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Dp412e: a novel human embryonic dystrophin isoform induced by BMP4 in early differentiated cells.

Massouridès E, Polentes J, Mangeot PE, Mournetas V, Nectoux J, Deburgrave N, Nusbaum P, Leturcq F, Popplewell L, Dickson G, Wein N, Flanigan KM, Peschanski M, Chelly J, Pinset C - Skelet Muscle (2015)

Bottom Line: Unexpectedly, 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.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.

View Article: PubMed Central - PubMed

Affiliation: I-STEM, CECS, Génopôle-Campus 1, 5 rue Henri Desbruères, 91030 Evry, Cedex France.

ABSTRACT

Background: Duchenne 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.

Methods: The 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.

Results: Unexpectedly, 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.

Conclusions: In 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.

No MeSH data available.


Related in: MedlinePlus

Correlation of BMP pathway activation with mesodermal commitment and Dp412e expression. a Microarray data showing 2-dCT of Human TGF-β/BMP Signaling Pathway genes in hiPSCs 1 without or 3 days following a single BMP4 treatment or with addition of NOG prior to BMP4 treatment (four technical duplicates/condition). Genes selected for display showed a minimum of ±2-fold change on RQs calculation in either BMP4-treated hiPSCs 1 or NOG/BMP4-treated hiPSCs 1, as compared to hiPSCs 1 without BMP4 treatment (p < 0.05; based on a Student’s t test). b Quantitative RT-PCR in hiPSCs 1 of Dp412e, BMP4, SOX2, and T expression after 3 days of BMP4 or Activin A treatment. Gene expression was normalized to peptidylprolyl isomerase A or cyclophilin A (PPIA) and plotted (log10 scale) relative to the mean expression of hiPSCs 1 at day 3 without treatment (n = 2). c Western blot analysis of phospho-SMAD 1/5 in hiPSCs 1 from days 0 through 4 after BMP4 treatment. α-Tubulin was used as loading control. d Quantitative RT-PCR in hiPSCs 1 of DMD transcripts with specific primers to exons 2–3, PAX3, T, PITX2, ISL1, ACTC1, ACTA2, GATA4, and BMP4 3 days after BMP4 treatment, with or without Noggin (NOG) pre-treatment. Gene expression was normalized to GAPDH and plotted (log10 scale) relative to hiPSCs 1 at D0
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Fig4: Correlation of BMP pathway activation with mesodermal commitment and Dp412e expression. a Microarray data showing 2-dCT of Human TGF-β/BMP Signaling Pathway genes in hiPSCs 1 without or 3 days following a single BMP4 treatment or with addition of NOG prior to BMP4 treatment (four technical duplicates/condition). Genes selected for display showed a minimum of ±2-fold change on RQs calculation in either BMP4-treated hiPSCs 1 or NOG/BMP4-treated hiPSCs 1, as compared to hiPSCs 1 without BMP4 treatment (p < 0.05; based on a Student’s t test). b Quantitative RT-PCR in hiPSCs 1 of Dp412e, BMP4, SOX2, and T expression after 3 days of BMP4 or Activin A treatment. Gene expression was normalized to peptidylprolyl isomerase A or cyclophilin A (PPIA) and plotted (log10 scale) relative to the mean expression of hiPSCs 1 at day 3 without treatment (n = 2). c Western blot analysis of phospho-SMAD 1/5 in hiPSCs 1 from days 0 through 4 after BMP4 treatment. α-Tubulin was used as loading control. d Quantitative RT-PCR in hiPSCs 1 of DMD transcripts with specific primers to exons 2–3, PAX3, T, PITX2, ISL1, ACTC1, ACTA2, GATA4, and BMP4 3 days after BMP4 treatment, with or without Noggin (NOG) pre-treatment. Gene expression was normalized to GAPDH and plotted (log10 scale) relative to hiPSCs 1 at D0

Mentions: PCR arrays were used to evaluate the impact of BMP4 treatment on signaling among the whole TGF-β superfamily, including the BMP pathway. They showed upregulation of BMP pathway actors, such as several ligands (BMP2, 4, and 7), type I and II receptors (BMPR1A, ACVR1A, and BMPR2, ACVR2A), SMAD1 and SMAD5, SMAD4 and inhibitors (NOG, CHRD, BAMBI, SMAD6, and 7; Fig. 4a). Concerning the TGF-β pathway, type I receptor was upregulated but not type II. The Activin pathway actors were also upregulated with SMAD2, SMAD3, and the receptors cited above. However, 72 h following Activin A treatment, unlike brachyury (T), a specific mesoderm marker, Dp412e was not induced by the Activin A pathway (Fig. 4b). Moreover, under this condition, the level of SOX2 remained high while endogenous BMP4 was not upregulated.Fig. 4


Dp412e: a novel human embryonic dystrophin isoform induced by BMP4 in early differentiated cells.

Massouridès E, Polentes J, Mangeot PE, Mournetas V, Nectoux J, Deburgrave N, Nusbaum P, Leturcq F, Popplewell L, Dickson G, Wein N, Flanigan KM, Peschanski M, Chelly J, Pinset C - Skelet Muscle (2015)

Correlation of BMP pathway activation with mesodermal commitment and Dp412e expression. a Microarray data showing 2-dCT of Human TGF-β/BMP Signaling Pathway genes in hiPSCs 1 without or 3 days following a single BMP4 treatment or with addition of NOG prior to BMP4 treatment (four technical duplicates/condition). Genes selected for display showed a minimum of ±2-fold change on RQs calculation in either BMP4-treated hiPSCs 1 or NOG/BMP4-treated hiPSCs 1, as compared to hiPSCs 1 without BMP4 treatment (p < 0.05; based on a Student’s t test). b Quantitative RT-PCR in hiPSCs 1 of Dp412e, BMP4, SOX2, and T expression after 3 days of BMP4 or Activin A treatment. Gene expression was normalized to peptidylprolyl isomerase A or cyclophilin A (PPIA) and plotted (log10 scale) relative to the mean expression of hiPSCs 1 at day 3 without treatment (n = 2). c Western blot analysis of phospho-SMAD 1/5 in hiPSCs 1 from days 0 through 4 after BMP4 treatment. α-Tubulin was used as loading control. d Quantitative RT-PCR in hiPSCs 1 of DMD transcripts with specific primers to exons 2–3, PAX3, T, PITX2, ISL1, ACTC1, ACTA2, GATA4, and BMP4 3 days after BMP4 treatment, with or without Noggin (NOG) pre-treatment. Gene expression was normalized to GAPDH and plotted (log10 scale) relative to hiPSCs 1 at D0
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig4: Correlation of BMP pathway activation with mesodermal commitment and Dp412e expression. a Microarray data showing 2-dCT of Human TGF-β/BMP Signaling Pathway genes in hiPSCs 1 without or 3 days following a single BMP4 treatment or with addition of NOG prior to BMP4 treatment (four technical duplicates/condition). Genes selected for display showed a minimum of ±2-fold change on RQs calculation in either BMP4-treated hiPSCs 1 or NOG/BMP4-treated hiPSCs 1, as compared to hiPSCs 1 without BMP4 treatment (p < 0.05; based on a Student’s t test). b Quantitative RT-PCR in hiPSCs 1 of Dp412e, BMP4, SOX2, and T expression after 3 days of BMP4 or Activin A treatment. Gene expression was normalized to peptidylprolyl isomerase A or cyclophilin A (PPIA) and plotted (log10 scale) relative to the mean expression of hiPSCs 1 at day 3 without treatment (n = 2). c Western blot analysis of phospho-SMAD 1/5 in hiPSCs 1 from days 0 through 4 after BMP4 treatment. α-Tubulin was used as loading control. d Quantitative RT-PCR in hiPSCs 1 of DMD transcripts with specific primers to exons 2–3, PAX3, T, PITX2, ISL1, ACTC1, ACTA2, GATA4, and BMP4 3 days after BMP4 treatment, with or without Noggin (NOG) pre-treatment. Gene expression was normalized to GAPDH and plotted (log10 scale) relative to hiPSCs 1 at D0
Mentions: PCR arrays were used to evaluate the impact of BMP4 treatment on signaling among the whole TGF-β superfamily, including the BMP pathway. They showed upregulation of BMP pathway actors, such as several ligands (BMP2, 4, and 7), type I and II receptors (BMPR1A, ACVR1A, and BMPR2, ACVR2A), SMAD1 and SMAD5, SMAD4 and inhibitors (NOG, CHRD, BAMBI, SMAD6, and 7; Fig. 4a). Concerning the TGF-β pathway, type I receptor was upregulated but not type II. The Activin pathway actors were also upregulated with SMAD2, SMAD3, and the receptors cited above. However, 72 h following Activin A treatment, unlike brachyury (T), a specific mesoderm marker, Dp412e was not induced by the Activin A pathway (Fig. 4b). Moreover, under this condition, the level of SOX2 remained high while endogenous BMP4 was not upregulated.Fig. 4

Bottom Line: Unexpectedly, 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.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.

View Article: PubMed Central - PubMed

Affiliation: I-STEM, CECS, Génopôle-Campus 1, 5 rue Henri Desbruères, 91030 Evry, Cedex France.

ABSTRACT

Background: Duchenne 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.

Methods: The 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.

Results: Unexpectedly, 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.

Conclusions: In 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.

No MeSH data available.


Related in: MedlinePlus