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

Ethics, consent, and permissions

All healthy individuals and DMD patients had written an informed consent before the
biopsy procedure.

At the Cochin Hospital-Cochin Institute, the collection of primary cultures of myoblasts
was established from patient muscle biopsies conducted as part of medical diagnostic
procedure of neuromuscular disorders. For each patient included in this study, signed
informed consent was obtained to collect and study biological resources, and establish
primary cultures of fibroblasts and myoblasts at the Hospital Cell Bank-Cochin Assistance
Publique—Hôpitaux de Paris (APHP) . This collection of myoblasts was declared to legal
and ethical authorities at the Ministry of Research (number of declaration, 701, n°
of the modified declaration, 701–1) via the medical hosting institution, APHP, and
to the “Commission Nationale de l’Informatique et des Libertés” (CNIL) (number of
declaration, 1154515).

The BMD muscle biopsy was carried out in accordance with the ethical rules of the
institutions involved under approvals of the Nationwide Children’s Hospital Institutional
Review Board.

Cell culture

Human adult myoblasts from healthy individuals and DMD patients were provided by Celogos
and Cochin Hospital-Cochin Institute (Additional file 1: Table S1). In Celogos laboratory, cell preparation was done according to patent
US2010/018873 A1.

Cells were maintained in a myoblast medium: DMEM/F-12, HEPES (31330–038, Thermo Fisher
Scientific, MA, USA) supplemented with 10 % fetal bovine serum (FBS, Hyclone, Logan,
UT), 10 U/mL penicillin/streptomycin (15140122, Thermo Fisher Scientific) on 0.1 %
gelatin (G1393, Sigma-Aldrich®, St. Louis, MO, USA) coated cultureware with 10 ng/mL
fibroblast growth factor 2 (FGF2) (100-18B, Peprotech, Rocky Hill, NY), and Dexamethasone
50 nM (D4902, Sigma-Aldrich®).

For hiPSCs generation, plasmid MSCV-IRES-GFP (pMIG) vectors containing complementary
deoxyribonucleic acids (cDNAs) of v-myc avian myelocytomatosis viral oncogene homolog
(C-MYC), POU class 5 homeobox 1 or OCT-4 (POU5F1), SRY (sex determining region Y)-box
2 (SOX2), and Kruppel-like factor 4 (KLF4) were kindly provided by George Q. Daley
32]. These plasmids were individually transfected using Lipofectamine® 2000 (11668–027,
Thermo Fisher Scientific) into platinum (PLAT)-A packaging cells (for amphotropic
retroviral production, RV-102, Cell Biolabs, San Diego, CA) in a biosafety level 3
laboratory or PLAT-E packaging cells (for ecotropic retroviral production, RV-101,
Cell Biolabs) in a biosafety level 2 laboratory. PLAT cells medium was replaced 24 h
post-transfection: Dulbecco’s modified Eagle medium (DMEM), high glucose, GlutaMAX™
(31965023), pyruvate sodium 1 mM (11360–039), ?-mercaptoethanol 50 ?M (31350–010)
(Thermo Fisher Scientific), and 10 % FBS (Hyclone).

At the same time, myoblasts were thawed and seeded at 1?×?10
⁵
cells/cm
²
in a 6-well plate.

One day after seeding, myoblasts for ecotropic reprogramming were treated for 1 h
with murine cationic amino acid transporter-1 (mCAT-1) gesicles 2.5?×?10
^?2
?g/?L final concentration 33], in 500 ?L/well of fresh myoblast medium. Viral supernatants were collected 48 h
post-transfection, filtered through a 0.45-?m filter (146561, Dutscher SA, Brumath,
France), and mixed at a 1:1:1:1 ratio in a tube already containing Polybren (4 ?g/mL
final concentration, H9268, Sigma-Aldrich®) and Hepes (0.01 M final concentration,
15630056, Thermo Fisher Scientific). One milliliter of this mix was added to each
well of mCAT-1 gesicles-treated myoblasts.

The medium was changed the following day. Four days post-transduction, myoblasts were
passaged and seeded at three densities, 1.5?×?10
⁴
, 3?×?10
⁴
, and 6?×?10
⁴
cells/cm
²
, on feeder layers of Zenith CF1 MEF (ZFVC-001, IVF Online, Toronto, Canada), mitomycin-C
(M4287, Sigma-Aldrich®) treated. After 24 h, reprogrammed myoblasts were shifted in
“hESCs medium”: Knockout™ DMEM (10829–018), 20 % KnockOut™ Serum Replacement (10828–028),
1X MEM Non-Essential Amino Acids Solution (11140–035), 50 ?M ?-mercaptoethanol (31350–010),
2 mM GlutaMAX™ Supplement (35050–038), and 10 U/mL penicillin/streptomycin (15140–122)
(Thermo Fisher Scientific), 10 ng/mL FGF2 (Peprotech), supplemented with 0.5 mM valproic
acid (P4543, Sigma-Aldrich®) during 10 days, with medium change every 2 days.

Human myoblasts for amphotropic reprogramming were transduced by the amphotropic retrovector
mix for 24 h. They were then passaged and seeded in myoblast medium at 3.4?×?10
³
cells/cm
²
per well in 0.1 % gelatin-coated 6-well plates. The following day, the reprogrammed
myoblasts were shifted to hESCs medium with valproic acid treatment during 10 days
and medium change every 2 days.

The hiPSCs colonies were picked between day 15 and day 40 after transduction. They
were subsequently expanded with manual passages as clumps and maintained on mitomycin-C
treated mouse embryonic fibroblasts (MEFs) in hESCs medium. StemMACSâ„¢ Y27632 (130-103-922,
Miltenyi biotech, Bologna, Italy) was used at 10 ?M to increase the seeding efficiency
of hiPSCs for the initial colony expansion after picking, for the first passage and
at thawing.

Human iPSCs and ESCs (Additional file 1: Table S1) were harvested for banking in single cell with StemPro® Accutase® (A11105-01,
Thermo Fisher Scientific) and froze in Cryostor® CS10 (210102, BioLife Solutions,
Inc., Bothell, WA).

Before our experiments, all hiPSCs and hESCs were adapted and maintained with mTeSRâ„¢1
culture medium (05850, Stemcell Technologies, Vancouver, Canada) on Corning® Matrigel®
Basement Membrane Matrix, lactose dehydrogenase elevating virus (LDEV)-Free-coated
cultureware (354234, Corning Incorporated, NY, USA).

For adaptation of hiPSCs and hESCs to mTeSRâ„¢1 culture system (05850, Stemcell Technologies),
cells were thawed on mitomycin-C treated MEFs in mTeSRâ„¢1 medium (05850, Stemcell Technologies)
with 10 ?M StemMACS™ Y27632. After 6 days, the adaptation consisted of three successive
passages on Corning® Matrigel®-coated (354234) cultureware with several differentiation
removal steps. The cells were first passaged with collagenase type IV (7909, Stemcell
Technologies), then with dispase (1 U/mL, 7923, Stemcell Technologies) and finally
in single cell with StemPro® Accutase® (A11105-01, Thermo Fisher Scientific) with
an average of 5 days between each passage. At this step, cells must be mainly undifferentiated
to be successfully adapted to mTeSRâ„¢1 culture system (05850, Stemcell Technologies)
and to be banked. Cells were then seeded, passaged and thawed each time with 10 ?M
StemMACSâ„¢ Y27632.

We used for Fig. 1a the AnalySIS getIT 5.1 software with an Olympus CKX41 microscope and SC30 digital
camera.

Fig. 1. BMP4 treatment induces mesoderm lineage markers in hiPSCs/hESCs. a Examples of morphology in hiPSCs 3 and DMD hiPSCs 1 at day 4 either without or after
a single BMP4 treatment. Scale bar?=?50 ?m. b TaqMan® Human Stem Cell Pluripotency Array data showing 2
^-dCT
of lineage marker genes in hiPSCs 1 at day 3 either without or after a single BMP4
treatment. Genes selected for display showed a minimum of ±2 fold change on relative
quantifications (RQs) calculation in BMP4-treated hiPSCs 1, as compared to hiPSCs
1 without BMP4 treatment (p??0.05; based on a Student’s t test). c and d Quantitative RT-PCR of c pluripotency and d early mesoderm specific genes. Curves represent the mean?±?SD (standard deviation)
from nine pluripotent stem cell lines (hPSCs) at days 0 through 4 after BMP4 treatment.
Gene expression was normalized to the mean of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and ubiquitin C (UBC) and plotted (log10 scale) relative to the mean expression of all pluripotent stem
cells at day 0 (D0)

Cell treatments

Pluripotent stem cells were thawed on Corning® Matrigel®-coated (354234) cultureware
in mTeSR™1 (05850, Stemcell Technologies) with 10 ?M StemMACS™ Y27632. When the cells
were close to 70 % confluence, they were passaged and seeded at 4?×?10
⁴
cells/cm
²
with 10 ?M StemMACS™ Y27632, with or without recombinant human BMP4 (314-BP-050, RD
Systems, Minneapolis, MN) at a final concentration of 5 ng/mL. Collections of RNAs
or proteins were done without any further medium change. The medium was changed at
day 4 only for collections between days 5 and 7.

Recombinant human noggin (NOG, 120-10C, Peprotech) was added for BMP4 inhibition at
the seeding step at 100 ng/mL final concentration, 1 h prior BMP4 addition.

For Activin A treatment, after seeding of human pluripotent stem cells (hPSCs) as
described above for BMP4 treatment, Activin A was added at 10 ng/mL (120–14; Peprotech).

RNA purification and qRT-PCR

After harvesting pluripotent stem cells using StemPro® Accutase® (A11105-01, Thermo
Fisher Scientific) and myoblasts/myotubes with Trypsin-ethylenediaminetetraacetic
acid (EDTA) (25300–054, Thermo Fisher Scientific), cell pellets were resuspended in
350 ?L of lysis buffer containing guanidine isothiocyanate (RLT) (79216, Qiagen, Hilden,
Germany) and stored at ?80 °C. Total RNA were extracted and purified according to
either the RNeasy Mini Kit or Micro Kit protocol (74104 and 74004, Qiagen). Several
human RNAs were used for the dystrophin protein 412 kDa embryonic (Dp412e) expression study (Testis 636533; Ovary 636555; Fetuses, 636185; Master Panel II,
636643; Cerebellum, 636535; Cerebral cortex 636561; Fetal heart, 636583; Smooth muscle,
636547; Heart, 636532; Skeletal muscle, 636534; Clontech, Palo Alto, CA). Muscle RNA
from healthy individual biopsy was provided by Cochin Hospital-Cochin Institute.

RNA level and quality were checked using a Nanodrop spectrophotometer (ND-1000, Thermo
Fisher Scientific Inc., USA). For each sample, 500 ng of total RNA were reverse transcribed
with random primers (48190–011), oligo(dT) (SO131), and deoxynucleotide (dNTP) (10297–018)
using Superscript® III reverse transcriptase (18080–044) (Thermo Fisher Scientific).
Thermocycling conditions were 10 min, 25 °C; 60 min, 55 °C; and 15 min, 75 °C.

We amplified for quantitative real-time polymerase chain reaction (qRT-PCR) these
cDNA using primers (Thermo Fisher Scientific) listed in Additional file 2: Table S2. They were designed using Primer 3 (http://primer3.ut.ee) 34], Primer blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast) and amplifX (v1.5.4, by Nicolas Jullien; CNRS; Aix-Marseille University; http://crn2m.univ-mrs.fr/pub/amplifx-dist). The amplification efficiency of each primer set (except for QT primer and Dp412e forward primer) was preliminarily determined by running a standard curve. Detection
was performed using a QuantStudioâ„¢ 12K Flex Real-Time PCR System (Thermo Fisher Scientific).
Reactions were carried out in a 384-well plate, with 10 ?L containing diluted cDNA
and primers, as well as Luminaris Color HiGreen qPCR Master Mixes Low Rox (K0973,
Thermo Fisher Scientific Inc.). Thermocycling conditions were 50 °C during 2 min,
95 °C during 10 min, followed by 45 cycles including 15 sec at 95 °C, 1 min at 60 °C
plus a dissociation stage. All samples were measured in triplicate.

Protein isolation and Western blot analyses

After three rinses with cold PBS 1X (w/o Ca
²⁺
and Mg
²⁺
, D8537, Sigma-Aldrich®), protein extracts were isolated from cultured cells by scraping
(010154, Dutscher) with an extraction protein buffer (75 mM Tris–HCl pH 6.8, 15 %
sodium dodecyl sulfate (SDS), 5 % ?-mercaptoethanol, 20 % glycerol, 4?×?10
^?4
mg/?L bromophenol blue, Protease Inhibitor Cocktail diluted 1:100 (P8340, Sigma-Aldrich®),
PhosSTOP tablet (04906845001, Roche Diagnostics Corp., Indianapolis, USA)). Protein
extracts were then heated once 5 min at 95 °C. Depending on the viscosity of the protein
extract, additional protein extraction buffer was needed for some samples before a
second and sometimes a third step of 5-min heating. Protein Extracts were centrifuged
7 min at 16,000 g, and supernatants were kept at ?80 °C. Muscle protein extracts from
healthy individual biopsy were provided by Cochin Hospital-Cochin Institute. The Western
blots were performed for dystrophin detection with Criterionâ„¢ XT Tris-Acetate Precast
Gels 3–8 % (345-0129/30, Bio-Rad, Hercules, CA) and XT Tricine running buffer (161–0790,
Bio-Rad). Samples were run at room temperature (RT) for 1 h and 15 min at 150 V (except
for samples in Fig. 3c: 2 h and 30 min at 150 V with running buffer replenishing after 1 h and 15 min) with
HiMarkâ„¢ Pre-Stained Protein Standard (LC5699, Thermo Fisher Scientific). Gels were
rinsed once in water and blotted for 11 min with “high molecular weight” program of
TransBlot® Turbo™ transfer system (Bio-Rad) using Trans-Blot®Turbo™ Midi Nitrocellulose
Transfer Packs (170–4159). Blots were blocked for 45 min with 5 % non-fat dried milk
(170–6404, Bio-Rad) in phosphate-buffered saline tween (PBST) buffer (Phosphate-buffered
saline 1X tablets, P4417, Sigma-Aldrich®; 0.1 % Tween® 20, 28829.296, VWR, West Chester,
PA) followed by an overnight incubation at 4 °C under agitation with either NCL-DYS1
Dy4/6D3 (RRID: AB_442080, Novocastra Laboratories, Newcastle, UK) 1:30 or Manex6 35] kindly provided by Prof. Glenn Morris (MDA Monoclonal Antibody Resource, Wolfson
Centre for Inherited Neuromuscular Disease, Oswestry, UK) 1:30, Mandra1 clone 7A10
(exon 77; Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA) 1:10, Manhinge4A
clone 5C11 (exon 62; DSHB) 1:30, Manex7374A clone 10A11 (exon 73–74; DSHB) 1:30, Mandys19
clone 8F6 (exon 21; DSHB) 1:30, and Mandys101 clone 7D12 (exon 40–41; DSHB) 1:30,
in 5 % non-fat dried milk in PBST buffer. After PBST rinses, horseradish peroxidase
(HRP)-conjugated polyclonal rabbit anti-mouse (P0260, DAKO, Glostrup, Denmark) 1:5000
was used as a secondary antibody after DYS1 or ECL Anti-Mouse IgG, horseradish peroxidase-linked
species-specific whole antibody from sheep 1:10,000 (NA931, GE Healthcare Life Sciences)
after all the other primary dystrophin antibodies in 5 % non-fat dried milk in PBST,
for 5 h at RT under agitation. The blots were rinsed with PBST before immuno-reactive
bands were visualized using Amersham ECL Select Western Blotting Detection Reagent
according to the manufacturer’s protocol (RPN2235, GE Healthcare Life Sciences, Buckinghamshire,
UK) and the ImageQuant LAS 4000 mini system (GE Healthcare Life Sciences).

After DYS1 visualization, blots were rinsed in PBST and stripped with Restoreâ„¢ Western
Blot Stripping buffer (21059, Thermo Fisher Scientific) for 15 min at RT. After additional
PBST rinses, blots were blocked again and incubated with mouse monoclonal anti-alpha
Tubulin antibody (DM1A) (ab7291, Abcam, Cambridge, UK) 1:2500 in 5 % non-fat dried
milk PBST, during 1 h at RT under agitation. After PBST rinses, ECL Anti-Mouse IgG,
horseradish peroxidase-linked species-specific whole antibody from sheep 1:10,000
(NA931, GE Healthcare Life Sciences) was used as a secondary antibody in 5 % non-fat
dried milk PBST, for 1 h at RT under agitation. Amersham ECL Prime Western Blotting
Detection Reagent (RPN2232, GE Healthcare Life Sciences) was used for immuno-reactive
bands visualization.

?-Tubulin detection was done either after DYS1 for the blots on Fig. 3, in Additional file 3: Figure S5a and Additional file 4: Figure S6b or in parallel to DYS1 and other dystrophin antibodies for all the other
blots presented in this study by cutting the membranes before primary antibody incubation.

Western blots were performed for Phospho-small mothers against decapentaplegic (SMAD)1/5
detection with Criterion™ TGX™ Precast Gels 4–15 % (5671084, Bio-Rad) and Tris/Glycine/SDS
running buffer (1610772, Bio-Rad). Samples were run at RT for 55 min at 200 V. Gels
were rinsed once in water and blotted for 7 min with “mixed MW” program of TransBlot®
Turboâ„¢ transfer system (Bio-Rad).

Blots were blocked for 1 h with 5 % non-fat dried milk in tris-buffered saline tween
(TBST) buffer at RT (137 mM NaCl, 20 mM Tris pH 7.6, 0.1 % Tween® 20 (28829.296, VWR))
followed by an overnight incubation at 4 °C under agitation with Phospho-SMAD1/5 antibody
(Ser463/465, 41D10; 9516P, Cell Signaling, Beverly, MA) 1:1000 in 10 % bovine serum
albumin (BSA) TBST. After TBST rinses, ECL Anti-Rabbit IgG, HRP-linked (NA934, GE
Healthcare Life Sciences) 1:10,000 was used as a secondary antibody in 5 % non-fat
dried milk TBST, for 1 h at RT under agitation. The blots were rinsed with TBST before
immuno-reactive bands visualization with Amersham ECL Prime. ?-Tubulin detection was
then done after a stripping step (see above).

Quantification showed in Additional file 4: Figure S6c was done with the ImageQuant TL 7.0 software.

5?RACE PCR

Characterization of transcripts by rapid amplification of cDNA 5? ends (5?RACE) polymerase
chain reaction (PCR) was performed using the SMARTer® RACE cDNA Amplification Kit
(634923, Clontech) according to the manufacturer instructions. RNA of the precursors
derived from hiPSCs 1 3 days after BMP4 treatment was used for this purpose. Briefly,
integrity of purified RNAs was checked upon migration in an agarose gel stained with
Ethidium Bromide. RNA (500 ng) was used as a template to produce the RACE-ready cDNAs
whose 5? extremities are modified to incorporate the Universal Primer (UPA) sequence.
A first round of cDNA amplification was performed using a first set of primers (Forward:
UPA long: 5?-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3?, Reverse: DP427R1: 5?-TGTAGGTCACTGAAGAGGTTCTCAA-3?
corresponding to a region in the third exon of DMD cDNA). Products of amplification were next amplified by nested primers (Forward:
NUPA: 5?-AAGCAGTGGTATCAACGCAGAGT-3?, Reverse: DP427R2: 5?-TGTGCATTTACCCATTTTGTG-3?
corresponding to a region in the second DMD exon). To increase the specificity of the RACE assay, priming of cDNAs synthesis
was also achieved using a DMD-specific primer in the fourth exon (Ex4R1: 5?-GGGCATGAACTCTTGTGGAT-3?) instead of
the polyT primer included in the SMARTer® RACE cDNA Amplification Kit. cDNAs obtained
with this modified protocol were next amplified by primers Forward: UPA long: 5?-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3?,
Reverse: 602Rex3: 5?-GGTCTAGGAGGCGCCTCCCATCCTGTAG-3?), followed by a nested PCR using
Forward: NUPA: 5?-AAGCAGTGGTATCAACGCAGAGT-3?, Reverse: 603Rexnew: 5?-TCCACACCAGGTGGGGACGGATGACCT-3?
corresponding to Dp412e exon 1.

Amplicons from the nested PCRs were finally cloned using the Zero Blunt® PCR cloning
kit (K2700-20, Thermo Fisher Scientific) prior sequencing (GATC Biotech, Konstanz,
Germany). Overlapping reads were assembled into contigs mapped on the DMD gene.

Immunolabeling

After 4 days with or without BMP4 treatment, slides for immunolabeling were prepared
with the Thermo Scientificâ„¢ Cytospinâ„¢ 4 Cytocentrifuge. Briefly, cultures were washed
once with PBS 1X (P4417, Sigma-Aldrich®), and StemPro® Accutase® (A11105-01, Thermo
Fisher Scientific) was added for 5 min at 37 °C. The cells were harvested and transferred
in a 15-mL tube, and the enzyme was diluted in DMEM/F-12, HEPES (31330–038, Thermo
Fisher Scientific). Cells were counted and resuspended at 400,000-cells/mL concentration.
Five hundred microliter of the cell suspension were loaded in each Shandon cytospin®
centrifuge funnels, such as a spot of 200,000-cells was available on each superfrost®
plus slide (631–0108, VWR) at the end of the centrifugation cycle (900 rpm, 4 min).
Cells were immediately fixed with 4 % paraformaldehyde (15710, Euromedex, Souffelweyersheim,
France) for 10 min at RT and then washed three times in PBS 1X for 15 min each.

Cell spots were delimited for immunolabeling with a PAP pen (Z672548-1EA, Sigma-Aldrich®) and then permeabilized with PBS 1X containing 0.3 % Triton™ X-100 (T8787, Sigma-Aldrich®) for 30 min at RT. Incubation with pure NCL-DYS1 Dy4/6D3 (Novocastra Laboratories)
was performed overnight at 4 °C, then slides were washed three times with PBS 1X for
30 min each wash. Incubation with a mix of Dylight 549-conjugated AffiniPure F(ab?)2
fragment Goat Anti-Mouse IgG (H+L) (1:1000; 115-506-003, Jackson ImmunoResearch laboratories
Inc., PA, USA) and DAPI (1:2000; 268298, Calbiochem, La Jolla, CA) was done for 30 min
at RT. Slides were finally washed three times with PBS 1X (30 min/wash) and mounted
with fluoromount (F4680; Sigma-Aldrich®). Observation and image captures were done on an AxioObserver Z1 Zeiss microscope
with Zen Blue software (Carl Zeiss Microscopy GmbH, Jena, Germany).

For Fig. 3d, 40 % of brightness were added in Microsoft® PowerPoint® 2010 after the TIFF image
insertion of hiPSCs 1 and DMD hiPSCs 2.

PCR and TaqMan® arrays analyses

TaqMan® Array Human Stem Cell Pluripotency Panel (4385344, Thermo Fisher Scientific)
was used to investigate the differentiation status of BMP4-treated hiPSCs 1 at day
3 as compared to untreated hiPSCs 1. According to TaqMan® Array protocol (Thermo Fisher
Scientific), instead of 500 ng, we used 1500 ng of total RNA for each sample to do
the reverse transcription (for details, see “RNA Purification and qRT-PCR” paragraph
above). Each cDNA sample was mixed with 2X TaqMan® Gene Expression Master Mix (4369016,
Thermo Fisher Scientific) and distributed in triplicates on the TaqMan® arrays. qRT-PCR
were performed on an Applied Biosystems 7900HT Fast Real-Time PCR System (2 min, 50 °C;
10 min, 95 °C; 40X (15 s, 95 °C; 60 s, 60 °C)), and raw data were formatted to be
analyzed using the online SAbioscience tool.

TGF-?/BMP signaling pathway study was performed using 384 wells RT
²
Profiler Plus PCR Array (PAHS-035YE, Qiagen) according to the Qiagen Handbook. After
total RNA isolation using RNeasy Mini kit (74104, Qiagen), 500 ng of RNA for each
sample were treated with Buffer GE (Qiagen; 5 min, 42 °C) to eliminate genomic DNA.
Purified RNAs were then reverse transcribed using the RT
²
First Strand Kit (330401, Qiagen; 5X Buffer BC3, Control P2, RE3 Reverse Transcriptase
Mix; 15 min, 42 °C then 5 min, 95 °C). Each cDNA sample was complemented with Qiagen
2X RT
²
SYBR Green Mastermix (330521, Qiagen) and distributed in quadruplicates in the PCR
arrays.

Three samples were tested at day 3: hiPSCs 1 without treatment, after BMP4, or NOG
+ BMP4 treatment. Analyses (?Ct, ??Ct, Fold Change, statistical analyses) were performed
using the online SAbioscience tool associated to the RT
²
PCR arrays (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php).

Embryoid bodies

We used the Stemcell Technologies protocol to produce embryoid bodies (EBs). Briefly,
pluripotent stem cells were thawed on Corning® Matrigel®-coated (354234) flasks in
mTeSR™1 (05850, Stemcell Technologies) with 10 ?M StemMACS™ Y27632. When the cells
were close to 70 % confluence, they were harvested using StemPro® Accutase® (A11105-01,
Thermo Fisher Scientific) and resuspended in AggreWellâ„¢ medium (05893, StemCell Technologies)
supplemented with 10 ?M StemMACS™ Y27632 and finally seeded on AggreWell™ TM800 culture
plates (27865, Stemcell Technologies). According to the StemCell procedure, three
million cells were thus distributed on each well to form a maximum of 300 EBs with
10,000 cells per EB. EBs were maintained in Aggrewell plates during the whole culture
duration to avoid EBs fusion and to preserve a homogenous differentiation. Half of
the culture media was gently replaced every 2 days. For BMP4 treatment, BMP4 was added
directly in Aggrewell™ medium at EBs seeding (30 ng/mL) and every medium change. To
harvest EBs, the whole culture medium of a well was pipetted up and down, transferred
to a 40 ?m strainer (352340, Dutscher) to eliminate any single cell, and each well
was washed twice with DMEM/F-12, HEPES (31330–038, Thermo Fisher Scientific). EBs
were then transferred to a new tube with DMEM/F-12, HEPES, centrifuged 5 min at 300 g,
resuspended in buffer RLT (79216, Qiagen) or protein extraction buffer (see “Protein
isolation and Western Blot analyses” section), and stored at ?80 °C for subsequent
analyses.

Exon skipping

Two days after induction, BMP4-treated hiPSCs 1 were transfected by electroporation
with a phosphorodiamidate morpholino oligomer (PMO) targeting exon 53 of the DMD gene, based on previously published data 36] at 1, 10, or 100 ?M with 200,000 cells in 20 ?L solution from the P3 Primary Cell
4D-Nucleofector® X Kit (V4XP-3032, Lonza, Basel, Switzerland) using the CB150 program
on the 4D-Nucleofectorâ„¢ System (Lonza). RNA extraction was carried on transfected
cells 24 h later followed by reverse transcription as described above. PCR was done
on 250 ng cDNA using forward and reverse primers (Fw 5?-TTACCGACTGGCTTTCTCTGC-3? and
Rv 5?-GTCTGCCACTGGCGGAGGTC-3?, Thermo Fisher Scientific) and Taq DNA polymerase (10342,
Thermo Fisher Scientific) as described by the manufacturer’s instructions, for a final
reaction volume of 50 ?L. PCR reaction started by a step at 94 °C for 3 min, followed
by 35 cycles at 94 °C for 45 s, 55 °C for 45 s and 72 °C for 45 s, and a final step
at 72 °C for 5 min. Exon skipping was analyzed using the DNA 1000 kit (5067, Agilent,
Santa Clara, CA, USA) on the Agilent 2100 Bioanalyzer. Full-length PCR product was
460 bp and exon skipped length PCR product was 248 bp. Results are displayed as a
gel-like image and computed by the Agilent 2100 Bioanalyzer software v3.81.

Statistical analyses

Except for the PCR arrays, all statistical analyses were done with JMP® (v9.0.2).
We used Wilcoxon’s test for qRT-PCR analyses. No statistical difference was found
between DMD hiPSCs and normal hPSCs at day 0 and every day after BMP4 induction until
day 4. Therefore, DMD patients and healthy individuals were analyzed together for
this study. Exceptionally, BMP4-treated DMD hiPSCs 2 sample was removed from the analysis
of the primer set specific to exons 20–21, as we did not detect any DMD expression due to the patient genetic mutation.