Generation of two genomic-integration-free DMD iPSC lines with mutations affecting all dystrophin isoforms and potentially amenable to exon-skipping

Duchenne muscular dystrophy (DMD) is the most common paediatric muscular dystrophy and is caused by mutations in the DYSTROPHIN gene. We generated two induced pluripotent stem cell (iPSC) lines from DMD patients with nonsense mutations in exons 68 (UCLi011-A) or 70 (UCLi012-A) by transfecting reprogramming mRNAs. Both mutations affect expression of all dystrophin isoforms. iPSCs expressed pluripotency-associated markers, differentiated into cells of the three germ layers in vitro and had normal karyotypes. The selected mutations are potentially amenable to read-through therapies, exon-skipping and gene-editing. These new iPSCs are also relevant to study DYSTROPHIN role in tissues other than skeletal muscle.


Resource utility
The new genomic-integration-free DMD iPSC lines UCLi011-A and UCLi012-A carry nonsense mutations beyond exon 63 of the dystrophin gene (Table 1). Although uncommon, mutations located between exon 63 and exon 79 cause loss of all the dystrophin isoforms including Dp71, the most abundant isoform in brain, and which deficiency is highly associated with cognitive impairment. These mutations are potentially amenable to therapeutic approaches based upon exon-skipping and read-through strategies (reviewed in Scoto et al., 2018) and are relevant to study dystrophin role both in muscle and extra-muscular tissues. Overall, UCLi011-A and UCLi012-A iPSCs will be useful to study the impact of dystrophin deficiency in multiple tissues and to screen possible therapies, particularly using recently-established platforms of complex muscle disease modelling in vitro (Maffioletti et al., 2018

Resource details
DMD is an inherited muscle-wasting disorder of childhood caused by mutations in the dystrophin gene (Mercuri and Muntoni 2013). Dystrophin is the largest gene in nature and has a very complex transcriptional regulation, with several tissue specific isoforms associated with their own promoter and unique first exon (Muntoni et al., 2003). The deficiency of Dp71, the shortest isoform, although ubiquitously expressed, has been linked to severe cognitive deficit, thus raising interest in the function it plays in the central nervous system. The promoter and unique first exons of this isoform is located in intron 62 of the dystrophin gene, so any mutations located towards the 3′ of exon 63 will affect Dp71 in addition to all the remaining isoforms.
The resulting iPSC lines showed the expected morphology of human pluripotent colonies (Fig. 1A) and expressed the pluripotency-associated markers OCT4, NANOG and SOX2 at mRNA and protein levels ( Fig. 1B,C). Both iPSCs UCLi011-A and UCLi012-A presented a normal karyotype (46,XY) with a correct ploidy and no major chromosomal abnormalities ( Fig. 1D; UCLi011-A tested at passage 8; UCLi012-A tested at passage 6). Sanger sequencing confirmed that after reprogramming the DMD iPSCs still harbour the disease-causing mutations located in exon 68 (c.9851G>A (p.Trp3284X) and 70 c.10141C>T (p.Arg3381X) of the dystrophin gene (Fig. 1D). Functional pluripotency was demonstrated by differentiation into cell types of the three germ layers in embryoid body formation assays (Fig. 1E).
Additionally, cell identity was confirmed by STR analysis, which demonstrated a 100% match in the analysed alleles of parental fibroblasts and derived iPSCs (available with the authors). Finally, both iPSC lines were negative for Mycoplasma contamination (characterization and validation summarized in Table 2).

Immunofluorescence
Cells were washed with PBS, fixed with 4% (w/v) PFA for 5 min, followed by a further PBS wash. Fixed cells were permeabilized for 1 hour with permeabilization solution (1% bovine serum albumin (BSA) + 0.2% Triton in PBS) at room temperature. Cells were then blocked for 30 min with 10% donkey or goat serum diluted in permeabilizing solution at room temperature. Cells were then incubated overnight at 4°C with the primary antibodies (Table 3) diluted in permeabilization solution. Unbound primary antibody was removed with three washes of 0.2% Triton in PBS. Cells were then incubated for 1 hour with secondary antibodies and Hoechst 33342 diluted in 0.2% Triton in PBS. Unbound secondary antibody was washed away with two washes of 0.2% Triton in PBS, followed by one rinse with PBS. Cells were imaged with an inverted fluorescence microscope (Leica DM16000B).

qPCR analysis
RNA was isolated from cell pellets using RNeasy Mini kit (Qiagen; 74,104) according to manufacturer's instructions. A DNase step was included to eliminate genomic contamination. RNA quality and yield was assessed using a Nanodrop. Retro-transcription to cDNA was conducted with the ImProm-II™ Reverse Transcription System kit (Promega; A3800) following manufacturer's instructions. qPCRs were performed with SYBR-Green Real Time Master Mix (Promega; A600A) according to manufacturer instructions using the BioRad CFX96 machine. A house keeping gene (GAPDH) reaction was included on each plate for all samples. The ΔCT method has been used to analyse the experimental CT values. A commercially-available human iPSC line (Gibco; cat. no. A13777) has been included as a positive control and human myoblasts provided a negative control. Primer sequences are listed in Table 3.

Embryoid body formation assay
iPSCs were dissociated into clumps using gentle cell dissociation reagent and embryoid bodies (EBs) were allowed to form and grow in suspension in TeSR™-E6 medium (Stemcell Technologies) in non-adhesive dishes. After 7 days EBs were transferred to standard 10 cm tissue cultures dishes to allow adhesion in DMEM (Sigma) with 20%(v/ v) FBS (Life technologies), 1% L-glutamine (Sigma), 1% PS (Sigma) in  G. Ferrari, et al. Stem Cell Research 43 (2020) 101688 (caption on next page) G. Ferrari, et al. Stem Cell Research 43 (2020) 101688 3% O 2 and 5% CO 2 to induce spontaneous differentiation. Media was changed every other day and plates were fixed in 4% PFA after 14-20 days of differentiation.

Sequencing, STR profiling and karyotype analysis
Genomic DNA was extracted from each cell line by DNeasy kit (Quiagen). 100 ng/μl of Gotaq® DNA polymerase (Promega) was used for amplification (35 cycles using a BioRad T100™ Thermal cycler). DMD specific primers upstream and downstream the point mutations were designed (Table 3) and purified PCR reactions sequenced via dideoxynucleoside Sanger sequencing by Source Biosciences (Cambridge). iPSCs UCLi011-A and UCLi012-A were authenticated by STR analysis performed by Source Biosciences (Nottingham) using Promega PowerPlex 16 HS assay (available with the authors). For each cell line karyotyped, a T25 flask of 80% confluent cells was sent to The Doctors Laboratory (TDL, London, UK) were G-band analysis was performed at a resolution of 6-10 MB (UCLi011-A: passage 8, 10 metaphase spreads analysed; UCLi012-A: passage 6, 20 metaphase spreads analysed).