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ERBB3 and NGFR mark distinct skeletal muscle progenitor cells in human development enabling enrichment and maturation of hPSC muscle

ABSTRACT: Abstract: Human pluripotent stem cells (hPSCs) can be directed to differentiate into skeletal muscle progenitor cells (SMPCs). However, the myogenic potential of hPSC-SMPCs compared to human fetal or adult satellite cells (Scs) remains unclear. This study demonstrates hPSC-SMPCs derived by commonly used protocols are functionally less mature than freshly-isolated human fetal or adult Scs. We utilized RNA-SEQ of human fetal Scs to identify differentially expressed genes including NGFR, ERBB3, which enriched for MYOD+ or PAX7+ cells. Furthermore, inhibition of TGFβ (TGFβi) improved hPSC-muscle maturation in three independent lines as measured by TEM and expression of myosins. Engraftment of CRISPR/Cas9-corrected Duchenne Muscular Dystrophy (DMD) hiPSC-SMPC subpopulations treated with TGFβi in vivo, increased dystrophin-positive fibers to levels of engrafted cultured fetal Scs in mdx mouse models of DMD. This work provides the first characterization of the developmental status of hPSC-SMPCs and identifies candidates to enable robust myogenic activity in vitro and in vivo.

ORGANISM(S): Homo sapiens

PROVIDER: GSE87365 | GEO | 2016/09/27

SECONDARY ACCESSION(S): PRJNA344494

REPOSITORIES: GEO

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Project description:The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to roughly 0.1% of the entire human DNA sequence. Mutations in this gene cause Duchenne muscular dystrophy and other milder X-linked, recessive dystrophinopathies. Using a custom-made tiling array, specifically designed for the DMD locus, we identified a variety of novel long non-coding RNAs (ncRNAs), both sense and antisense oriented, whose expression profiles mirror that of DMD gene. Importantly, these transcripts are intronic in origin and specifically localized to the nucleus and are transcribed contextually with dystrophin isoforms or primed by MyoD-induced myogenic differentiation. Furthermore, their forced ectopic expression in both human muscle and neuronal cells causes a specific and negative regulation of endogenous dystrophin full length isoforms and significantly down-regulate the activity of a luciferase reporter construct carrying the minimal promoter regions of the muscle dystrophin isoform. Consistent with this apparently repressive role, we found that, in muscle samples of DMD symptomatic female carriers, lncRNAs expression levels inversely correlate with those of muscle full length DMD isoforms. Overall these findings unveil an unprecedented complexity of the transcriptional pattern of the DMD locus and reveal that DMD-specific lncRNAs may contribute to the orchestration and homeostasis of the muscle dystrophin expression pattern by selective targeting and down-modulation of dystrophin promoter transcriptional activity. We tiled the entire DMD gene, in both sense and antisense directions, using the web-based Agilent eArray database, Version 4.5 (Agilent Technologies), with 60-mer oligos every 66 bp of repeat-masked genome sequence. We defined probe sets for both orientations, encompassing the DMD exons, promoters, introns, predicted MiRNA (identified by PromiRII) and conserved non-coding sequences (CNSs) identified within dystrophin introns using the VISTA programme (http://genome.lbl.gov/vista/index.shtml). Two specific sets of probes were designed to cover, in both directions, the cDNA sequences of a group of control genes identified in the Gene Expression Omnibus (GEO) database http://www.ncbi.nlm.nih.gov/geo/) and expressed equally in both normal and dystrophic muscles. Each probe set was opportunely distributed and replicated several times in order to obtain two 4x44k microarrays, referred to as DMD GEx Sense and DMD GEx Antisense, respectively, able to detect transcripts in the same and opposite directions as that of DMD gene transcription. Three commercial poly A+ RNAs from normal human brain, heart and skeletal muscle tissues were utilised (Ambion). Skin poly A+ RNA was isolated from total Skin RNA (Stratagene) using the Qiagen Oligotex kit. All RNA samples were checked for purity using a ND-1000 spectrophotometer (NanoDrop Technologies), and for integrity by electrophoresis on a 2100 BioAnalyzer (Agilent Technologies). Sample labelling and hybridisation were performed according to the protocols provided by Agilent (One-Color Microarray-Based Gene Expression Analysis version 5.0.1). The array was analysed using the Agilent scanner and Feature Extraction software (v9.1).

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ERBB3 and NGFR mark distinct skeletal muscle progenitor cells in human development enabling enrichment and maturation of hPSC muscle

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