Abstract
Mesenchymal stem cells (MSCs) derived from human embryonic stem cells (hESCs) have significant potential for cell-mediated bone regeneration. Our recent study revealed that inhibiting the epigenetic regulator EZH2 plays a key role in promoting the mesodermal differentiation of hESCs. In this study, an epigenome-wide analysis of hESCs and MSCs revealed that growth differentiation factor 6 (GDF6), which is involved in bone formation, was the most upregulated gene associated with MSCs compared to hESCs. Furthermore, we identified GDF6 as a repressive target of EZH2 and found that ectopic GDF6 selectively promoted hESC differentiation towards the mesodermal lineage and enriched the MSC population. Our results provide molecular insights governing the mesenchymal commitment of hESCs and identify an inducing factor that offers strong promise for the future of regenerative medicine.
Highlights
We discovered that growth differentiation factor 6 (GDF6; known as BMP13) was the most upregulated gene in Mesenchymal stem cells (MSCs) compared to human embryonic stem cells (hESCs)
GDF6 promotes hESC differentiation into MSCs Since GDF6 was a repressive target of enhancer of zeste homolog 2 (EZH2), we assessed whether GDF6 could promote hESC differentiation into MSCs by treating hESCs with GDF6 for 3 days and examining the expression levels of both mesodermal and MSC markers
We identified GDF6 as a repressive target of EZH2 that facilitated hESC differentiation into the mesodermal lineage and subsequently generated a greater number of MSCs with high osteogenic potential
Summary
Growth differentiation factor 6, a repressive target of EZH2, promotes the commitment of human embryonic stem cells to mesenchymal stem cells. We compared epigenomic changes relating to histone modifications and the transcriptome between hESCs and MSCs using publicly available RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data This epigenome-wide map revealed that EZH2 was enriched at the promoters of genes belonging to the Wnt and TGF-β signaling superfamily in hESCs, but this enrichment was significantly reduced in MSCs. We discovered that growth differentiation factor 6 (GDF6; known as BMP13) was the most upregulated gene in MSCs compared to hESCs. GDF6 is a member of the TGF-β superfamily, which is associated with the maintenance and differentiation of ESCs. Interestingly, GDF6 was expressed in the nucleus pulposus of intervertebral discs and hypertrophic chondrocytes during early ossification of vertebrae.[17] Mutations in GDF6 are associated with vertebral segmentation defects in Klippel-Feil syndrome and multiple synostoses syndrome.[18,19] In mice, GDF6 is expressed in frontal bone primordia from embryonic day (E) 10.5 through E12.5 and in a striped pattern across developing skeletal condensations at E13.5.20,21. Our findings with GSK126, a selective small-molecule-inhibitor of EZH2, and suggest that H3K27me[3] enrichment at the GDF6 promoter is observed that GDF6 expression was significantly increased by dependent on EZH2 enzymatic activity
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