title: Induction of specific neuron types by overexpression of single transcription factors.
abstract: Specific neuronal types derived from embryonic stem cells (ESCs) can facilitate mechanistic studies and potentially aid in regenerative medicine. Existing induction methods, however, mostly rely on the effects of the combined action of multiple added growth factors, which generally tend to result in mixed populations of neurons. Here, we report that overexpression of specific transcription factors (TFs) in ESCs can rather guide the differentiation of ESCs towards specific neuron lineages. Analysis of data on gene expression changes 2 d after induction of each of 185 TFs implicated candidate TFs for further ESC differentiation studies. Induction of 23 TFs (out of 49 TFs tested) for 6 d facilitated neural differentiation of ESCs as inferred from increased proportion of cells with neural progenitor marker PSA-NCAM. We identified early activation of the Notch signaling pathway as a common feature of most potent inducers of neural differentiation. The majority of neuron-like cells generated by induction of Ascl1, Smad7, Nr2f1, Dlx2, Dlx4, Nr2f2, Barhl2, and Lhx1 were GABA-positive and expressed other markers of GABAergic neurons. In the same way, we identified Lmx1a and Nr4a2 as inducers for neurons bearing dopaminergic markers and Isl1, Fezf2, and St18 for cholinergic motor neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific messenger RNA (mRNA) and microRNAs (miRNAs). Sets of Ascl1-induced mRNAs and miRNAs were enriched in Ascl1 targets. In further studies, enrichment of cells obtained with the induction of Ascl1, Smad7, and Nr2f1 using microbeads resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and expressed markers of GABAergic neurons. In summary, this study indicates that induction of transcription factors is a promising approach to generate cultures that show the transcription profiles characteristic of specific neural cell types.
authors.affiliation: Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, 21224, USA.|Department of Psychology, Center for Neuroscience, UC Davis, Davis, 95616, USA.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Teratani-Ota
authors.affiliation: Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, 21224, USA.|Laboratory of Stem Cell Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Yamamizu
authors.affiliation: Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, 21224, USA.
authors.forename: Yulan
authors.initials: Y
authors.lastname: Piao
authors.affiliation: Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, 21224, USA.
authors.forename: Lioudmila
authors.initials: L
authors.lastname: Sharova
authors.affiliation: Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, 21224, USA.|Elixirgen, LLC, Baltimore, MD, 21205, USA.
authors.forename: Misa
authors.initials: M
authors.lastname: Amano
authors.affiliation: Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, 21224, USA.|Elixirgen, LLC, Baltimore, MD, 21205, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Yu
authors.affiliation: Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, 21224, USA.
authors.forename: David
authors.initials: D
authors.lastname: Schlessinger
authors.affiliation: Department of Systems Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Tokyo, 160-8582, Japan.
authors.forename: Minoru S H
authors.initials: MS
authors.lastname: Ko
authors.affiliation: Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, 21224, USA. sharoval@mail.nih.gov.
authors.forename: Alexei A
authors.initials: AA
authors.lastname: Sharov
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D051838:COUP Transcription Factor I / Q000378:metabolism; D065150:Cellular Reprogramming / Q000235:genetics; D020869:Gene Expression Profiling; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D055495:Neurogenesis; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D051906:Smad7 Protein / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D059467:Transcriptome / Q000235:genetics; D015854:Up-Regulation / Q000235:genetics
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D051838:COUP Transcription Factor I; D051906:Smad7 Protein; D014157:Transcription Factors; C499317:Ascl1 protein, mouse; C497590:Nr2f1 protein, mouse
keywords: Ascl1; Cholinergic neurons; Dopaminergic neurons; ESCs; GABAergic neurons; Gene expression profiling; miRNA