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

IntroductionPluripotent 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.MethodsWe 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.ResultsDLK1-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.ConclusionsRepression of ncRNAs derived from the DLK1-DIO3 imprinted locus is associated with reduced neural lineage differentiation potential in hESCs.Electronic supplementary materialThe online version of this article (doi:10.1186/scrt535) contains supplementary material, which is available to authorized users.

Identification of long non-coding RNAs involved in neuronal development and intellectual disability

“…This set includes almost all lncRNAs that have been demonstrated in literature to play a role in neuronal development, such as MIAT, TUG1, DGCR5, MEG3, and TUNA 263743444546. One lncRNA with validated function during neurogenesis that is notably absent in our candidate list is RMST 35.…”

Dynamic behaviors of astrocytes in chemically modified fibrin and collagen hydrogels

“…After the cells were cultured for 24 hours, the migration of astrocytes was recorded with a time-lapse microscope (Zeiss Axio Observer microscope) placed in a plastic incubator with 5% CO 2 at 37°C. 69, 70 Sterile conditions were maintained throughout. The time-lapse image recording was performed to record cell migration using ZEN 2011 imaging microscope software.…”

Dnmt3l-knockout donor cells improve somatic cell nuclear transfer reprogramming efficiency

“…DNMT3L mutations have been associated with schizophrenia, subtelomeric hypomethylation, lower intelligence and ovarian endometriosis (El-Maarri et al 2009, Haggarty et al 2010, Borghese et al 2012, Saradalekshmi et al 2014. Recent studies have shown that the loss of Dnmt3l expression correlates with failed TE silencing and defective genomic imprinting at the Dlk1-Dio3 locus in induced pluripotent stem cells (iPSCs) (Stadtfeld et al 2012, Tang et al 2012, which might correlate with reduced neural lineage differentiation potential (Stadtfeld et al 2012, Mo et al 2015. Dnmt3l-mutant iPSCs are therefore not suitable for biomedical research.…”