Loss of non-coding RNA expression from the DLK1-DIO3 imprinted locus correlates with reduced neural differentiation potential in human embryonic stem cell lines

Stem Cell Res Ther. 2015 Jan 5;6(1):1. doi: 10.1186/scrt535.


Introduction: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Differentiation
  • Cell Line
  • Cell Lineage
  • Embryoid Bodies / metabolism
  • Eye Proteins / metabolism
  • Genetic Loci
  • Genomic Imprinting*
  • Homeodomain Proteins / metabolism
  • Human Embryonic Stem Cells / cytology
  • Human Embryonic Stem Cells / metabolism*
  • Humans
  • Immunohistochemistry
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / metabolism
  • Iodide Peroxidase / genetics*
  • MicroRNAs / metabolism
  • Neurons / cytology
  • Neurons / metabolism*
  • Oligonucleotide Array Sequence Analysis
  • PAX6 Transcription Factor
  • Paired Box Transcription Factors / metabolism
  • RNA Interference
  • RNA, Long Noncoding / antagonists & inhibitors
  • RNA, Long Noncoding / genetics
  • RNA, Long Noncoding / metabolism
  • RNA, Small Interfering / metabolism
  • RNA, Untranslated / genetics
  • RNA, Untranslated / metabolism*
  • Real-Time Polymerase Chain Reaction
  • Repressor Proteins / metabolism
  • Sequence Analysis, DNA
  • Transcriptome


  • Eye Proteins
  • Homeodomain Proteins
  • MEG3 non-coding RNA, human
  • MicroRNAs
  • PAX6 Transcription Factor
  • PAX6 protein, human
  • Paired Box Transcription Factors
  • RNA, Long Noncoding
  • RNA, Small Interfering
  • RNA, Untranslated
  • Repressor Proteins
  • iodothyronine deiodinase type III
  • Iodide Peroxidase