondence: Mark_Zervas@brown.edu Diane_Hoffman-Kim@brown.edu
Mark Zervas
3Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island, USA
5Brown Institute for Brain Science, Providence, Rhode Island, USA
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  • For correspondence: Mark_Zervas@brown.edu Diane_Hoffman-Kim@brown.edu
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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.

Note All research was planned and conducted while members were at Brown University

Research funding NIH/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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Posted February 05, 2017.
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Quantitative Analysis of Dopamine Neuron Subtypes Generated from Mouse Embryonic Stem Cells
Yu-Ting L. Dingle, Katherine B. Xiong, Jason T. Machan, Kimberly A. Seymour, Debra Ellisor, Diane Hoffman-Kim, Mark Zervas
bioRxiv 093419; doi: https://doi.org/10.1101/093419
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Quantitative Analysis of Dopamine Neuron Subtypes Generated from Mouse Embryonic Stem Cells
Yu-Ting L. Dingle, Katherine B. Xiong, Jason T. Machan, Kimberly A. Seymour, Debra Ellisor, Diane Hoffman-Kim, Mark Zervas
bioRxiv 093419; doi: https://doi.org/10.1101/093419

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