The study was approved by The Johns Hopkins Medicine Institutional Review Board.
Human subjects and tissue collection
The male proband (subject 01A) first exhibited weakness in the legs as a teenager. At time of enrollment, he was 42 years old, and exhibited mild facial weakness, scapular winging and asymmetric weakness in the limbs, consistent with the FSHD phenotype. Molecular diagnosis of FSHD was confirmed by the University of Iowa Diagnostic Laboratories, which identified a HindIII 4qA allele of 26 kb in length. An open muscle biopsy was taken from the biceps muscle (01Abic), which had its strength rated as 4+ out of 5 on the modified Medical Research Council scale. Tissue was also obtained from the biceps muscle (01Ubic) of the proband's 46-year-old brother (01U), who had full muscle strength and no disease allele found on gene testing. Approximately 300 mg of tissue was reserved for histological and biochemical assays, and ~500 to 700 mg tissue was used for primary cell isolation.
Primary cell isolation and cell culture
The biceps muscle tissue was minced into fragments of <5 mm2, and stored at 4°C in a Ham's F10 medium containing 20% fetal bovine serum (FBS; Hyclone, Thermo Scientific, Logan, UT, USA), 2% chick embryo extract and 2.5 ng/ml basic fibroblast growth factor (Biopioneer, San Diego, CA, USA), supplemented with 1% antibiotic/antimycotic (Cellgro, Manassas, VA, USA) until processed for cell isolation (within approximately 24 hours of biopsy) (viable cells have been successfully isolated from tissues stored in this manner up to 6 days after biopsy). Tissue was cleaned and minced in Hanks' balanced salt solution (HBSS, catalogue number 14175; Gibco, Grand Island, NY, USA) and dissociated in 5 ml of enzyme solution (1 mg/ml collagenase IV, 2.4 U/ml dispase (both Worthington, Lakewood, NJ, USA) and 2.5 mmol/l CaCl2 in HBSS] for 45 minutes at 37°C, triturating every 15 minutes with a 5 ml serological pipette (moving it up and down ~10 to 15 times), and filtered through 100 μm and 40 μm cell sieves (BD Biosciences, Bedford, MA, USA). Cells were pelleted by spinning in a centrifuge at 1000 g for 5 minutes, and resuspended in 2 ml growth medium (Ham's F-10 supplemented with 20% FBS (Hyclone), 0.5% chick embryo extract, 1.2 mmol/l CaCl2 and 1% antibiotic/antimycotic (Cellgro), prepared as fresh stock at least every 7 days) and counted with a hemocytometer. The 01Abic cells were seeded in 2 ml growth medium on a 35 mm gelatin-coated dish, and the 01Ubic cells were seeded on a 60 mm dish in 5 ml growth medium. Cells were cultured undisturbed for 48 hours, after which they were given fresh growth medium daily for 2 to 4 days. When approximately 50 to 70% confluent, cells were treated with trypsin (TrypLE Express/Gibco) for 5 minutes at 37°C, neutralized with an equal volume of growth medium, and counted with a hemocytometer. For expansion, cells were seeded at ~2000 to 3000 cells/cm2 in growth medium. For freezing, an equal volume of ice-cold 2 × freezing medium (20% dimethysulfoxide, 50% FBS, 30% growth medium) was added to cells and mixed well, then cells were incubated in the liquid nitrogen vapor phase for at least 1 hour before storage at -140°C.
To standardize conditions for growth of primary and immortalized cells, primary myoblasts were subsequently cultured in a 4:1 mixture of Dulbecco modified Eagle medium (DMEM) and Medium 199 (Hyclone), supplemented with 15% FBS (Hyclone), 0.02 M HEPES buffer, 1.4 mg/l vitamin B12 (both Sigma-Aldrich, St. Louis, MO, USA), 0.03 mg/l ZnSO4
(Fisher Scientific, Fair Lawn, NJ, USA), 0.055 mg/l dexamethasone (Sigma-Aldrich), 2.5 μg/l hepatocyte growth factor (Chemicon International, Temecula, CA, USA) and 10 μg/l basic fibroblast growth factor (BioPioneer), on dishes coated with 0.1% pigskin gelatin (Sigma-Aldrich) in a 2-5% oxygen environment [34
]. Hepatocyte and fibroblast growth factors were added to a volume of medium that was used within 1 week. Cells were routinely grown in 100 mm tissue culture dishes (BD Falcon, Franklin Lakes, NJ, USA) covered by 10 ml medium, with a weekly medium change if cells were not passaged before that (usually one passage every 5 to 7 days). Cells were passaged at 50 to 90% confluency using 0.05% trypsin/EDTA (Gibco), and cell numbers determined (Z1 Coulter Particle Counter; Beckman Coulter, Miami, FL, USA). PD was calculated using the formula: PD = ln[(final number of cells)/(initial number of cells)]/In(2).
For differentiation, 1.2 × 105 myoblasts were seeded in six-well dishes, then 24 hours later, cells were washed twice with phosphate-buffered saline (PBS) and fed with differentiation medium (DMEM and Medium 199 in a ratio of 4:1, supplemented either with 2% horse serum (Gibco) or with 0.02 mol/l HEPES plus 10 mg/l insulin and 100 mg/l apo-transferrin (both Sigma-Aldrich)). Results obtained were similar with both differentiation media, although a delay of ~1 day was observed with horse serum relative to insulin/transferrin.
The construction of the vectors used for immortalization (CDK4-pBabe-neo and hTERT-pBabe-Hygro), has been described previously [7
]. In brief, mouse CDK4 (kindly provided by Charles J. Sherr) and hTERT cDNAs were inserted into pBabe vectors [35
] containing neomycin- and hygromycin-resistance genes, respectively. Additionally, loxP sites were placed internal to the long terminal repeats of the hTERT expression vector, to allow for excision of the entire expression cassette by Cre recombinase. These vectors were transfected into the Phoenix ecotropic packaging cell line using the calcium phosphate technique [36
], and the virus-containing supernatant was used to infect the amphotropic packaging cell line PA317 [37
] to obtain stable virus-producing cell lines after selection with 0.5 mg/ml G418 or hygromycin (EMD Biosciences, San Diego, CA, USA). All infections were performed in the presence of 2 μg/ml polybrene (Sigma-Aldrich).
For infection of myoblasts, 5 × 104 cells were seeded in six-well dishes 24 hours before infection. Selection was started 48 hours after infection using 300 mg/l hygromycin and 400 mg/l G418 for 1 and 2 weeks, respectively. Depending on growth rates and infection efficiency, cells were passaged during the selection period before becoming confluent.
Isolation of myogenic immortalized clones
Immortalized populations were seeded at low densities, (50, 100, 200 or 400 cells per 100 mm dish). About 10 days after seeding, clones were isolated using cloning rings and seeded into 48-well dishes, followed by six well and 100 mm dishes. The exact time point for clone isolation was chosen to maximize the cell number and minimize cell density of individual clones, to facilitate growth after passage but avoid fusion of myoblasts, respectively. We estimate that clones contained 100 to 1000 cells at the time of isolation. All clones were analyzed for CD56 expression by flow cytometry (see below) and fusion potential in differentiation medium. The percentage of cell nuclei in multinucleated and/or MF20-positive cells was determined for 10 random fields after 3 or 7 days in differentiation medium.
Terminal restriction fragment assay
Terminal restriction fragment assay was performed as previously described [38
Cells were detached with trypsin, counted, and sedimented by centrifugation, then 1.5 × 105
cells were resuspended in 0.2 ml DMEM with 10% calf serum (Cosmic Calf Serum; Hyclone) containing 5% supernatant from the hybridoma line 5.1H11 [39
], which recognizes CD56 (kindly provided by Helen Blau). As a control, primary antibody was omitted. Cells were incubated for 30 min at room temperature, washed twice with 10% calf serum (Cosmic Calf Serum; Hyclone) in PBS, and incubated with secondary Alexa488-conjugated goat anti-mouse antibody (Invitrogen, Eugene, OR, USA) for 30 min at room temperature. After washing with PBS, cells were resuspended in 0.2 ml PBS and analyzed on a flow cytometer (FACSCalibur; Becton Dickinson). A second flow cytometer (FACSAria; Becton Dickinson) was used for sorting, with 5 × 106
cells stained in upscaled volumes.
Cells on gelatin-coated glass chamber slides (Thermo Scientific) were fixed with cold (-20°C) ethanol for 5 minutes, air-dried, and rehydrated with PBS. After blocking with 10% calf serum (Cosmic Calf Serum; Hyclone) in PBS, slides were incubated with primary antibody (1:100 monoclonal mouse anti-desmin clone D33 (Thermo Scientific, Rochester, NY, USA) or MF20 supernatant (myosin heavy chain, 1:30; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) for 1 hour at room temperature. Slides were washed three times with PBS for 5 min, each and incubated with secondary Alexa488-conjugated goat anti-mouse antibody (Invitrogen) for 1 hour at room temperature. After three washes with PBS, slides were covered with mounting medium containing 4',6-diamidino-2-phenylindole (Vectashield; Vector Laboratories, Burlingame, CA, USA) and imaged with a fluorescence microscope (Axiovert 200 M; Zeiss).
Tissues were frozen in liquid nitrogen-cooled isopentane, and cut on a cryostat. Frozen sections (0.8 μm) were collected on microscope slides (Fisherbrand Superfrost Plus; Fisher Scientific), air-dried, and fixed with 4% formaldehyde in PBS for 15 minutes at room temperature. Slides were washed three times with PBS and stained with hematoxylin and eosin. Sections were imaged under a microscope (AxioImager M2, Zeiss) and images captured with a camera (Digital microscopy camera Axio Cam, ICC3, Zeiss).