Generation of Isogenic D4Z4 Contracted and Noncontracted Immortal Muscle Cell Clones from a Mosaic Patient: A Cellular Model for FSHD

Yvonne D Krom; Julie Dumonceaux; Kamel Mamchaoui; Bianca den Hamer; Virginie Mariot; Elisa Negroni; Linda N Geng; Nicolas Martin; Rabi Tawil; Stephen J Tapscott; Baziel GM van Engelen; Vincent Mouly; Gillian S Butler-Browne; Silvère M van der Maarel

doi:10.1016/j.ajpath.2012.07.007

. 2012 Oct;181(4):1387–1401. doi: 10.1016/j.ajpath.2012.07.007

Generation of Isogenic D4Z4 Contracted and Noncontracted Immortal Muscle Cell Clones from a Mosaic Patient

A Cellular Model for FSHD

Yvonne D Krom ^⁎, Julie Dumonceaux ^†,^‡,^§, Kamel Mamchaoui ^†,^‡,^§, Bianca den Hamer ^⁎, Virginie Mariot ^†,^‡,^§, Elisa Negroni ^†,^‡,^§,^¶, Linda N Geng ^∥, Nicolas Martin ^†,^‡,^§, Rabi Tawil ^⁎⁎, Stephen J Tapscott ^∥,^††, Baziel GM van Engelen ^‡‡, Vincent Mouly ^†,^‡,^§, Gillian S Butler-Browne ^†,^‡,^§, Silvère M van der Maarel ^⁎,^⁎

PMCID: PMC3463638 PMID: 22871573

Abstract

In most cases facioscapulohumeral muscular dystrophy (FSHD) is caused by contraction of the D4Z4 repeat in the 4q subtelomere. This contraction is associated with local chromatin decondensation and derepression of the DUX4 retrogene. Its complex genetic and epigenetic cause and high clinical variability in disease severity complicate investigations on the pathogenic mechanism underlying FSHD. A validated cellular model bypassing the considerable heterogeneity would facilitate mechanistic and therapeutic studies of FSHD. Taking advantage of the high incidence of somatic mosaicism for D4Z4 repeat contraction in de novo FSHD, we have established a clonal myogenic cell model from a mosaic patient. Individual clones are genetically identical except for the size of the D4Z4 repeat array, being either normal or FSHD sized. These clones retain their myogenic characteristics, and D4Z4 contracted clones differ from the noncontracted clones by the bursts of expression of DUX4 in sporadic nuclei, showing that this burst-like phenomenon is a locus-intrinsic feature. Consequently, downstream effects of DUX4 expression can be observed in D4Z4 contracted clones, like differential expression of DUX4 target genes. We also show their participation to in vivo regeneration with immunodeficient mice, further expanding the potential of these clones for mechanistic and therapeutic studies. These cell lines will facilitate pairwise comparisons to identify FSHD-specific differences and are expected to create new opportunities for high-throughput drug screens.

Facioscapulohumeral muscular dystrophy (FSHD) affects approximately 1:20,000 people and is thereby the third most common myopathy after Duchenne muscular dystrophy and myotonic dystrophy. FSHD is clinically characterized by progressive weakness and wasting of the facial, shoulder, and upper arm muscles, often followed by a widespread dystrophy during adulthood.¹

In most cases, FSHD shows an autosomal dominant pattern of inheritance being caused by contraction of the D4Z4 macrosatellite repeat in the subtelomeric region of chromosome 4q (FSHD1).² Normal alleles contain between 11 and 100 D4Z4 repeat units ordered head-to-tail, whereas, in patients with FSHD1, one of the chromosomes 4 carries an array of 1 to 10 D4Z4 units.³ Contraction of the D4Z4 repeat is associated with reduced levels of repressive chromatin markers; although D4Z4 is normally densely CpG methylated, D4Z4 at the disease allele is hypomethylated in FSHD and contains reduced levels of the heterochromatic modification histone 3 lysine 9 trimethylation.^4–6 There is a second, less common form of FSHD, FSHD2, whereby patients are phenotypically indistinguishable from FSHD1 and display a similar loss of repressive chromatin markers, but in the absence of D4Z4 repeat contraction.^5,7

Each D4Z4 unit contains a copy of the DUX4 retrogene,^8,9 which led to the hypothesis that incomplete chromatin silencing at D4Z4 causes inappropriate expression of DUX4 in FSHD muscle. Initially, this hypothesis could not be verified; DUX4 transcripts could not be detected in FSHD muscle, shifting the focus to other chromosome 4qter genes such as FRG1 and FRG2 as potential candidate genes. Recent genetic and molecular data, however, provide strong evidence for DUX4 expression in skeletal muscle being causally related to FSHD pathogenesis.

The genetic evidence that supports a role for DUX4 comes from the observation that the subtelomere of chromosome 10q contains an almost identical D4Z4-repeat array, but contractions of this repeat are generally nonpathogenic. In addition, different haplotypes of 4q have been identified on the basis of the presence of sequence variants in and around the D4Z4 repeat, and only D4Z4 contractions on specific genetic backgrounds of 4q, the so-called permissive chromosomes 4, such as 4A161, cause FSHD.¹⁰ FSHD-permissive chromosomes contain an additional DUX4 exon with a polyadenylation signal immediately distal to the most telomeric D4Z4 unit.^11,12 Nonpathogenic haplotypes, on the contrary, do not have this exon or polyadenylation signal. Because this polyadenylation signal enables the stabilization of the DUX4 transcript generated from the distal D4Z4 unit in skeletal muscle of patients with FSHD1, these data strongly support a causative role for the stabilized DUX4 transcript expressed from the telomeric D4Z4 unit in the pathogenesis of FSHD.

Molecular data support the genetic and epigenetic data for a pathogenic role for the DUX4 gene in FSHD. Overexpression models show that DUX4 is toxic to many cells.^13–16 DUX4 protein expression causes cell death in a variety of cell types with skeletal muscle being highly sensitive. Although early studies did not detect endogenous DUX4 mRNA in FSHD skeletal muscle, more sensitive PCR-based assays have identified FSHD-associated transcripts from the telomeric D4Z4 unit.¹² More specifically, expression of a full-length DUX4 transcript in muscle biopsies and muscle cell cultures of patients with D4Z4 contraction (FSHD1) and patients without D4Z4 contraction (FSHD2) has been identified, which could not be detected in muscle or in muscle cell cultures from control subjects.^11,17 DUX4 is normally expressed as a germline transcription factor, and its activity in skeletal muscle of patients with FSHD leads to the activation of germline and early developmental programs, as well as the activation of retrotransposons and suppression of innate immune genes.¹⁸

Somatic mosaicism for the D4Z4 contraction can be observed in almost 50% of de novo FSHD1 families.¹⁹ These persons have experienced a postzygotic contraction of the D4Z4 repeat, generally resulting in the presence of two cell populations: one population of cells with normal-sized D4Z4 repeats (noncontracted) and one population of cells with one FSHD-sized D4Z4 repeat (D4Z4 contracted) because of a contraction. Thus, for a mosaic person, the genome of the D4Z4 contracted and the noncontracted cells are identical except for the presence or absence of a contracted allele. Mosaicism for D4Z4 repeat sizes in these persons can be observed at roughly equal frequency in blood, muscle, and skin.²⁰ Taking advantage of the high frequency of somatic mosaicism for the D4Z4 repeat contraction in patients with de novo FSHD1, we generated and characterized clonal immortalized myogenic cell lines from a mosaic patient with FSHD1. With the use of a well-established immortalization strategy,²¹ we have overcome the low proliferative potential of primary myoblasts. Hereby, we have created isogenic FSHD and normal myogenic cell lines from a single person which can be used for molecular and drug development studies. With the use of cell lines derived from these patients with mosaic FSHD, we expect to overcome some of the genetic and phenotypic variability often observed in human studies because these cell lines are isogenic except for the D4Z4 repeat size.

Materials and Methods

Patient

A needle muscle biopsy from a patient with mosaic FSHD was obtained after informed consent. Mosaicism was determined by genetic tests, and FSHD was clinically confirmed, as described in Lemmers et al^22,23 (see family 24). Briefly, the patient was seen at 37 years of age for progressive weakness of proximal lower limb muscles which had lasted for 2 years with a recent onset of foot extensor weakness. On physical examination, shoulder girdle weakness was present as well, and, with hindsight, the patient recalled decreased strength in his arms from the age of 28 years. Considerable asymmetrical facial weakness was present. Pulsed field gel electrophoresis (PFGE) analysis of DNA isolated from peripheral blood cells showed mosaicism for a D4Z4 contraction. The patient had two equally prevalent cell populations, one allele with a repeat of 13 D4Z4 units and one in which this allele was contracted to 3 D4Z4 repeat units. As confirmed by simple sequence length polymorphism analysis performed as previously described,¹⁰ the mosaicism in this mosaic patient occurred on a permissive 4A161 chromosome. Within this context, a needle muscle biopsy was obtained from the biceps of this mosaic patient at 54 years of age.

Generating Immortalized Myoblast Cell Lines

Immortal clones were generated as previously described.²¹ Briefly, the muscle biopsy was minced, and the explants were plated onto culture dishes coated with fetal calf serum and cultivated in growth medium [one-quarter Medium 199 (Life Technologies, Carlsbad, CA) plus three-quarters Dulbecco's modified Eagle's medium (DMEM) high glucose supplemented with 20% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% glucose, 1% GlutaMAX and dexamethazone (10⁻⁷ mol/L final volume), and 2.5 ng/mL recombinant hepatocyte growth factor (Life Technologies)]. The myogenic cells were allowed to migrate out of the explants and were further expanded and enriched with the use of CD56-magnetic microbeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. The purified myoblasts were then transduced by two retroviruses carrying hTERT or Cdk4 genes containing either the puromycin or neomycin resistance gene, respectively. After selection, the immortalized population was cloned with the use of glass cylinders. The proportion of myoblasts was established by desmin labeling (first antibody: desmin clone D33, mouse IgG1; 1:50; Dako; secondary antibody: Alexa-555 goat anti-mouse, 1:400; Life Technologies).

Genetic Characterization of Clonal Myoblast Cell Lines

The clonal myoblast cell lines were tested for the presence or absence of the D4Z4 contraction by PFGE, essentially as previously described.¹⁰ Complete genetic testing results, including D4Z4 repeat sizes and chromosomes 4 and 10 haplotypes of the patient participating in this study, was already available.^22,23 Briefly, 5 × 10⁵ cells of each myogenic clone were embedded in agarose plugs and after pronase (Roche Applied Science, Indianapolis, IN) treatment, digested with EcoRV (Fermentas, Hanover, MD). As a control, agarose-embedded DNA plugs derived from peripheral blood lymphocytes from this patient were used. EcoRV was used instead of the combination of EcoRI and BlnI, because previous genotyping studies on the peripheral blood cells of the patient showed that the D4Z4 repeat array on chromosome 4B and chromosome 10 were equal in size, both being 80 kb. EcoRV can discriminate between these repeat arrays because of the different position of the distal EcoRV restriction site.²² Digested DNA was separated by PFGE, and D4Z4 repeat sizes were visualized by sequential hybridization with radioactive probes p13E-11 and D4Z4.

Immunofluorescence Staining

The myogenic purity of the clones was determined by counting desmin-positive cells as a percentage of total cells. Cells were cultivated in growth medium, fixed in 95% ethanol, and incubated in 5% fetal calf serum in PBS for 30 minutes. The cultures were then incubated at 37°C for 2 hours with the primary antibody specific for desmin (clone D33, mouse igG1, 1:50; Dako). Specific antibody binding was shown with Alexa-555 goat anti-mouse (1:400; Life Technologies). To visualize nuclei, cells were mounted in medium (Mowiol; Calbiochem-Novabiochem, Nottingham, United Kingdom) containing bis-benzimide (0.0001% w/v; Hoechst; Sigma-Aldrich, Indianapolis, IN). All images were visualized with an Olympus BX60 microscope (Olympus Optical, Tokyo, Japan), digitalized with the Photometrics CoolSNAP fx CCD camera (Roper Scientific, Trenton, NJ), and analyzed with the MetaVue image analysis software version 7.0r4 (Molecular Devices, Sunnyvale, CA).

For coimmunofluorescence staining of DUX4 and pan-myosin heavy chain (MHC), cells were fixed in 2% paraformaldehyde for 7 minutes at room temperature and then washed twice with PBS. Cells were permeabilized with 1% Triton X-100 (Sigma-Aldrich) in PBS for 10 minutes at room temperature with gentle rocking. Primary rabbit-DUX4 antibody directed against the C-terminal region of human DUX4 (E5-5; 1:100)²⁴ and antibody against MHC (MF20, mouse IgG2b, 1:100; Developmental Studies Hybridoma Bank, Iowa City, IA) were diluted in PBS, and cells were incubated overnight at 4°C with the first antibody, washed three times with PBS, followed by incubation with 1:400 diluted Alexa Fluor 488-conjugated donkey anti-rabbit and Alexa Fluor 594-conjugated donkey anti-mouse polyclonal antibody for 1 hour, gently rocking in the dark. Next, cells were washed three times with PBS/0.025% Tween before they were mounted on microscope slides with the use of Aqua Poly/Mount (Polysciences, Inc., Warrington, PA) containing 500 ng/mL DAPI. Stained cells were analyzed on a Leica DMRA2 microscope (Leica, Wetzlar, Germany).

Fusion Index

Cells (7 × 10⁵) were grown in a 60-mm plate in growth medium. One day later, the medium was removed, the cells were washed with PBS, and differentiation medium (DMEM supplemented with 10 μg/mL insulin, 100 μg/mL transferring, and 50 μg/mL gentamicin; Sigma-Aldrich) was added. The cultures were fixed at day 3 or day 5 after induction of differentiation with 100% ethanol, and MHC labeling was performed (MF20, mouse IgG2b, 1:20 dilution; Developmental Studies Hybridoma Bank). The efficiency of the fusion was assessed by counting the number of nuclei in differentiated myotubes (>3 myonuclei) as a percentage of the total number of nuclei (mononucleated and plurinucleated). Statistical analysis was performed by two-sample t-test with P < 0.01 considered to be significant.

RNA Isolation and cDNA Synthesis

For RNA isolation, five D4Z4 contracted and five noncontracted myogenic clones were grown in 100-mm diameter Petri dishes until 40% to 50% confluence to minimize spontaneous differentiation. Myotubes were obtained by growing the cells until 80% confluence, then switching them to serum-deprived conditions (DMEM supplemented with 10 μg/mL insulin, 100 μg/mL transferrin, and 50 μg/mL gentamicin) for 5 days. Total RNA was isolated from proliferating and differentiated cultures with the use of miRNeasy kit (Qiagen, Valencia, CA), including DNaseI treatment according to the instructions of the manufacturer. The RNA concentration of the samples was determined with a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA), and the quality of the RNA was analyzed with a RNA 6000 nanochip on an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA). RNA samples having a RNA integrity number score between 9.5 and 10 were used in this study.

cDNA was synthesized with 1 or 3 μg of total RNA (depending on the gene to be amplified; Table 1) with the use of the Revert Aid H Minus first-strand cDNA synthesis kit with oligo dT-primed primers (Fermentas) according to the manufacturer's instructions. The cDNA was subsequently treated with 0.5 U RNase H for 20 minutes at 37°C, and total cDNA was diluted in 50 μL of water. For real-time RT-PCR analysis, cDNA was diluted 10-fold.

Table 1.

List of Primers and Corresponding Sequencing Used for qPCR Analysis

Primer	Sequence	Temperature (°C)	RNA input (μg)
GUS		60 and 62	1 or 3
Forward	5′-CTCATTTGGAATTTTGCCGATT-3′
Reverse	5′-CCGAGTGAAGATCCCCTTTTTA-3′
MYOG		60	1
Forward	5′-GCCAGACTATCCCCTTCCTC-3′
Reverse	5′-GGGGATGCCCTCTCCTCTAA-3′
MYH2A		60	1
Forward	5′-TGGCAAAATACAGGGGACTC-3′
Reverse	5′-CCAAAGCGAGAGGAGTTGTC-3′
MYOD1		60	1
Forward	5′-TACGAAGGCGCCTACTACAAC-3′
Reverse	5′-AGGCAGTCTAGGCTCGACAC-3′
FRG1		60	1
Forward	5′-GGCGGGTTCTACAGAGACG-3′
Reverse	5′-TTCTGGACGAGTATGTGAGTCG-3′
FRG2		60	3
Forward	5′-GGGAAAACTGCAGGAAAA-3′
Reverse	5′-CTGGACAGTTCCCTGCTGTGT-3′
DUX4		62	3
Forward	5′-CCCAGGTACCAGCAGACC −3′
Reverse	5′-TCCAGGAGATGTAACTCTAATCCA-3′
fllDUX4		60	3
Forward	5′-CCTGGGATTCCTGCCTTCTA-3′
Reverse	5′-AGCCAGAATTTCACGGAAGA-3′
PITX1		58	1
Forward	5′-ACATGAGCATGAGGGAGGAG-3′
Reverse	5′-GTTACGCTCGCGCTTACG-3′
RFPL2		60	1
Forward	5′-CCCACATCAAGGAACTGGAG-3′
Reverse	5′-TGTTGGCATCCAAGGTCATA-3′
TRIM43		60	1
Forward	5′-ACCCATCACTGGACTGGTGT-3′
Reverse	5′-CACATCCTCAAAGAGCCTGA-3′
DEFB103		60	1
Forward	5′-TGTTTGCTTTGCTCTTCCTG-3′
Reverse	5′-CGCCTCTGACTCTGCAATAA-3′
KHDC1		60	1
Forward	5′-ACCAATGGTGTTTCACATGG-3′
Reverse	5′-TGAATAAGGGTGTGGCTGTG-3′
MURF1		58	1
Forward	5′-CTTGACTGCCAAGCAACTCA-3′
Reverse	5′-CAAAGCCCTGCTCTGTCTTC-3′
ATROGIN-1		58	1
Forward	5′-AAAGAGCGCCATGGATATTG-3′
Reverse	5′-CTCAGGGATGTGAGCTGTGA-3′
TP53		58	1
Forward	5′-GTTCCGAGAGCTGAATGAGG-3′
Reverse	5′-TCTGAGTCAGGCCCTTCTGT-3′
MBD3L2		58	1
Forward	5′-CGTTCACCTCTTTTCCAAGC-3′
Reverse	5′-AGTCTCATGGGGAGAGCAGA-3′
PRAMEF1		58	3
Forward	5′-CTCCAAGGACGGTTAGTTGC-3′
Reverse	5′-AGTTCTCCAAGGGGTTCTGG-3′
ZSCAN4		60	1
Forward	5′-AACAAGTGAATGCCCAAACC-3′
Reverse	5′-TGTTCCAGCCATCTTGTTCA-3′
CCNA1		58	1
Forward	5′-CAACTTCCTGGACAGGTTCC-3′
Reverse	5′-TTCGAAGCCAAAAGCATAGC-3′
GAPDH		60	1
Forward	5′-AAGGTCGGAGTCAACGGATTT-3′
Reverse	5′-ACCAGAGTTAAAAGCAGCCCTG-3′

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Quantitative RNA Expression Analysis

All quantitative RT-PCR (qPCR) analyses were performed in duplicate with the use of SYBR green mastermix on the MyIQ or CFX system (Bio-Rad, Hercules, CA), using 0.5 pmol/L of each primer in a final volume of 10 μL per reaction. For gene expression analysis 3 μL of diluted cDNA was used per reaction. Cycling conditions were as follows: initial denaturation step at 95°C for 3 minutes, followed by 40 cycles of 10 seconds at 95°C and 45 seconds at 58°C, 60°C, or 62°C according to the primer used (see Table 1). Specificity of all reactions was monitored by standard gel electrophoresis and/or melting curve analysis: initial denaturation step at 95°C, followed by 1 minute of incubation at 65°C and sequential temperature increments of 0.5°C every 10 seconds up to 95°C. All primer sets (Table 1) were designed with Primer3 software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) and, for cDNA analysis, designed to span at least one intron. Data were analyzed with Bio-Rad CFX manager version 2.0. Relative expression was calculated, using β-glucuronidase (GUS) as a reference for cDNA input, using the CFX manager software version 2.1 (Bio-Rad). Statistical analyses were performed by two-sample t-tests with P < 0.05 considered to be significant.

Regeneration Potential in Vivo

To determine the in vivo regeneration capacity of these isogenic clones with or without D4Z4 contraction, cells were injected into the tibialis anterior (TA) of 3-month-old female immunodeficient mice Rag2^−/− γC^−/− C5^−/− (n = 4 for each clone). TAs were cryo-damaged before injection to induce muscle regeneration, and 5 × 10⁵ cells were injected at a single point, as described previously.²⁵ One month later, the mice were sacrificed, and the muscles were removed and frozen in isopentane cooled in liquid nitrogen and stored at −80°C. Serial transverse sections (5 μm) of the muscles on glass slides were generated with a cryotome (Thermo Shandon, Inc., Pittsburgh, PA). Muscle was sectioned every 100 μm along the length of the muscle, and, in between, cryosections were conserved at −80°C for further RNA analysis. The frozen sections were stained with human-specific lamin A/C antibody (clone JOL2, mouse IgG1; 1:300; Abcam, Inc., Cambridge, MA) and human-specific spectrin antibody (NCL-Spec1, clone RBC2/3D5, mouse IgG2b, 1:50, Novocastra, Newcastle, United Kingdom) for 1 hour. The secondary antibodies used were cyanine 3-conjugated goat anti-mouse IgG1 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) and Alexa Fluor 488 goat anti-mouse IgG2b (Molecular Probes, Eugene, OR), respectively. The number of human spectrin-positive fibers and lamin A/C-positive nuclei were calculated on the cryosection with the best number of spectrin-positive fibers.

The RNA extraction was performed after completed dissociation of muscle sections with the use of 1 mL of TRIzol according to the manufacturer's protocol (Life Technologies). The RNA pellets were resuspended in 10 μL of RNase-free water.

Results

Generation and Characterization of Immortalized Isogenic Human Myoblast Clones

To overcome the limited life span and genetic variability of primary myoblast cultures, we have immortalized primary myoblasts obtained from a biceps muscle biopsy of a patient with mosaic FSHD1. By using CD56 magnetic-activated cell sorting, the myoblast cell population was enriched before immortalization by stable transduction with a hTERT- and a Cdk4-expressing retroviral vector.^26,27 hTERT- and Cdk4-positive cells were cloned and individual clones were expanded. In patients with mosaic FSHD, contraction of D4Z4 occurs after fertilization; thus, two types of myogenic clones are to be expected from the immortalized population: those with D4Z4 contraction and those without a contraction of the D4Z4 repeat array. With the use of PFGE, individual clones were thus screened for the presence or absence of D4Z4 contraction. The percentage of mosaicism was expected to be 50%, based on the frequencies observed in peripheral blood cells of this patient. The repeat sizes of the allele that underwent a somatic contraction are 13 D4Z4 units for the noncontracted cells and 3 D4Z4 units for the D4Z4 contracted cells. Hybridization of EcoRV-digested gDNA with probe p13E-11 showed that individual clones displayed either a contracted D4Z4 repeat array of 17 kb (3 units) or the original array of 48 kb (13 units). A representative PFGE blot of a few selected clones is shown in Figure 1. In total, five D4Z4 noncontracted and five D4Z4 contracted clones were used for further characterization (see Supplemental Table S1 at http://ajp.amjpathol.org). Thus, by clonal expansion, we established a panel of immortalized muscle cell clones that are genetically identical, with the exception of the D4Z4 repeat size and of the integration sites of the immortalization genes.

PFGE analysis of isogenic myogenic clones of a patient with mosaic FSHD. High-quality DNA isolated from the peripheral blood lymphocytes (P) and isogenic myogenic clones of a patient with mosaic FSHD were digested with EcoRV and hybridized with probe p13E-11. The **arrows** depict the mosaic alleles and the **asterisk** indicates chromosome 10-specific bands. Myogenic clones A2 and A5 contain only the contracted allele.

Morphology and Myogenic Characteristics of Clones with or without D4Z4 Contraction

The proportion of myogenic cells in all immortalized muscle cell clones was determined by the percentage of desmin-positive cells. Immunofluorescence analysis showed 100% desmin-positive cells in all clones (data not shown). Next, the fusion index was determined, and steady state expression levels of differentiation markers were quantified during proliferation and after several days in differentiation conditions, that is, serum starvation. The fusion index was determined in D4Z4 contracted and noncontracted clones at days 3 and 5 of differentiation (Figure 2; n = 3). At day 3, the fusion index was significantly higher in the D4Z4 contracted clones than in the D4Z4 noncontracted clones (75% ± 7% vs 52% ± 4%; P = 0.006), with low intervariability within the three D4Z4 contracted and noncontracted clones. However, after 5 days of differentiation, the enhanced fusion index observed in D4Z4 contracted clones did not reach significance (78% ± 14% vs 64% ± 8% for D4Z4 contracted and noncontracted clones, respectively; P = 0.19). At the transcriptional level, expression levels of the myogenic commitment gene MYOD1 and the early and late myogenic differentiation markers myogenin (MYOG) and fast myosin heavy chain 2A (MYH2A) were evaluated in the D4Z4 contracted and noncontracted clones during proliferation and 5 days after differentiation (Figure 3). In proliferating cells, MYOD1 levels were abundantly present with no difference between the D4Z4 contracted and noncontracted clones (Figure 3A). However, the myogenic differentiation markers MYOG and MYH2A were significantly increased in D4Z4 contracted clones compared with the noncontracted clones (P = 0.04 and P = 0.02, respectively; Figure 3, C and E). Although being isogenic, the expression levels of these myogenic markers differ between the D4Z4 contracted clones with clone 12 displaying the greatest increase. Because the high increase in clone 12 was observed in three independent cell-culturing experiments, its response appears to be cell autonomous and not because of differences in cell culture conditions.

Fusion index of isogenic myogenic clones of a patient with mosaic FSHD. Cells were differentiated and fixed at day 3 or day 5 of differentiation and stained for myosin. The efficiency of the fusion was determined by counting the number of nuclei in differentiated myotubes (>3 myonuclei) as a percentage of the total number of nuclei. Values represent the means ± SDs; n = 3 D4Z4 contracted versus n = 3 D4Z4 noncontracted clones. **P < 0.01.

Expression levels of the myogenic markers, *MYOD1, MyoG*, and *MYH2* during proliferation and after differentiation. Transcript levels were quantified in five D4Z4 contracted and five D4Z4 noncontracted clones during proliferation (A, C, and E) and 5 days after induction of differentiation (B, D, and F). Values represent the means ± SEMs. Clone A1 was used as the reference, and its expression level was set at an arbitrary value of 1. The calculations were independently done for myoblasts and myotubes. Values represent the means ± SEMs. *P <0.05.

As expected, higher MYOG and MYH2A transcript levels were observed in differentiated cells compared with the level detected during proliferation (data not shown). MYOD1 levels were comparable in the proliferative and differentiation phase. In myotubes, no significant difference in MYOD1 and MYOG levels could be detected between D4Z4 contracted and noncontracted clones (Figure 3, B and D), whereas MYH2A showed significantly lower transcript levels in the myotubes from the D4Z4 contracted clones (P = 0013; Figure 3F). Overall, these data indicate that proliferating cells from the contracted clones enter the myogenic differentiation program prematurely.

Expression of 4qter Genes

To investigate the direct effect of the D4Z4 contraction on 4qter gene expression, transcript levels of FRG2, FRG1, and DUX4 were determined by qPCR with the use of intron-spanning primers (Table 1), during proliferation and after 5 days of differentiation. Overall, FRG2 levels in proliferating clones were low and only detectable when increasing RNA input. Although not significant, because one noncontracted clone showed low FRG2 transcript levels, the contracted clones showed increased FRG2 levels (Figure 4A). During differentiation, FRG2 expression levels were shown to be significantly enhanced in the myotubes derived from D4Z4 contracted clones compared with the noncontracted clones (P = 0.022; Figure 4B). However, absolute levels were still low. FRG1 transcript levels remain stable during myogenesis (see Supplemental Figure S1 at http://ajp.amjpathol.org), and the level of FRG1 transcripts did not differ between D4Z4 contracted and noncontracted clones both during proliferation and after differentiation (Figure 4, C and D). To quantify DUX4 levels during proliferation, the RNA input levels needed to be increased. DUX4 was expressed in all D4Z4 contracted clones, whereas it was absent or barely detectable at the RNA level in the noncontracted clones (Figure 5, A and B). In differentiated cells, DUX4 expression was increased in the D4Z4 contracted clones and remained absent in noncontracted clones (Figure 5B). As for the myogenic markers, substantial variability in DUX4 expression levels was observed between the D4Z4 contracted clones in both proliferating and differentiating conditions.

Expression levels of 4qter genes *FRG1* and *FRG2* during proliferation and after differentiation. Transcript levels were quantified in five D4Z4 contracted and five D4Z4 noncontracted clones during proliferation (A and C) and 5 days after induction of differentiation (B and D). Clone A1 was used as the reference, and its expression level was set at an arbitrary value of 1. Values represent the means ± SEMs. *P < 0.05.

DUX4 transcript and protein levels during proliferation and after differentiation. DUX4 transcript levels were quantified by qPCR in five D4Z4 contracted and five D4Z4 noncontracted clones during proliferation (A) and 5 days after induction of differentiation (B). The lowest expression level was set at an arbitrary value of 1. Values represent the means ± SEMs. D4Z4 contracted proliferating cells (C) and differentiated cells (D) were costained with DUX4 and Myosin. A representative immunofluorescent picture of a D4Z4 contracted clone is depicted. **Left panels** show staining with DUX4. **Right panels** show a merged picture of myoblast (C) and myotubes (D) costained with DUX4 (in green) and MYH (in red) and counterstained with DAPI (in blue). Only contracted clones show the characteristic pattern with a subset of myonuclei expressing abundant levels of DUX4 protein. *P < 0.05.

Although DUX4 expression was significantly up-regulated in the D4Z4 contracted clones, the abundance of DUX4 mRNA transcripts remained low, similar to what has been described in primary FSHD myotubes.^12,17 To test whether all nuclei of D4Z4 contracted clones express small amounts of DUX4 protein or whether a subset of myonuclei express DUX4 at appreciable levels, DUX4 immunofluorescence was performed. Three D4Z4 noncontracted (3, A1, and A10) and three D4Z4 contracted (12, A5, and A11) clones were cultured, fixed, and costained with DUX4 and the myogenic marker pan-MHC, both when proliferating and at day 5 of differentiation. In agreement with previous data, in clonal cells DUX4 protein is only expressed in a small proportion of myonuclei in the D4Z4 contracted muscle clones. In accordance with our observations of DUX4 transcript levels, DUX4-positive nuclei could already be detected in the D4Z4 contracted clones during proliferation, but only in the highest expressers (clones A5 and 12), showing 1 DUX4-positive nucleus per 8000 nuclei (Figure 5C). Five days after induction of differentiation, DUX4-positive myonuclei could be detected in all three D4Z4 contracted clones with an incidence that ranged between 1:1000 and 1:3000 positive myonuclei (Figure 5D). DUX4 protein–expressing myonuclei were never observed in any of the noncontracted clones (data not shown).

Expression of Downstream Targets

Recently Geng et al¹⁸ described a DUX4 transcriptional network that was based on transcriptome analysis combined with chromatin immunoprecipitation studies of human myoblasts after lentiviral DUX4 transduction. They identified a number of germline genes as direct or indirect DUX4 targets. In our cellular model, we therefore investigated the expression of a number of these and previously identified direct or indirect DUX4 targets as well as other genes that have been proposed to be involved in FSHD etiology: PITX1, MURF1, ATROGIN1, TP53, CCNA1, MBD3L2, PRAMEF1, ZSCAN4, TRIM43, RFPL2, KHDC1, and DEFB103.^{12,18,28–30} Their expression levels were evaluated during proliferation and at day 5 of differentiation by qPCR. For PITX1 and TP53, we observed no quantitative differences in mRNA levels between D4Z4 contracted and noncontracted clones during proliferation (Figure 6, A and C) or differentiation (Figure 6, B and D).

Expression levels of potential DUX4 downstream targets, *PITX1* and *TP53*, during proliferation and after differentiation. Transcript levels were quantified by qPCR in five D4Z4 contracted and five D4Z4 noncontracted clones during proliferation (A and C) and 5 days after induction of differentiation (B and D). The lowest expression level was set at an arbitrary value of 1. Values represent the means ± SEMs. P < 0.05.

Of the muscle atrophy F-box (ATROGIN1) and muscle ring-finger protein 1 (MURF1) genes, only MURF1 was significantly up-regulated in the D4Z4 contracted clones 5 days after induction of differentiation (Figure 7, A–D). The DUX4 target genes that are involved in germ cell and early development, ZSCAN4, TRIM43, RFLP2, and PRAMEF1,^31–35 were all shown to be significantly increased in the D4Z4 contracted cells during proliferation (Figure 8,A, C, E, and G). The Methyl-CpG binding domain protein 3-like 2 (MBD3L2) gene that is also expressed in germ cells^36,37 was not significantly deregulated during proliferation (Figure 9C), but it did show a significant increase in the D4Z4 contracted myotubes (Figure 9D). In addition, ZSCAN4 and TRIM43 were significantly up-regulated in the D4Z4 contracted clones 5 days after induction of differentiation (Figure 8, B and D). During differentiation, RFPL2, PRAMEF1, CCNA1 encoding cyclin 1A, which is highly expressed in testis,^38,39 and KHDC1, another gene early expressed in development,⁴⁰ also seemed to be at higher abundance in the D4Z4 contracted clones, but, because their levels are also high in one noncontracted clone (A1), it did not reach significance (Figure 8, F and H, and Figure 9, B and F).

Expression levels of muscle-specific E3 ubiquitin ligases, muscle ring finger 1 (MURF1) and Atrogin-1 during proliferation and after differentiation. Transcript levels were quantified by qPCR in five D4Z4 contracted and five D4Z4 noncontracted clones during proliferation (A and C) and 5 days after induction of differentiation (B and D). The lowest expression level was set at an arbitrary value of 1. Values represent the means ± SEMs. *P < 0.05.

Expression levels of DUX4 downstream targets, which normally are expressed in germline, during proliferation and after differentiation. Transcript levels were quantified in five D4Z4 contracted and five D4Z4 noncontracted clones during proliferation (A, C, E, and G) and 5 days after induction of differentiation (B, D, F, and H). The lowest expression level was set at an arbitrary value of 1. Values represent the means ± SEMs. *P < 0.05.

Expression levels of DUX4 downstream targets, which normally are expressed in germline or early during development, during proliferation and after differentiation. Transcript levels were quantified in five D4Z4 contracted and five D4Z4 noncontracted clones during proliferation (A, C, and E) and 5 days after induction of differentiation (B, D, and F). The lowest expression level was set at an arbitrary value of 1. Values represent the means ± SEMs. *P < 0.05.

Previously, it was shown that DUX4 inhibits the innate immune response, at least partly, through the induction of DEFB103 (β-defensin 3) expression.¹⁸ Therefore, we also analyzed the expression of DEFB103 in our clones, indicating a noticeable increase in the proliferating D4Z4 contracted clones, which was not significant because of the increase in one of the noncontracted clones (A1) (Figure 10A). In myotubes, no difference in DEFB103 expression between D4Z4 contracted and noncontracted clones was observed (Figure 10B). DEFB103 has been recently shown to affect myogenesis partly by increasing myostatin (MSTN) expression levels.¹⁸ Therefore, we also evaluated MSTN mRNA levels in the D4Z4 contracted and noncontracted clones. Although we have not observed a correlation between DEFB103 and MSTN expression levels, both showed a trend toward up-regulation in the proliferated (Figure 10C) but not in the differentiated D4Z4 contracted clones (Figure 10D).

Expression levels of *DEFB103* and Myostatin in myoblasts and myotubes. Transcript levels were quantified in five D4Z4 contracted and five D4Z4 noncontracted clones during proliferation (A and C) and 5 days after induction of differentiation (B and D). The lowest expression level was set at an arbitrary value of 1. Values represent the means ± SEMs. P < 0.05.

Contribution to Muscle Regeneration

Next, we determined the in vivo regeneration capacity of the D4Z4 contracted and noncontracted clones and assessed whether we could monitor expression of the DUX4 responsive genes in the mouse muscle that was transplanted. D4Z4 contracted (54.12) and noncontracted (54.6) cells were injected into cryo-damaged TA of 3-month-old female immunodeficient Rag2^−/− γC^−/− C5^−/− mice. One month after injury, the mice were sacrificed, and the presence of newly generated human fibers was identified by human spectrin (expressed in differentiated fibers) staining. Human lamin A/C was used to count the number of human nuclei within the muscle. As depicted in Figure 11, A–C, both D4Z4 contracted and noncontracted clones were efficiently incorporated into the regenerating fibers with similar efficiency as described previously for normal immortal cell clones.²⁷ Interestingly, although only a fraction of the TA mouse muscle fibers have been reconstituted with human muscle fibers and considering that the proportion of DUX4-positive cells will most likely be exceedingly low, because only 1 of 1000 human myonuclei will express DUX4, we did observe increased levels of the DUX4 responsive gene, TRIM43, in the TA transplanted with the D4Z4 contracted clones (Figure 11D). Although the levels were too low to be quantified, in an independent experiment that used a different set of D4Z4 contracted and noncontracted clones, increased levels of DUX4 responsive genes, such as TRIM43, ZSCAN4, and RFPL2, could be identified in the D4Z4 contracted mouse muscle that was transplanted (see Supplemental Figure S2 at http://ajp.amjpathol.org).

In vivo muscle formation with the use of noncontracted and contracted clones. The TA of immunodeficient mice were cryodamaged before being transplanted with 500,000 cells of either D4Z4 contracted or noncontracted clones. One month after injury and implantation, mice (n = 4) were sacrificed, and TA was costained with human specific lamin A/C (in red) and spectrin antibodies (in green). DAPI (in blue) was used to visualize all of the nuclei. **Panel A** shows a representative image of TA either being transplanted with noncontracted (**left panel**) or D4Z4 contracted clones (**right panel**). The histograms show the number of spectrin-positive fibers (B) and the number of Lamin A/C-positive nuclei (C). No significant difference in regeneration potential of D4Z4 noncontracted and contracted clones was observed. Values represent the means ± SEMs. P < 0.05. D: TRIM43 transcript levels were quantified in TA muscle transplanted with either D4Z4 contracted (54.12) or noncontracted (54.6) clone. Values represent the means ± SEMs.

Discussion

Our understanding of the pathogenic mechanism in FSHD has greatly advanced in recent years. Leaky expression of the normally repressed DUX4 retrogene in muscle appears to be a main hallmark of FSHD.^11,17,41 The availability of this uniform disease model⁴² is expected to shift the research focus toward studies that address the function and regulation of DUX4 and to launch new incentives to identify molecules that can suppress DUX4 activity. Elucidation of downstream targets of DUX4 could also identify new therapeutic targets. These studies will require validated cellular and animal model systems.

Many biological variables affect the outcome of molecular studies that use human biomaterials. These include, but are not limited to, the genetic and phenotypic heterogeneity of individuals, age, sex, and tissue heterogeneity, factors all known to influence the outcome of a study. Indeed, a recent report indicates that in FSHD the factor family of origin contributes significantly to cellular properties of myoblast cultures such as fusion rate and stress response.⁴³ Most likely these factors have contributed to the inconsistent expression data reported in various FSHD studies.^28,30,44,45 Here, we attempted to overcome some of these limitations by the generation of immortalized isogenic FSHD muscle cell lines that genetically differ only by the presence or absence of the disease-associated D4Z4 contraction and the sites of transgene integration. The generation of this cellular model opens the possibility to investigate the direct effects of D4Z4 contraction with a minimum of genomic variables.

Previous reports showed that by the use of hTERT and CDK4, immortalized human FSHD cell lines with a greatly extended proliferative life span can be generated, and, importantly, they were shown to maintain their capacity to differentiate.^21,26 In the present study, besides assessing the in vitro and in vivo differentiation potential, the preservation of FSHD-specific transcriptional signatures in the D4Z4 contracted cell clones also has been addressed. Of interest, the immortalization and clonal expansion procedure did not affect the molecular hallmark of FSHD myoblast cultures; abundant amounts of DUX4 protein could be detected in a small subset of myonuclei of clones containing the contracted allele only. Moreover, the overall low DUX4 expression levels in the D4Z4 contracted clones significantly induced the expression of established DUX4 target genes such as ZSCAN4, TRIM43, RFPL2, PRAMEF1, MBD3L2,¹⁸ and MURF1.²⁸ We also observed that in our D4Z4 contracted clones P53, PITX1, and ATROGIN1, previously proposed to be involved in FSHD,^12,28,29 were not differentially expressed during either proliferation or after differentiation.

One of the five D4Z4 noncontracted clones (A1) shows an atypical expression pattern. Although DUX4 protein could not be detected 5 days after induction of differentiation, DUX4 target genes such as PRAMEF1, ZSCAN4, RFPL2, CCNA1, KHDC1, and DEFB103 are expressed in this particular clone. This could be because of heterogeneity of the transduced population or more likely because of the number and/or integration sites of the transgene. This observation also emphasizes the importance of extensive screening of the clones before using them in further studies. When removing clone A1 from the analysis, two additional genes that recently have been identified in the DUX4 transcriptional network, CCNA1 and KHDC1,¹⁸ are significantly increased in the D4Z4 contracted clones. Overall, this isogenic immortalized cellular model retains the FSHD-specific characteristics, including robust up-regulation of the recently identified DUX4 target genes that could serve as good biomarkers in future therapeutic intervention studies.

D4Z4 contracted clones have a significantly higher fusion index concomitant with increased expression of the early and late myogenic differentiation markers MYOG and MYH2A, respectively, at early stage of differentiation. Differences in fusion index between FSHD and control myoblasts to our knowledge have not been reported before, possibly because of the high variation in fusion rate between muscle cell cultures obtained from different persons, especially when comparing FSHD muscle cultures.^43,46 This reinforces the strength of our model; comparing D4Z4 contracted and noncontracted clones obtained from a single person allowed us to uncover differences in fusion rate between D4Z4 contracted or noncontracted clones in early stages of differentiation. This is in agreement with reports of a defect in the myogenic pathway in FSHD myoblasts.^47,48 For example, Cheli et al⁴⁷ showed increased MYOD1 transcripts in proliferating FSHD myoblast. In accordance, Winokur et al⁴⁸ detected an increase in the early direct targets of MYOD1. MYOD1 has been defined as one of the earliest markers of myogenic commitment, triggering myogenic differentiation, concomitant with an exit of the cell cycle,⁴⁹ followed by an increase in MYOG expression. Although we did not assess a difference in MYOD1 levels, the myogenic markers induced by MYOD1 during normal myogenesis were significantly increased. So presumably, the isogenic immortalized clones are in a more activated state compared with the primary myoblast studied in previous reports. Taking the kinetics of the myogenic markers during the myogenic program into consideration, all these data are in agreement with the premature entry into differentiation that we observed in the contracted FSHD clones.

The DUX4 target gene DEFB103, formerly shown to be involved in the host defense mechanism, seems also to regulate myogenesis.¹⁸ In the isogenic clones, DEFB103 levels seem to be increased in the D4Z4 contracted clones during proliferation and not after differentiation. In support, MSTN, induced by DEFB103,¹⁸ was also increased in proliferating D4Z4 contracted clones. Intriguingly, in primary myoblast cultures DEFB103 supplementation only alters MSTN expression in proliferating myoblasts,¹⁸ consistent with elevated expression of MSTN levels in the proliferating D4Z4 contracted clones. Five days after induction of differentiation significantly reduced MYH2A levels were apparent in the D4Z4 contracted clones, which is in agreement with previous data.⁴⁷ Because myoblasts that were cultured for 3 days in the presence of DEFB103 showed a decline in fusion,¹⁸ our data might suggest that increased DUX4, possibly in part through increased DEFB103 levels, and increased MSTN levels hinder the myogenic program at later stages that involve muscle maturation, as indicated by reduced MYH2A levels in the D4Z4 contracted clones.

FRG1 has been proposed as an FSHD candidate gene on the basis of its transcriptional up-regulation in FSHD muscle, and muscle-specific overexpression of FRG1 in a transgenic mouse model leads to muscle dystrophy.^50,51 However, expression studies have yielded inconsistent results as to whether FRG1 is indeed up-regulated in FSHD.^44,45,52 Interestingly, FRG1 expression is not significantly different between D4Z4 contracted and noncontracted clones, both during proliferation and after differentiation. Thus, its role in FSHD pathogenesis remains speculative. In contrast, FRG2 mRNA levels are specifically increased in D4Z4 contracted clones, consistent with published data.^44,53 However, their expression levels remain low, and patients with FSHD with a classical disease phenotype have been reported in which the partial deletion of the D4Z4 repeat extended proximally and also included the FRG2 locus.⁵⁴

A more likely pathogenic factor in FSHD is DUX4 expressed from the telomeric D4Z4 unit. DUX4 induces apoptosis in a p53-dependent manner in somatic cells, including skeletal muscle, and interferes with myogenic pathways as was shown in several overexpression models.^14–16 DUX4 activates expression of early stem cell and germline programs in skeletal muscle.¹⁸ Full-length DUX4 (flDUX4) transcripts have only been detected in FSHD myoblasts, myotubes, and skeletal muscle.¹⁷ FlDUX4 expression levels are low and not always detectable in FSHD muscle. We confirmed low flDUX4 expression in all contracted clones already during proliferation, both by qPCR and immunofluorescence. However, on differentiation, we observe higher DUX4 expression as evidenced by an increased number of DUX4-positive myonuclei. This observation supports a direct link between the genetic lesion and transcriptional derepression of DUX4. It also argues that the pattern of occasional DUX4-expressing nuclei cannot be explained by a specific subtype of myoblasts that express DUX4 or by an effect of the local milieu, but that this burst-like phenotype is a locus-intrinsic property.

Unexpectedly, in proliferating cultures of D4Z4 noncontracted clone A1 and in myotubes of D4Z4 noncontracted clone 3, low levels of DUX4 transcripts could be detected by qPCR. By RT-PCR we confirmed that these clones express low levels of flDUX4 (see Supplemental Figure S3 at http://ajp.amjpathol.org). Possibly, CDK4, hTERT expression, or the integration event itself in these noncontracted clones affects DUX4 expression. However, because we did not observe DUX4 protein or a consistent increase in expression of DUX4 target genes in these clones, the biological relevance of this observation remains unclear.

Characteristic for FSHD is the clinical variability in distribution and progression of muscle weakness. Although some of these differences can be ascribed to the number of residual D4Z4 units,^55–57 the clinical variability between close relatives having the same disease allele and the variable clinical presentation in monozygotic twins^58–60 suggest additional (epigenetic) factors. One possibility is that the regulation of this characteristic phenotype of bursts of DUX4 expression contributes to the variable clinical presentation. The data from our molecular profiling of our D4Z4 contracted clones, in this respect, is remarkable. Although isogenic, the expression levels of DUX4 and the coregulation of its targets in the D4Z4 contracted clones are highly variable. In accordance, the proportion of DUX4-positive myonuclei similarly varied between the D4Z4 contracted clones. Differences in the molecular signature of isogenic cells, cultured under the same environmental conditions and sharing the same replicative history, have been documented, and stochastic gene expression has been broadly explored for its biological implications. Variation in expression levels can originate from mRNAs that are synthesized in short but intense bursts of transcription.⁶¹ For DUX4, activation of the DUX4 promoter might be slow and unstable, but producing many mRNA copies on each activation event. Consequently, small differences in the number of activation events will propagate to larger fluctuations in DUX4 mRNA transcripts. Of interest, some evidence exist that chromatin remodeling can contribute to stochastic changes in gene expression. With the use of chromatin-remodeling agents^62,63 and of reporter constructs, it was shown that two identically regulated reporter genes integrated in two distinct chromosomes showed uncorrelated bursts of expression. However, when moved to adjacent sites in a single locus, their mRNA expression pattern became almost fully correlated.⁶¹ Because FSHD has an epigenetic etiology and because the D4Z4-adjacent FRG2 gene showed the same variation in mRNA transcript levels in the D4Z4 contracted clones as DUX4, it is possible that the chromatin remodeling machinery drives this stochastic expression pattern of DUX4 as against the transcription machinery. Therefore, it will be important in future studies to identify the factors that regulate DUX4 expression and to extend these studies in clonal myogenic cells of additional patients.

Previous transplantation experiments showed that immortalized human myoblasts participate with higher efficiency in regenerating mouse muscle than parental primary myoblasts.²⁷ In the present report, we show that both D4Z4 contracted and noncontracted immortalized clones share the ability to participate in the regeneration of new mature muscle in vivo. This provides a new model to study the in vivo behavior of D4Z4 contracted and noncontracted clones and the effect of gene deregulation in this context. DUX4 is a primate-specific retrogene that likely has acquired primate-specific functions and perhaps cannot exert the same functions in mouse as it can in humans. Therefore, it might be challenging to unravel the consequences of DUX4 expression in skeletal muscle in a mouse model that overexpresses DUX4. Transplantation of D4Z4 contracted and noncontracted human myoblast clones into injured mouse muscle, as we have done in this study, while maintaining the unique FSHD molecular phenotype of bursts of DUX4 expression in a subset of human myonuclei will overcome this limitation; in this experimental set up, DUX4 expression can be examined in its natural context in vivo. In addition, it will be possible to select the clones presenting a normal karyotype, which retain their capacity to differentiate and have integrated the hTERT and CDK4 inserts at an innocuous location. Therefore, with current knowledge, transplantation of immortalized D4Z4 contracted and noncontracted FSHD clones into a mouse model will represent a dedicated system to assess the short-term consequence of DUX4 expression during muscle regeneration in the absence or presence of drugs that augment or suppress DUX4 levels. Indeed our preliminary data suggest that human DUX4 targets can be detected in this model when D4Z4 contracted clones participate in regeneration.

In summary our data show that we have generated a panel of isogenic immortal muscle cell lines that differ only by the presence or absence of the D4Z4 contraction. In addition to their capacity to differentiate both in vitro and in vivo, these muscle cell lines have maintained the molecular fingerprint of FSHD by displaying differential expression of DUX4, FRG2, ZSCAN4, TRIM43, RFPL2, PRAMEF1, MDB3L2, and MURF1, which could be used as FSHD biomarkers. It is to be expected that this panel of muscle cell lines will be a valuable source for future FSHD studies and high-throughput screenings.

Acknowledgment

We thank the patient for his cooperation in this study.

Footnotes

Supported by the Fields Center for FSHD and Neuromuscular Research (R.T. and S.M.M.), NIH grants R21 AR059966 (S.M.M. and G.S.B.B.) and P01NS069539 (S.J.T., R.T., S.M.M.), Muscular Dystrophy Association grant 173202 (S.M.M.), the Geraldi Norton Foundation and Eklund family (R.T. and S.M.M.), the Association Française contre les Myopathies (E.N., G.S.B.B., V.M.), and the Émergence-UPMC-2010 research program (V.M.).

Y.D.K. and J.D. contributed equally to this work.

Supplemental material for this article can be found at http://ajp.amjpathol.org or at http://dx.doi.org/10.1016/j.ajpath.2012.07.007.

Supplementary data

Supplemental Figure S1

Expression levels of the 4qter gene FRG1 during differentiation. Transcript levels were quantified in five D4Z4 contracted and five D4Z4 noncontracted clones during proliferation and 5 days after induction of differentiation. To compare FRG1 levels during differentiation, the levels were normalized to the myogenic stable reference gene, GAPDH. Expression levels in the proliferating 54.A1 clone is set at 1. Values represent the means ± SEMs. *P < 0.05.

mmc1.pdf^{(10.5KB, pdf)}

Supplemental Figure S2

Expression levels of DUX4 downstream targets in vivo in mouse muscle transplanted with D4Z4 noncontracted and contracted clones. The TA of immunodeficient mice were cryodamaged before being transplanted with 500,000 cells of either D4Z4 contracted (54.A5) or noncontracted (54.3) clones. Transcript levels of RFPL2, ZSCAN4, and TRIM43 were amplified by qPCR and were shown to be expressed in the TA muscle transplanted with D4Z4 contracted clone only (54.A5) by gel analysis. A technical PCR duplicate was loaded on gel with an asterisk referring to the water control. Human GUS and GAPDH were used as internal controls, thus showing that graft efficiencies were comparable for contracted and noncontracted clones.

mmc2.pdf^{(39.7KB, pdf)}

Supplemental Figure S3

Full-length (fl)DUX4 transcript levels during proliferation and after differentiation. A: Design of the flDUX4 qPCR primers avoiding the amplification of DUX4s. flDUX4 transcript levels were quantified in five D4Z4 contracted and five D4Z4 noncontracted clones during proliferation (B) and 5 days after induction of differentiation (C). Values represent the means ± SEMs. *P < 0.05.

mmc3.pdf^{(53.8KB, pdf)}

Supplemental Table S1

mmc4.doc^{(148.5KB, doc)}

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Associated Data

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Supplementary Materials

Supplemental Figure S1

mmc1.pdf^{(10.5KB, pdf)}

Supplemental Figure S2

mmc2.pdf^{(39.7KB, pdf)}

Supplemental Figure S3

mmc3.pdf^{(53.8KB, pdf)}

Supplemental Table S1

mmc4.doc^{(148.5KB, doc)}

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PERMALINK

Generation of Isogenic D4Z4 Contracted and Noncontracted Immortal Muscle Cell Clones from a Mosaic Patient

Yvonne D Krom

Julie Dumonceaux

Kamel Mamchaoui

Bianca den Hamer

Virginie Mariot

Elisa Negroni

Linda N Geng

Nicolas Martin

Rabi Tawil

Stephen J Tapscott

Baziel GM van Engelen

Vincent Mouly

Gillian S Butler-Browne

Silvère M van der Maarel

Abstract

Materials and Methods

Patient

Generating Immortalized Myoblast Cell Lines

Genetic Characterization of Clonal Myoblast Cell Lines

Immunofluorescence Staining

Fusion Index

RNA Isolation and cDNA Synthesis

Table 1.

Quantitative RNA Expression Analysis

Regeneration Potential in Vivo

Results

Generation and Characterization of Immortalized Isogenic Human Myoblast Clones

Figure 1.

Morphology and Myogenic Characteristics of Clones with or without D4Z4 Contraction

Figure 2.

Figure 3.

Expression of 4qter Genes

Figure 4.

Figure 5.

Expression of Downstream Targets

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Contribution to Muscle Regeneration

Figure 11.

Discussion

Acknowledgment

Footnotes

Supplementary data

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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