title: Loss of non-coding RNA expression from the DLK1-DIO3 imprinted locus correlates with reduced neural differentiation potential in human embryonic stem cell lines.
abstract: BACKGROUND
  Pluripotent stem cells are increasingly used to build therapeutic models, including the transplantation of neural progenitors derived from human embryonic stem cells (hESCs). Recently, long non-coding RNAs (lncRNAs), including delta-like homolog 1 gene and the type III iodothyronine deiodinase gene (DLK1-DIO3) imprinted locus-derived maternally expressed gene 3 (MEG3), were found to be expressed during neural development. The deregulation of these lncRNAs is associated with various neurological diseases. The imprinted locus DLK1-DIO3 encodes abundant non-coding RNAs (ncRNAs) that are regulated by differential methylation of the locus. We aim to study the correlation between the DLK1-DIO3-derived ncRNAs and the capacity of hESCs to differentiate into neural lineages.
  
  
  METHODS
  We classified hESC sublines into MEG3-ON and MEG3-OFF based on the expression levels of MEG3 and its downstream microRNAs as detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). A cDNA microarray was used to analyze the gene expression profiles of hESCs. To investigate the capacity of neural differentiation in MEG3-ON and MEG3-OFF hESCs, we performed neural lineage differentiation followed by neural lineage marker expression and neurite formation analyses via qRT-PCR and immunocytochemistry, respectively. MEG3-knockdown via small interfering RNA (siRNA) and small hairpin RNA (shRNA) was used to investigate the potential causative effect of MEG3 in regulating neural lineage-related gene expression.
  
  
  RESULTS
  DLK1-DIO3-derived ncRNAs were repressed in MEG3-OFF hESCs compared with those in the MEG3-ON hESCs. The transcriptome profile indicated that many genes related to nervous system development and neural-type tumors were differentially expressed in MEG3-OFF hESCs. Three independent MEG3-knockdown assays using different siRNA and shRNA constructs consistently resulted in downregulation of some neural lineage genes. Lower expression levels of stage-specific neural lineage markers and reduced neurite formation were observed in neural lineage-like cells derived from MEG3-OFF-associated hESCs compared with those in the MEG3-ON groups at the same time points after differentiation.
  
  
  CONCLUSIONS
  Repression of ncRNAs derived from the DLK1-DIO3 imprinted locus is associated with reduced neural lineage differentiation potential in hESCs.
authors.affiliation: Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan. d98642005@ntu.edu.tw.
authors.forename: Chu-Fan
authors.initials: CF
authors.lastname: Mo
authors.affiliation: Department of Obstetrics & Gynecology, College of Medicine and the Hospital, National Taiwan University Hospital, Taipei, 100, Taiwan. fanggrass39@gmail.com.
authors.forename: Fang-Chun
authors.initials: FC
authors.lastname: Wu
authors.affiliation: Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National Taiwan University, Taipei, 100, Taiwan. d99b48002@ntu.edu.tw.|Genome and Systems Biology Degree Program, National Taiwan University, Taipei, 106, Taiwan. d99b48002@ntu.edu.tw.|Genome and Systems Biology Degree Program, Academia Sinica, Taipei, 115, Taiwan. d99b48002@ntu.edu.tw.
authors.forename: Kang-Yu
authors.initials: KY
authors.lastname: Tai
authors.affiliation: Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan. b87606029@ntu.edu.tw.
authors.forename: Wei-Chun
authors.initials: WC
authors.lastname: Chang
authors.affiliation: Genome and Systems Biology Degree Program, National Taiwan University, Taipei, 106, Taiwan. kai-wei.chang@hotmail.com.|Genome and Systems Biology Degree Program, Academia Sinica, Taipei, 115, Taiwan. kai-wei.chang@hotmail.com.
authors.forename: Kai-Wei
authors.initials: KW
authors.lastname: Chang
authors.affiliation: Genomic Research Center, Academia Sinica, Taipei, 115, Taiwan. kuohuch@gate.sinica.edu.tw.|Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, 115, Taiwan. kuohuch@gate.sinica.edu.tw.
authors.forename: Hung-Chih
authors.initials: HC
authors.lastname: Kuo
authors.affiliation: Department of Obstetrics & Gynecology, College of Medicine and the Hospital, National Taiwan University Hospital, Taipei, 100, Taiwan. hnho@ntu.edu.tw.
authors.forename: Hong-Nerng
authors.initials: HN
authors.lastname: Ho
authors.affiliation: Department of Obstetrics & Gynecology, College of Medicine and the Hospital, National Taiwan University Hospital, Taipei, 100, Taiwan. hfchen@ntu.edu.tw.|Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National Taiwan University, Taipei, 100, Taiwan. hfchen@ntu.edu.tw.
authors.forename: Hsin-Fu
authors.initials: HF
authors.lastname: Chen
authors.affiliation: Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan. shaupinglin@ntu.edu.tw.|Agricultural Biotechnology Research Centre, Academia Sinica, Taipei, 115, Taiwan. shaupinglin@ntu.edu.tw.|Research Centre for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, 106, Taiwan. shaupinglin@ntu.edu.tw.|Centre for Systems Biology, National Taiwan University, Taipei, 106, Taiwan. shaupinglin@ntu.edu.tw.
authors.forename: Shau-Ping
authors.initials: SP
authors.lastname: Lin
mesh_terms: D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D058732:Embryoid Bodies / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D056426:Genetic Loci; D018392:Genomic Imprinting; D018398:Homeodomain Proteins / Q000378:metabolism; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D007453:Iodide Peroxidase / Q000235:genetics*; D035683:MicroRNAs / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000378:metabolism; D034622:RNA Interference; D062085:RNA, Long Noncoding / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D022661:RNA, Untranslated / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D012097:Repressor Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D059467:Transcriptome
chemical_list: D005136:Eye Proteins; D018398:Homeodomain Proteins; C498907:MEG3 non-coding RNA, human; D035683:MicroRNAs; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; D062085:RNA, Long Noncoding; D034741:RNA, Small Interfering; D022661:RNA, Untranslated; D012097:Repressor Proteins; C117274:iodothyronine deiodinase type III; D007453:Iodide Peroxidase