title: A Knockin Reporter Allows Purification and Characterization of mDA Neurons from Heterogeneous Populations.
abstract: Generation of midbrain dopaminergic (mDA) neurons from human pluripotent stem cells provides a platform for inquiry into basic and translational studies of Parkinson's disease (PD). However, heterogeneity in differentiation in vitro makes it difficult to identify mDA neurons in culture or in vivo following transplantation. Here, we report the generation of a human embryonic stem cell (hESC) line with a tyrosine hydroxylase (TH)-RFP (red fluorescent protein) reporter. We validated that RFP faithfully mimicked TH expression during differentiation. Use of this TH-RFP reporter cell line enabled purification of mDA-like neurons from heterogeneous cultures with subsequent characterization of neuron transcriptional and epigenetic programs (global binding profiles of H3K27ac, H3K4me1, and 5-hydroxymethylcytosine [5hmC]) at four different stages of development. We anticipate that the tools and data described here will contribute to the development of mDA neurons for applications in disease modeling and/or drug screening and cell replacement therapies for PD.
authors.affiliation: Department of Cell Biology and Neurosciences, Montana State University, Bozeman, MT 59717, USA; Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA; Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address: nxia007@gmail.com.
authors.forename: Ninuo
authors.initials: N
authors.lastname: Xia
authors.affiliation: Department of Cell Biology and Neurosciences, Montana State University, Bozeman, MT 59717, USA; Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA; Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Fang
authors.initials: F
authors.lastname: Fang
authors.affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Pengbo
authors.initials: P
authors.lastname: Zhang
authors.affiliation: Department of Cell Biology and Neurosciences, Montana State University, Bozeman, MT 59717, USA; Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA; Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Cui
authors.affiliation: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Chhavy
authors.initials: C
authors.lastname: Tep-Cullison
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA.
authors.forename: Tim
authors.initials: T
authors.lastname: Hamerley
authors.affiliation: Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Hyun Joo
authors.initials: HJ
authors.lastname: Lee
authors.affiliation: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Theo
authors.initials: T
authors.lastname: Palmer
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Bothner
authors.affiliation: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Jin Hyung
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Cell Biology and Neurosciences, Montana State University, Bozeman, MT 59717, USA; Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA; Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Renee Reijo
authors.initials: RR
authors.lastname: Pera
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D059290:Dopaminergic Neurons / Q000378:metabolism*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D020869:Gene Expression Profiling; D055879:Gene Knock-In Techniques; D017930:Genes, Reporter; D000066449:Human Embryonic Stem Cells / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008636:Mesencephalon / Q000166:cytology*; D017423:Sequence Analysis, RNA; D014158:Transcription, Genetic; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D044503:5-Methylcytosine; D008164:Luminescent Proteins; D014446:Tyrosine 3-Monooxygenase; D000097573:Red Fluorescent Protein; C011865:5-hydroxymethylcytosine
keywords: 5-hydroxymethylcytosine; dopaminergic neurons; enhancers; gene editing; reporter; stem cell; transcriptome profiling; tyrosine hydroxylase