Epigenetic Manipulation Facilitates the Generation of Skeletal Muscle Cells from Pluripotent Stem Cells

Abstract

Human pluripotent stem cells (hPSCs) have the capacity to differentiate into essentially all cell types in the body. Such differentiation can be directed to specific cell types by appropriate cell culture conditions or overexpressing lineage-defining transcription factors (TFs). Especially, for the activation of myogenic program, early studies have shown the effectiveness of enforced expression of TFs associated with myogenic differentiation, such as PAX7 and MYOD1. However, the efficiency of direct differentiation was rather low, most likely due to chromatin features unique to hPSCs, which hinder the access of TFs to genes involved in muscle differentiation. Indeed, recent studies have demonstrated that ectopic expression of epigenetic-modifying factors such as a histone demethylase and an ATP-dependent remodeling factor significantly enhances myogenic differentiation from hPSCs. In this article, we review the recent progress for in vitro generation of skeletal muscles from hPSCs through forced epigenetic and transcriptional manipulation.

Figure 1

The chromatin regulation of developmental genes in PSCs. In PSCs, developmental genes are marked by bivalent domains containing both H3K4me3 and H3K27me3, which are associated with the transcriptional silencing in the undifferentiated state. The removal of H3K27me3 and the binding of transcription factors (TFs) allow rapid transcriptional activation upon differentiation stimuli.

Figure 2

The states of H3K4me3 and H3K27me3 near myogenic genes in hESCs and myoblasts. The ChIP-sequencing peaks of H3K4me3 and H3K27me3 at the MYOD1, PAX3, and PAX7 genes are shown. The data were generated in the Bernstein laboratory [59].

Figure 3

H3K27me3 demethylation in hESCs by forced expression of JMJD3c. (a) Immunostaining analysis showing the H3K27me3 demethylation in whole nuclei of hESCs by JMJD3c overexpression (arrows). (b) ChIP-sequencing analysis showing the significant reduction of H3K27me3 at the PAX7 gene in hESCs by JMJD3c overexpression. H3K4me3 enrichment remains at the PAX7 gene after JMJD3c overexpression.

Figure 4

Efficient myogenic differentiation of hESCs by synRNA-mediated overexpression of JMJD3c and MYOD1. Transfection of synRNAs encoding JMJD3c and MYOD1 directly converts hESCs into MHC-positive myogenic cells for 5 days post differentiation. The differentiation efficiency was much higher in the JMJD3c/MYOD1-overexpressing hESCs compared with the MYOD1-overexpressing hESCs. MHC, green. DAPI, blue.