Epigenetic reprogramming of human embryonic stem cells into skeletal muscle cells and generation of contractile myospheres

Direct generation of a homogeneous population of skeletal myoblasts from human embryonic stem cells (hESCs) and formation of three-dimensional contractile structures for disease modeling in vitro are current challenges in regenerative medicine. Previous studies reported on the generation of myoblasts from ESC-derived embryoid bodies (EB), but not from undifferentiated ESCs, indicating the requirement for mesodermal transition to promote skeletal myogenesis. Here, we show that selective absence of the SWI/SNF component BAF60C (encoded by SMARCD3) confers on hESCs resistance to MyoD-mediated activation of skeletal myogenesis. Forced expression of BAF60C enables MyoD to directly activate skeletal myogenesis in hESCs by instructing MyoD positioning and allowing chromatin remodeling at target genes. BAF60C/MyoD-expressing hESCs are epigenetically committed myogenic progenitors, which bypass the mesodermal requirement and, when cultured as floating clusters, give rise to contractile three-dimensional myospheres composed of skeletal myotubes. These results identify BAF60C as a key epigenetic determinant of hESC commitment to the myogenic lineage and establish the molecular basis for the generation of hESC-derived myospheres exploitable for 'disease in a dish' models of muscular physiology and dysfunction

Dynamics of cellular states of fibro-adipogenic progenitors during myogenesis and muscular dystrophy

Fibro-adipogenic progenitors (FAPs) are currently defined by their anatomical position, expression of non-specific membrane-associated proteins, and ability to adopt multiple lineages in vitro. Gene expression analysis at single-cell level reveals that FAPs undergo dynamic transitions through a spectrum of cell states that can be identified by differential expression levels of Tie2 and Vcam1. Different patterns of Vcam1-negative Tie2highor Tie2lowand Tie2low/Vcam1-expressing FAPs are detected during neonatal myogenesis, response to acute injury and Duchenne Muscular Dystrophy (DMD). RNA\ua0sequencing analysis identified cell state-specific transcriptional profiles that predict functional interactions with satellite and inflammatory cells. In particular, Vcam1-expressing FAPs, which exhibit a pro-fibrotic expression profile, are transiently activated by acute injury in concomitance with the inflammatory response. Aberrant persistence of Vcam1-expressing FAPs is detected in DMD muscles or upon macrophage depletion, and is associated with muscle fibrosis, thereby revealing how disruption of inflammation-regulated FAPs dynamics leads to a pathogenic outcome

Epigenetic Reprogramming of Human Embryonic Stem Cells into Skeletal Muscle Cells and Generation of Contractile Myospheres

Direct generation of a homogeneous population of skeletal myoblasts from human embryonic stem cells (hESCs) and formation of three-dimensional contractile structures for disease modeling in vitro are current challenges in regenerative medicine. Previous studies reported on the generation of myoblasts from ESC-derived embryoid bodies (EB), but not from undifferentiated ESCs, indicating the requirement for mesodermal transition to promote skeletal myogenesis. Here, we show that selective absence of the SWI/SNF component BAF60C (encoded by SMARCD3) confers on hESCs resistance to MyoD-mediated activation of skeletal myogenesis. Forced expression of BAF60C enables MyoD to directly activate skeletal myogenesis in hESCs by instructing MyoD positioning and allowing chromatin remodeling at target genes. BAF60C/MyoD-expressing hESCs are epigenetically committed myogenic progenitors, which bypass the mesodermal requirement and, when cultured as floating clusters, give rise to contractile three-dimensional myospheres composed of skeletal myotubes. These results identify BAF60C as a key epigenetic determinant of hESC commitment to the myogenic lineage and establish the molecular basis for the generation of hESC-derived myospheres exploitable for “disease in a dish” models of muscular physiology and dysfunction

Data from: TBP/TFIID-dependent activation of MyoD target genes in skeletal muscle cells

Change in the identity of the components of the transcription pre-initiation complex is proposed to control cell type-specific gene expression. Replacement of the canonical TFIID-TBP complex with TRF3/TBP2 was reported to be required for activation of muscle-gene expression. The lack of a developmental phenotype in TBP2 null mice prompted further analysis to determine whether TBP2 deficiency can compromise adult myogenesis. We show here that TBP2 null mice have an intact regeneration potential upon injury and that TBP2 is not expressed in established C2C12 muscle cell or in primary mouse MuSCs. While TFIID subunits and TBP are downregulated during myoblast differentiation, reduced amounts of these proteins form a complex that is detectable on promoters of muscle genes and is essential for their expression. This evidence demonstrates that TBP2 does not replace TBP during muscle differentiation, as previously proposed, with limiting amounts of TFIID-TBP being required to promote muscle-specific gene expression

IMR90 overexpressing MyoD in differentiation medium

Human fibroblasts IMR90 transfected with doxycycline inducible mouse MYOD were cultured in growth media (GM, DMEM high glucose, 10% FBS). MYOD was induced with doxycycline treatment 24 hrs in GM and 72-96 hrs in differentiation media (DM, DMEM high glucose, 2% HS, ITS) prior collection

IMR90 overexpressing MyoD in growth medium

Human fibroblasts IMR90 transfected with doxycycline inducible mouse MYOD were cultured in growth media (DMEM high glucose, 10% FBS). MYOD was induced with doxycycline treatment 24 hrs prior collection

IMR90 overexpressing MyoD in growth medium, replicate

Human fibroblasts IMR90 transfected with doxycycline inducible mouse MYOD were cultured in growth media (DMEM high glucose, 10% FBS). MYOD was induced with doxycycline treatment 24 hrs prior collection

Muscle-relevant genes marked by stable H3K4me2/3 profiles and enriched MyoD binding during myogenic differentiation

<div><p>Post-translational modifications of histones play a key role in the regulation of gene expression during development and differentiation. Numerous studies have shown the dynamics of combinatorial regulation by transcription factors and histone modifications, in the sense that different combinations lead to distinct expression outcomes. Here, we investigated gene regulation by stable enrichment patterns of histone marks H3K4me2 and H3K4me3 in combination with the chromatin binding of the muscle tissue-specific transcription factor MyoD during myogenic differentiation of C2C12 cells. Using <i>k</i>-means clustering, we found that specific combinations of H3K4me2/3 profiles over and towards the gene body impact on gene expression and marks a subset of genes important for muscle development and differentiation. By further analysis, we found that the muscle key regulator MyoD was significantly enriched on this subset of genes and played a repressive role during myogenic differentiation. Among these genes, we identified the pluripotency gene <i>Patz1</i>, which is repressed during myogenic differentiation through direct binding of MyoD to promoter elements. These results point to the importance of integrating histone modifications and MyoD chromatin binding for coordinated gene activation and repression during myogenic differentiation.</p></div

Cluster 1 genes bound by MyoD.

<p>(A) Overlap of common H3K4me2/3 cluster 1 genes in Undiff and Diff C2C12 cells. The number of genes, which gain, loose or have constitutive MyoD binding are indicated, including their respective number of differentially expressed genes (fold change (FC) ≥ 2). (B) Percentage of genes in the common H3K4me2/3 clusters bound by MyoD in Undiff and Diff C2C12 cells. Highest enrichment in Undiff and Diff (each in cluster 1) is indicated by the two red lines. The <i>P</i> values are based on two-sided Fisher's exact test. (C) Heatmap of differentially expressed genes in H3K4me2/3 cluster1, which gain MyoD during differentiation (22 genes with fold change ≥ 2 out of 95 genes). The numbers in the heatmap represent the FPKM (fragments per kilo bases of exons for per million mapped) values. Gene names in blue indicate the genes with the E-box motif (CANNTG) within a 30 bp region centered on the peak summit. Gene names underlined in blue are genes with the MyoD preferred E-box motif (CAGCTG) within a 30 bp region centered on the peak summit.</p

Clustering analysis of H3K4me3 profile in undifferentiated C2C12 cells and comparison to H3K4me2 profiles.

<p>(A) H3K4me3 profiles identified by <i>k</i>-means clustering. The clustering is based on TSS and the corresponding number of genes is given for each cluster. Genes with multiple TSS can be present in more than one cluster. (B) The box plot (25% to 75% quartile) shows the levels of gene expression from the different H3K4me3 clusters in Undiff C2C12 cells. The expression of cluster 1 and cluster 2 genes was compared using the Mann-Whitney U test. (C) Overlap of H3K4me2 and H3K4me3 cluster 1 genes in Undiff C2C12 cells. The <i>P</i> value is based on a hypergeometric test. (D) GO enrichment analysis of common cluster 1 genes using the DAVID functional annotation tool. Top ten biological process terms with an adjusted (Benjamini-Hochberg) <i>P</i> value ≤ 0.01 are indicated. GO terms related to muscle development are highlighted in red. (E) H3K4me2 and H3K4me3 enrichment profiles of selected muscle-relevant cluster 1 genes. The TSS is marked by an arrow. The y-axis indicates the ChIP-seq signal.</p