Epigenetic deregulation of DLK-DIO3 imprinted locus and the differentiation potential of human embryonic stem cells
Date Issued
2015
Date
2015
Author(s)
Mo, Chu-Fan
Abstract
Selection of high-quality human embryonic stem cell (hESC) lines is crucial for successful hESC-based research and therapeutic models; however, some genetic and epigenetic instability, including imprinting instability, have been frequently found in cultured hESCs. Imprinted genes should be correctly regulated by Epigenetic machinery. Defective dosages of imprinted gene expression are associated with various growth defects and cancers. When screening the imprinting status in prolonged cultured hESCs by bisulfite sequencing, we observed that the DLK1-DIO3 imprinted locus was more susceptible to abnormal hypermethylation than other examined imprinted loci. Also, repression of maternally expressed non-coding RNAs (ncRNAs) from this locus was one of the first signs of epigenetic deregulation in the prolonged cultured hESCs. To investigate whether this locus correlates with hESC properties, we classified hESC sublines into “MEG3-ON” and “MEG3-OFF” primarily based on the expression levels of a long non-coding RNA from this locus (lncRNA), named MEG3, as well as its down-stream lncRNA and microRNAs (miRNAs). Through embryoid body (EB) formation, we found that the 12-Day-old EBs derived from MEG3-OFF hESCs, where DLK1-DIO3 ncRNAs were repressed, displayed smaller size and aberrant expressions of three germ layer genes. Transcriptome profiling showed that many genes involved in developmental events and different cancer types were deregulated in undifferentiated MEG3-OFF hESCs, compared with MEG3-ON hESCs. Notably, most of these deregulated genes are associated with neural lineage. When validating array results in three hESC lines and two hiPSC lines, we found an association between MEG3 repression and downregulation of several neural lineage-related genes. Direct manipulation of MEG3 via siRNA and shRNA knockdown approach further suggested a potential causative effect that MEG3 reduction leads to a decrease in these neural lineage gene expressions. When performing neural differentiation, lower expression levels of stage-specific neural lineage markers and reduced neurite formation were observed in the neural lineage-like cells derived from MEG3-OFF hESCs compared with those in MEG3-ON groups. Taken together, this study suggested that prevention of using MEG3-OFF hESCs where DLK1-DIO3 locus was deregulated may maintain the experimental efficiency with proper neural differentiation potential, which will benefit basic research and further therapeutic applications in this field.
Subjects
Non-coding RNA
DLK1-DIO3 imprinted locus
human embryonic stem cell
neural lineage differentiation
MEG3
long non-coding RNA
microRNA
neurite formation
SDGs
Type
thesis
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