Abstract
BackgroundDuchenne muscular dystrophy (DMD) is a devastating X-linked recessive genetic myopathy. DMD physiopathology is still not fully understood and a prenatal onset is suspected but difficult to address.MethodsThe bone morphogenetic protein 4 (BMP4) is a critical signaling molecule involved in mesoderm commitment. Human induced pluripotent stem cells (hiPSCs) from DMD and healthy individuals and human embryonic stem cells (hESCs) treated with BMP4 allowed us to model the early steps of myogenesis in normal and DMD contexts.ResultsUnexpectedly, 72h following BMP4 treatment, a new long DMD transcript was detected in all tested hiPSCs and hESCs, at levels similar to that found in adult skeletal muscle. This novel transcript named “Dp412e” has a specific untranslated first exon which is conserved only in a sub-group of anthropoids including human. The corresponding novel dystrophin protein of 412-kiloDalton (kDa), characterized by an N-terminal-truncated actin-binding domain, was detected in normal BMP4-treated hiPSCs/hESCs and in embryoid bodies. Finally, using a phosphorodiamidate morpholino oligomer (PMO) targeting the DMD exon 53, we demonstrated the feasibility of exon skipping validation with this BMP4-inducible hiPSCs model.ConclusionsIn this study, the use of hiPSCs to analyze early phases of human development in normal and DMD contexts has led to the discovery of an embryonic 412 kDa dystrophin isoform. Deciphering the regulation process(es) and the function(s) associated to this new isoform can contribute to a better understanding of the DMD physiopathology and potential developmental defects. Moreover, the simple and robust BMP4-inducible model highlighted here, providing large amount of a long DMD transcript and the corresponding protein in only 3 days, is already well-adapted to high-throughput and high-content screening approaches. Therefore, availability of this powerful cell platform can accelerate the development, validation and improvement of DMD genetic therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0062-6) contains supplementary material, which is available to authorized users.
Highlights
Duchenne muscular dystrophy (DMD) is a devastating X-linked recessive genetic myopathy
We demonstrated using a phosphorodiamidate morpholino oligomer (PMO) targeted to skip the DMD exon 53 that this robust bone morphogenetic protein 4 (BMP4)-inducible Human induced pluripotent stem cells (hiPSCs) model, providing large amount of a long DMD transcript and the corresponding protein, can be an efficient tool to accelerate the development of DMD genetic therapeutic approaches
BMP4-treated human pluripotent stem cells express a long DMD transcript with a novel exon 1 conserved in a sub-group of anthropoids After BMP4 treatment, all the primers designed along the DMD gene (Additional file 2: Table S2) demonstrated the robust expression of long DMD transcripts in all tested hPSCs, rising from day 1 to day 3, with high levels comparable to the levels of dystrophin protein 427 kDa muscular (Dp427m) expressed in normal adult skeletal muscle [37] (Fig. 2a)
Summary
Duchenne muscular dystrophy (DMD) is a devastating X-linked recessive genetic myopathy. The Duchenne muscular dystrophy (DMD) gene (ENSG00000198947; MIM 300377) is located on the X chromosome spanning more than two million base pairs [1, 2]. Other DMD transcripts and isoforms are expressed due to alternative promoters and splicing with specific pattern of expression along the development [3,4,5,6,7]. Mutations in the DMD gene cause Duchenne (MIM 310200) and Becker (MIM 300376) muscular dystrophies (BMD). The first symptoms of DMD usually appear between the ages of 2 and 5 years [8, 9]. Progressive muscle weakness typically leads to wheelchair dependency by the age of 12 years. There is no curative treatment but there are several therapeutic approaches in progress [10,11,12,13,14]
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