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The new DMD exon 1 belongs to a retrovirus-like sequence. (a) Alignment of the new exon 1 region (highlighted in light blue) among 100 vertebrate species with the conserved upstream/downstream sequence marked by red arrows ( https://genome.ucsc.edu ). (b) Schematic representation of the approximately 8 kb region in the DMD gene that is conserved among a sub-group of anthropoids. It is composed of simple repeats, Alu sequences and the whole human endogenous retrovirus-like sequence HuERS-P1 ( http://www.dfam.org/entry/DF0000214 ) flanked by two LTR8 elements.

Dystrophin protein expression. (a) Western blot analyses of protein extracts from hiPSCs 1 at day 4 either without or after BMP4 treatment using antibodies directed against different regions of dystrophin. (Dystrophin antibodies: Manex6 (exon 6); Mandys19 (exon 21), Mandys101 (exon 40–41), Manhinge4A (exon 62), Manex7374A (exon 73–74), Mandra1 (exon 77). (b) Western blot with DYS1 antibody in three pluripotent stem cell lines (hiPSCs 2 and 4, hESCs 2) from days 3 through 7 following BMP4 treatment. (c) Quantification of dystrophin protein levels from the Western blots (b) in Fig. 3 and (b) in Fig. S6 (Muscle biopsy protein extract from a healthy individual serves as a control and α-tubulin was used as loading control).

Dp412e expression study. Fold change obtained by quantitative RT-PCR using primers specific for Dp427m, Dp427c and Dp412e on RNAs from either human adult tissues, 7–11 weeks old fetuses or human 25–40 weeks old fetal tissues. Gene expression was normalized to UBC and relative to BMP4-treated hiPSCs 1 at day 3.

5′RACE reveals the presence of a novel exon 1. (a, b) Two sequences found several times by 5′RACE PCR on RNA from hiPSCs 1 three days after BMP4 treatment. In black are represented the nucleotides found to be spliced to the DMD gene exon 2 by 5′RACE PCR, with in (a), the 164 last nucleotides of Dp427c exon 1 and in (b), the new exon 1. In red are the parts of the RACE sequence corresponding to DMD gene exon 2. The black box in Dp427c sequence points out a nucleotide that does not match with Dp427c exon 1 reference sequence. The BLAT analyses were done on the web site https://genome.ucsc.edu .

BMP4-treated hiPSCs/hESCs express dystrophin protein. (a) Western blot in six pluripotent stem cell lines (hiPSCs 1, 3 and 4, DMD hiPSCs 1 and 3, hESCs 2) at day 4 either without or after BMP4 treatment. (b) Western blot in hiPSCs 1 from days 0 through 4 after a single BMP4 treatment. (Dystrophin antibody: DYS1; Muscle biopsy protein extract from a healthy individual serves as a control, α-tubulin was used as loading control).

The new DMD exon 1 belongs to a retrovirus-like sequence. (a) Alignment of the new exon 1 region (highlighted in light blue) among 100 vertebrate species with the conserved upstream/downstream sequence marked by red arrows ( https://genome.ucsc.edu ). (b) Schematic representation of the approximately 8 kb region in the DMD gene that is conserved among a sub-group of anthropoids. It is composed of simple repeats, Alu sequences and the whole human endogenous retrovirus-like sequence HuERS-P1 ( http://www.dfam.org/entry/DF0000214 ) flanked by two LTR8 elements.

BMP4-treated hPSCs express a new transient long DMD transcript. qRT-PCR of DMD transcripts using primers specific to exons 2–3 in hiPSCs 1 and DMD hiPSCs 3 at days 3 through 7 after BMP4 treatment. For each cell line, gene expression was normalized to GAPDH and plotted (log10 scale) relative to the expression at D0.

In silico translation of the novel DMD transcript. Representation of the open reading frame (ORF) resulting in translation of the novel DMD transcript. Possible translated proteins from this ORF are represented in red. The largest predicted protein is composed of 3562 amino acids (≈412 kDa) ( http://web.expasy.org/translate ).

Primer list. Primers used in the present study.

BMP4-treated hPSCs express a new transient long DMD transcript. qRT-PCR of DMD transcripts using primers specific to exons 2–3 in hiPSCs 1 and DMD hiPSCs 3 at days 3 through 7 after BMP4 treatment. For each cell line, gene expression was normalized to GAPDH and plotted (log10 scale) relative to the expression at D0.

In silico translation of the novel DMD transcript. Representation of the open reading frame (ORF) resulting in translation of the novel DMD transcript. Possible translated proteins from this ORF are represented in red. The largest predicted protein is composed of 3562 amino acids (≈412 kDa) ( http://web.expasy.org/translate ).

Dp412e expression study. Fold change obtained by quantitative RT-PCR using primers specific for Dp427m, Dp427c and Dp412e on RNAs from either human adult tissues, 7–11 weeks old fetuses or human 25–40 weeks old fetal tissues. Gene expression was normalized to UBC and relative to BMP4-treated hiPSCs 1 at day 3.

BMP4-treated hiPSCs/hESCs express dystrophin protein. (a) Western blot in six pluripotent stem cell lines (hiPSCs 1, 3 and 4, DMD hiPSCs 1 and 3, hESCs 2) at day 4 either without or after BMP4 treatment. (b) Western blot in hiPSCs 1 from days 0 through 4 after a single BMP4 treatment. (Dystrophin antibody: DYS1; Muscle biopsy protein extract from a healthy individual serves as a control, α-tubulin was used as loading control).

Human embryonic and induced pluripotent stem cell line list. Details concerning the stem cell lines used in the present study.

Dystrophin protein expression. (a) Western blot analyses of protein extracts from hiPSCs 1 at day 4 either without or after BMP4 treatment using antibodies directed against different regions of dystrophin. (Dystrophin antibodies: Manex6 (exon 6); Mandys19 (exon 21), Mandys101 (exon 40–41), Manhinge4A (exon 62), Manex7374A (exon 73–74), Mandra1 (exon 77). (b) Western blot with DYS1 antibody in three pluripotent stem cell lines (hiPSCs 2 and 4, hESCs 2) from days 3 through 7 following BMP4 treatment. (c) Quantification of dystrophin protein levels from the Western blots (b) in Fig. 3 and (b) in Fig. S6 (Muscle biopsy protein extract from a healthy individual serves as a control and α-tubulin was used as loading control).

5′RACE reveals the presence of a novel exon 1. (a, b) Two sequences found several times by 5′RACE PCR on RNA from hiPSCs 1 three days after BMP4 treatment. In black are represented the nucleotides found to be spliced to the DMD gene exon 2 by 5′RACE PCR, with in (a), the 164 last nucleotides of Dp427c exon 1 and in (b), the new exon 1. In red are the parts of the RACE sequence corresponding to DMD gene exon 2. The black box in Dp427c sequence points out a nucleotide that does not match with Dp427c exon 1 reference sequence. The BLAT analyses were done on the web site https://genome.ucsc.edu .

Primer list. Primers used in the present study.