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Establishment of clonal myogenic cell lines from severely affected dystrophic muscles - CDK4 maintains the myogenic population

Establishment of clonal myogenic cell lines from severely affected dystrophic muscles - CDK4... Background: A hallmark of muscular dystrophies is the replacement of muscle by connective tissue. Muscle biopsies from patients severely affected with facioscapulohumeral muscular dystrophy (FSHD) may contain few myogenic cells. Because the chromosomal contraction at 4q35 linked to FSHD is thought to cause a defect within myogenic cells, it is important to study this particular cell type, rather than the fibroblasts and adipocytes of the endomysial fibrosis, to understand the mechanism leading to myopathy. Results: We present a protocol to establish clonal myogenic cell lines from even severely dystrophic muscle that has been replaced mostly by fat, using overexpression of CDK4 and the catalytic component of telomerase (human telomerase reverse transcriptase; hTERT), and a subsequent cloning step. hTERT is necessary to compensate for telomere loss during in vitro cultivation, while CDK4 prevents a telomere-independent growth arrest affecting CD56 + myogenic cells, but not their CD56- counterpart, in vitro. Conclusions: These immortal cell lines are valuable tools to reproducibly study the effect of the FSHD mutation within myoblasts isolated from muscles that have been severely affected by the disease, without the confounding influence of variable amounts of contaminating connective-tissue cells. Background (FSHD), but the technique is also applicable to other Most muscular dystrophies result from a defect within muscular dystrophies. myogenic cells that leads to progressive muscle weak- FSHD has been linked to deletions of D4Z4 tandem ness and wasting, and in severe cases, to the replace- repeats at chromosome 4q [1], but it is still unclear how ment of muscle fibers by connective tissue and/or fat. this deletion causes disease. Primary skeletal-muscle cul- At advanced stages, skeletal muscle is replaced by tures have been used in attempts to model the disease, fibroblasts and adipocytes. Although these cell types and several FSHD-specific phenotypes, such as increased probably promote disease in later stages, it is generally vacuolization and sensitivity to oxidative stress [2] have believed that the root cause leading to most muscular been described. In addition, there have been reports dystrophiesisadefect originating in myogenic cells. describing incorrect expression of various FSHD candi- We therefore developed a protocol to enrich, immorta- date genes in muscles and myogenic cells from patients lizeand isolateraremuscleprogenitorcells from with FSHD, including FSHD region gene (FRG)1 and severely affected dystrophic muscles to obtain clonal the double homeobox (DUX)4 gene [3-6]. However, in myogenic cell lines. In this report, the protocol is many instances, these findings have not been consistent described for cells isolated from skeletal muscle of between different laboratories, perhaps as a result of people with facioscapulohumeral muscular dystrophy variables such as muscle type, disease progression, cul- ture purity, in vitro culture conditions or replicative age * Correspondence: Woodring.Wright@UTSouthwestern.edu of the cells. In particular, because of their low replicative Department of Cell Biology, UT Southwestern Medical Center at Dallas, potential, it is difficult to use the same purified primary Dallas, TX, USA cultures for multiple large-scale experiments in different Full list of author information is available at the end of the article © 2011 Stadler et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Stadler et al. Skeletal Muscle 2011, 1:12 Page 2 of 10 http://www.skeletalmusclejournal.com/content/1/1/12 laboratories to reproduce the findings with the same cells and validated this by several observations. First, material. Immortal cell lines provide a solution to this CD56+ and CD56- cells were distinct and stable popula- problem, and are a useful and unlimited resource for tions (Figure 1C), and skeletal-muscle cells did not the research community. switch between a CD56+ and CD56- phenotype. Second, To immortalize FSHD muscle-derived myogenic CD56+, but not CD56- cells, expressed the intermediate cells, we used the same strategy as previously filament protein, desmin, another marker of skeletal- described for normal skeletal-muscle cells; that is, muscle cells (Figure 1D). Third, purified CD56+ but not ectopic expression of human telomerase reverse tran- CD56- populations fused into multinucleated myotubes when changed to differentiation medium (Figure 1E). scriptase (hTERT) to overcome replicative senescence, and of CDK4 to block the growth arrest due to cell- culture stress (stress or aberrant signaling-induced CDK4 maintains the myogenic population senescence (stasis)) [7]. Replicative senescence is an To test the hypothesis that stasis of CD56+ cells is irreversible growth arrest triggered by a DNA damage responsible for their overgrowth by CD56- cells in vitro, signal from critically short telomeres [8,9]. Telomere we infected primary 01Abic cells with a retrovirus con- shortening occurs during each cell division, because of taining CDK4 at the fifth population doubling (PD5), the end replication problem [10], end processing [11] when the fraction of CD56+ cells was about 20%. and oxidative damage [12], and can be prevented by Whereas the percentage of CD56+ cells quickly dropped addition of telomeric repeats by the reverse transcrip- towards zero in uninfected cells (Figure 2A, blue line tase telomerase [13,14]. Overexpression of hTERT, the with yellow circles), CDK4 overexpression maintained catalytic subunit of telomerase, has been shown to the CD56+ cells at roughly 20% for an additional 50 prevent senescence of a variety of human somatic days, corresponding to about 25 PDs (Figure 2A, blue cells that do not express this gene endogenously line with blue squares). The effect of CDK4 on mainte- [15-17]. However, inadequate in vitro culture condi- nance of the CD56+ population was also dramatic in tions can cause accumulating stress to certain cell the primary cells cultured from the 01U biceps (desig- types, which may lead to stasis, a premature growth nated 01Ubic) (Figure 2A, red lines). At early PDs, this arrest independent of telomere length. We have cell strain consisted of a seemingly pure myogenic popu- shown that overexpression of CDK4 is able to bypass lation, which nonetheless was overgrown by initially rare stasis in many cell types, including myoblasts, while CD56- cells (Figure 2A, red line with yellow circles) at maintaining normal phenotypes and cell cycle control between 30 and 50 days of culture. CDK4 overexpres- [7,18]. We show in this paper that overexpression of sion prevented this overgrowth, and maintained the CDK4 delays or even prevents overgrowth of myo- CD56+ population for the entire culture period (Figure genic CD56+ cells by their non-myogenic CD56- 2A, red line with red squares). These experiments sug- counterparts, and therefore facilitates the isolation of gest that CDK4 overexpression confers a selective clonal myogenic cell lines from severely affected dys- growth advantage on CD56+ cells compared with their trophic muscles. CD56- counterparts. This hypothesis was confirmed by infection of CD56 sorted 01Abic cells with CDK4 Results (Figure 2B). Overexpression of CDK4 led to a doubling Primary cultures from severely affected FSHD muscles of the replicative lifespan of CD56+ cells, from about 40 primarily consist of non-myogenic cells to 80 PDs. In the absence of CDK4, the growth rate of During progression of FSHD, the muscle fibers of cer- the CD56+ cells began to slow down as early as PD20, tain skeletal muscles are progressively replaced by con- contributing to the overgrowth by CD56- cells long nective tissue. The biceps is one of the muscles before the CD56+ cells actually had their growth preferentially affected by FSHD (Figure 1A). Despite arrested. By contrast, CDK4 had only a minor effect on good strength in the biopsied biceps, muscle fibers from the lifespan of CD56- cells. Telomeres of CD56- cells the biceps of a 42-year-old man with FSHD (subject were shorter than those of their CD56+ counterparts at 01A) were found to be in close association with fibrotic growth arrest in the absence of CDK4, consistent with tissue and surrounded by large pockets of adipose cells. the interpretation that CD56- cells arrested because of By contrast, the biceps muscle from his 46-year-old replicative aging, whereas CD56+ cells arrested because healthy brother (subject 01U, inset) exhibited no endo- of stasis before reaching the telomere-based replicative mysial fibrosis, and was virtually free of adipocytes. The limits. CDK4 overexpression led to further telomere percentage of myogenic cells in the primary cell cultures shortening during the increased lifespan of the CD56+ from the biceps muscle of 01A (designated 01Abic) was population, reaching lengths similar to those of the senescent CD56- population when they finally became only 20%, and decreased rapidly during in vitro cultiva- senescent (not shown). tion (Figure 1B). We used CD56 to identify myogenic Stadler et al. Skeletal Muscle 2011, 1:12 Page 3 of 10 http://www.skeletalmusclejournal.com/content/1/1/12 Figure 1 Replacement of FSHD muscle by non-myogenic cells. (A) Transverse section of the biceps muscle from subject 01A with facioscapulohumeral muscular dystrophy (FSHD), showing extensive fibrosis and large pockets of adipocytes; muscle fibers displayed variable diameter. By contrast, biceps muscle from his brother (subject 01U, inset) had a relatively uniform array of myogenic fibers with no endomysial fibrosis or fatty infiltration. Hematoxylin and eosin, original magnification 20 ×. (B) Percentage of CD56+ cells in primary cell cultures isolated from FSHD biceps muscle shown in (A) during in vitro cultivation. PD = population doubling. (C) Growth curves of sorted CD56+ (red squares) and CD56- (green squares) populations from the primary 01A biceps cell culture. The percentage of CD56+ cells in sorted, CD56+ and CD56- cultures over time are also displayed (circles outlined in red (CD56+) or green (CD56-)). (D) Desmin immunostaining (green) of sorted CD56+ and CD56- cells. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (blue). (E) Sorted CD56+ and CD56- cells after 3 days in differentiation medium. Stadler et al. Skeletal Muscle 2011, 1:12 Page 4 of 10 http://www.skeletalmusclejournal.com/content/1/1/12 Figure 2 CDK4 maintains the myogenic population. (A) Percentage of CD56+ cells in primary cultures from biceps muscle of a subject with facioscapulohumeral muscular dystrophy (FSHD) (01Abic) and his unaffected brother (01Ubic) with and without overexpression of CDK4. (B) Growth curves of CD56 sorted 01Abic cells with and without overexpression of CDK4. Myogenic clones uniformly expressing CD56 can be which survived the subsequent expansion to the ~2 × isolated after immortalization of primary cultures 10 cells necessary for cryopreservation and analysis for consisting mainly of CD56- cells myogenicity (CD56 expression and fusion competence). Even though pure myogenic populations can be isolated Of 15 randomly analyzed clones, four (27%) were myo- from mixed primary cultures using antibody based genic. The higher percentage of myogenic clones than methods, such as fluorescence or magnetic-activated cell CD56+ cells in the immortal population (27% vs. 14%) sorting(FACS or MACS), thosecells haveavery is consistent with the observed growth advantage for restricted replicative lifespan, and therefore can be used CD56+ over CD56- cells at clonal density (data not for only a limited number of experiments. We thus shown). Growth curves of three immortal myogenic established the following protocol to isolate immortal clones are shown in Figure 3C, in comparison with the clonal myogenic cell lines from severely affected dys- CD56 sorted primary culture, with and without CDK4. trophic muscle. Primary cultures (in this case, 01Abic, Immortalized myoblasts had a doubling time of about containing only 20% CD56+ cells) were consecutively 24 to 30 hours, which we found was only slightly higher infected with retroviruses for overexpression of CDK4 than that of primary cultures at early PDs (~20 to 26 and hTERT, and then uniformly CD56-expressing hours). All clones underwent >200 PDs without any sign clones were isolated (Figure 3A). In our hands, differ- of growth retardation. Whereas telomere restriction ences in viral titers, multiplicity of infection (MOI) and fragment analysis showed that telomeres were extended virus type have not had significant effects on rate of by ectopic hTERT for most myoblast clones (Figure 3B, immortalization, and we have obtained similar results immortal clone number 6 as a representative example), using low MOI retroviruses or high MOI lentiviruses. some clones maintained telomere lengths at levels simi- For the experiments published here, about 1% of the lar to that of the primary culture (Figure 3B, compare cells survived selection and therefore had been trans- immortal clone number 2 with the primary CD56+ duced, corresponding to an MOI of ~0.01. As CDK4 population). The different telomere dynamics may be expression maintains the myogenic population, the due to a variety of factors, including hTERT integration immortal population had an increased and stable per- site-specific hTERT transcription, or differences in fac- centage of CD56+ cells after drug selection compared tors affecting telomerase enzyme assembly, recruitment with the parental population at the same time point or activity at telomeres. (14% vs. 2%), and no sorting step before infection was All immortal clones stably expressed CD56 and des- necessary to isolate myogenic clones, which could be min, and retained their ability to fuse into myosin- carried out easily and with little effort. Healthy-looking expressing, multinucleated myotubes when moved to and well-separated clones were picked, roughly 90% of differentiation medium (Figure 4, 01Abic immortal Stadler et al. Skeletal Muscle 2011, 1:12 Page 5 of 10 http://www.skeletalmusclejournal.com/content/1/1/12 Figure 3 Isolation of immortal CD56+ clones. (A) FACS histograms of primary 01Abic cells at PD7 (left) and two clones at PD35, one CD56+ and the other CD56-, after immortalization with CDK4 and human telomerase reverse transcriptase (hTERT) (right). Red lines represent cells treated with anti-CD56 antibody; green lines represent controls where the primary antibody was omitted, and hence they correspond to nonspecific background fluorescence. The percentage of CD56+ cells (fluorescence greater than background) is indicated. (B) Telomere restriction fragment analysis gel showing telomere length dynamics of the CD56+ population and two immortalized CD56+ clones at different PDs. (C) Growth curves of primary 01Abic cells sorted for CD56 (CD56+ population, red squares), cells infected with CDK4 (CD56+ population CDK4, red diamonds) and three CD56+ immortalized clones (blue lines). clone number 6 is shown as a representative example); Discussion however, the differentiation kinetics were found to slow Since the description of the first cancer cell line [19], down with time in culture. Immortal clones at early PDs infinitely growing human cell lines have been invaluable (Figure 4, PD35) readily formed very large myotubes tools in biology. In contrast to most cancer cells that after only 3 days in differentiation medium; ~70% of are immortal, human somatic cells have a finite lifespan nuclei were present in multinucleated and/or myosin- and have to be ‘immortalized’ to obtain unlimited prolif- positive cells, detected by immunostaining with MF20 erative capacity. Historically, this was initially achieved antibody. By contrast, after >100 PDs (Figure 4, PD120 by overexpression of viral oncogenes such as simian and PD234), fusion was delayed and resulting myotubes virus(SV)40[20], whichisalwaysassociatedwithat were thinner, with <10% of nuclei present in MF20- least some degree of neoplastic transformation. By con- positive cells on day 3, and approximately 50% (PD120) trast, hTERT overexpression not only immortalizes a and 30% (PD234) appearing in multinucleated/MF20- variety of human primary cultures, but has been positive cells at day 7. reported to do this while also maintaining the cellular Stadler et al. Skeletal Muscle 2011, 1:12 Page 6 of 10 http://www.skeletalmusclejournal.com/content/1/1/12 PD 35 PD 120 PD 234 100% 100% CD56 100% CD56 CD56 CD56 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 fluorescence intensity fluorescence intensity fluorescence intensity desmin differentiation (brightfield) differentiation (myosin heavy chain in green, DAPI in blue) Figure 4 Characterization of an immortal CD56+ clone during in vitro propagation. Three time points are shown: population doubling (PD) 35, 120 and 234 (see growth curve in Figure 3C). Top row: flow cytometry histograms of CD56 stained cells (red lines) and control cells without the primary antibody (green lines, representing background fluorescence). Uniform expression of CD56 was maintained throughout >200 PDs of in vitro cultivation (percentage of CD56+ cells is indicated). Second row: cells stained for expression of the muscle-specific intermediate-filament protein, desmin (green) and counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). Third and fourth rows: phase (third row) and fluorescence (fourth row) images of cells in differentiation medium after 3 days (PD 35) or 7 days (PD 120 and PD234) and stained for myosin heavy chain (MF20, green) and DNA (DAPI, blue). phenotype [15,21,22]. However, this latter notion has recombinase-mediated excision of the hTERT cassette been challenged by the observation of putative non-telo- (Stadler and Wright, unpublished data). The slower dif- meric activities of hTERT [23-25], with strong evidence ferentiation kinetics and formation of thinner myotubes seen in high passage hTERT cells (Figure 4) were not for its potential to modulate the Wnt pathway in mice [26]. In most myoblast clones, expression of hTERT for due to a direct effect of ectopic hTERT, as this pheno- just a few PDs is sufficient to elongate telomeres by sev- type was not seen with lower passage cells, in which eral kilobases (Figure 3C, clone number 6). Although hTERT had already been expressed for >20 PDs. We the importance of the Wnt pathway in skeletal-muscle suggest that this change in differentiation kinetics is differentiation is well documented [27], we have not instead due to prolonged in vitro cultivation and its found any obvious phenotypic change between the associated potential for epigenetic drift and constant hTERT-expressing cells described here and cell models selection for proliferation. Hence, it is crucial to take that only transiently overexpress hTERT before Cre the replicative history of a cell into account, even for counts 0 40 80 counts 0 40 80 counts 0 40 80 Stadler et al. Skeletal Muscle 2011, 1:12 Page 7 of 10 http://www.skeletalmusclejournal.com/content/1/1/12 immortal cell lines, and to perform parallel analyses only is myostatin, which has been shown to be expressed by on cell lines at roughly similar PD levels. In situations both skeletal-muscle fibroblasts and myoblasts [31], and where this is not practical, it may be necessary to con- to inhibit proliferation of myoblasts [32]. Our immorta- firm that myogenic potential and differentiation kinetics lized CD56+ and CD56- clones from skeletal muscle have not changed with additional culturing. These are provide a model system to study the relationship also important variables to consider when comparing between them and to help elucidate the intercellular crosstalk and regulatory pathways between different cell different cell lines and cell clones with one another. types. Although there may be insertion-specific variation, our Although we cannot exclude the possibility that pri- findings link changes in the differentiation ability of a cell line with the time spent by that cell line in culture. mary cultures from skeletal muscle contain non-myo- The conditions for immortalization of human myo- genic CD56+ cells, such as infiltrating immune cells blasts were initially established using primary cultures [33], all CD56+ immortal clones analyzed to date in our from the pectoralis major muscle[7].Inadditionto laboratory (>20) were desmin-positive and fusion- cell-culture supplements, such as dexamethasone and competent. hepatocyte growth factor, ectopic CDK4 was necessary to prevent telomere-independent growth arrest (stasis). Conclusions Fetal lung fibroblasts require the spin-trap compound n- In summary, we describe a simple and efficient protocol tert-butyl hydroxylamine in addition to low oxygen to to establish immortal myogenic cell lines from dys- avoid stasis [28], but this substance was extremely toxic trophicmusclethatconsistsmainlyofnon-muscletis- to myoblasts (unpublished observations). This might sue. Additionally, we provide evidence that stasis of reflect the consequences of blocking nitric oxide signal- myoblasts and not their CD56- counterpart is responsi- ing in myoblasts [29]. ble for overgrowth of the former by the latter in primary In fetal fibroblasts and epithelial cells, stasis is often cultures, which can be prevented by ectopic CDK4. To associated with an increase in p16 [18,28], and therefore our knowledge, this is the first report of immortal myo- ectopic expression of CDK4 was chosen to titer out the genic human cell lines harboring the FSHD mutation, upregulation of this cell cycle inhibitor. Whereas others and will be useful in investigating this enigmatic disease. have shown that p16 is increased in growth-arresting myoblasts [30], we found that p16 levels are not Methods increased in human myoblasts during stasis [7]. Differ- The study was approved by The Johns Hopkins Medi- ential regulation of cell cycle inhibitors in vitro may be cine Institutional Review Board. explained by different culture conditions, or by develop- mental differences of fetal versus adult myoblasts (used Human subjects and tissue collection in the former and latter studies, respectively), as has Themaleproband (subject 01A) firstexhibited weak- been shown for fibroblasts [28]. We are currently inves- ness in the legs as a teenager. At time of enrollment, he tigating which factors prevent adult myoblasts from was 42 years old, and exhibited mild facial weakness, being immortalized by hTERT in the absence of CDK4 scapular winging and asymmetric weakness in the limbs, and are hence responsible for stasis. consistent with the FSHD phenotype. Molecular diagno- In contrast to CD56+ myoblasts, CD56- cells did not sis of FSHD was confirmed by the University of Iowa exhibit stasis under our culture conditions, which were Diagnostic Laboratories, which identified a HindIII 4qA originally optimized for myoblasts. This is supported by allele of 26 kb in length. An open muscle biopsy was the following observations: First, CDK4 overexpression taken from the biceps muscle (01Abic), which had its in CD56- cells did not lead to a significant increase in strength rated as 4+ out of 5 on the modified Medical their proliferative potential. Second, CD56- cells could Research Council scale. Tissue was also obtained from be immortalized by hTERT alone in conditions of low the biceps muscle (01Ubic) of the proband’s 46-year-old oxygen (data not shown), suggesting that CD56+ cells brother (01U), who had full muscle strength and no dis- are more vulnerable to ‘stress’ (which triggers stasis) ease allele found on gene testing. Approximately 300 mg compared with CD56- cells, and therefore CD56- cells of tissue was reserved for histological and biochemical may have a growth advantage in ‘stressful’ situations, assays, and ~500 to 700 mg tissue was used for primary such as during pathological regeneration of muscle in cell isolation. vivo. Additionally, it seems that at low passage, sorted CD56+ cells have growth rates similar to those of Primary cell isolation and cell culture CD56-, whereas the former are rapidly overgrown when The biceps muscle tissue was minced into fragments of unsorted, arguing that CD56- cells might inhibit growth <5 mm , and stored at 4°C in a Ham’s F10 medium con- of CD56+ cells. One candidate for this inhibitory activity taining 20% fetal bovine serum (FBS; Hyclone, Thermo Stadler et al. Skeletal Muscle 2011, 1:12 Page 8 of 10 http://www.skeletalmusclejournal.com/content/1/1/12 Scientific, Logan, UT, USA), 2% chick embryo extract NJ, USA) covered by 10 ml medium, with a weekly and 2.5 ng/ml basic fibroblast growth factor (Biopioneer, medium change if cells were not passaged before that San Diego, CA, USA), supplemented with 1% antibiotic/ (usually one passage every 5 to 7 days). Cells were pas- antimycotic (Cellgro, Manassas, VA, USA) until pro- saged at 50 to 90% confluency using 0.05% trypsin/ cessed for cell isolation (within approximately 24 hours EDTA (Gibco), and cell numbers determined (Z1 Coul- of biopsy) (viable cells have been successfully isolated ter Particle Counter; Beckman Coulter, Miami, FL, from tissues stored in this manner up to 6 days after USA). PD was calculated using the formula: PD = ln biopsy). Tissue was cleaned and minced in Hanks’ [(final number of cells)/(initial number of cells)]/In(2). For differentiation, 1.2 × 10 balanced salt solution (HBSS, catalogue number 14175; myoblasts were seeded in Gibco, Grand Island, NY, USA) and dissociated in 5 ml six-well dishes, then 24 hours later, cells were washed of enzyme solution (1 mg/ml collagenase IV, 2.4 U/ml twice with phosphate-buffered saline (PBS) and fed with dispase (both Worthington, Lakewood, NJ, USA) and differentiation medium (DMEM and Medium 199 in a 2.5 mmol/l CaCl in HBSS] for 45 minutes at 37°C, tri- ratio of 4:1, supplemented either with 2% horse serum turating every 15 minutes with a 5 ml serological pipette (Gibco) or with 0.02 mol/l HEPES plus 10 mg/l insulin (moving it up and down ~10 to 15 times), and filtered and 100 mg/l apo-transferrin (both Sigma-Aldrich)). through 100 μmand 40 μm cell sieves (BD Biosciences, Results obtained were similar with both differentiation Bedford, MA, USA). Cells were pelleted by spinning in a media, although a delay of ~1 day was observed with centrifuge at 1000 g for 5 minutes, and resuspended in horse serum relative to insulin/transferrin. 2mlgrowthmedium(Ham’s F-10 supplemented with 20% FBS (Hyclone), 0.5% chick embryo extract, 1.2 Retroviral infection mmol/l CaCl and 1% antibiotic/antimycotic (Cellgro), The construction of the vectors used for immortaliza- prepared as fresh stock at least every 7 days) and tion (CDK4-pBabe-neo and hTERT-pBabe-Hygro), has counted with a hemocytometer. The 01Abic cells were been described previously [7,18]. In brief, mouse CDK4 seeded in 2 ml growth medium on a 35 mm gelatin- (kindly provided by Charles J. Sherr) and hTERT coated dish, and the 01Ubic cells were seeded on a 60 cDNAs were inserted into pBabe vectors [35] containing mm dish in 5 ml growth medium. Cells were cultured neomycin- and hygromycin-resistance genes, respec- undisturbed for 48 hours, after which they were given tively. Additionally, loxP sites were placed internal to fresh growth medium daily for 2 to 4 days. When the long terminal repeats of the hTERT expression vec- approximately 50 to 70% confluent, cells were treated tor, to allow for excision of the entire expression cas- with trypsin (TrypLE Express/Gibco) for 5 minutes at sette by Cre recombinase. These vectors were 37°C, neutralized with an equal volume of growth med- transfected into the Phoenix ecotropic packaging cell ium, and counted with a hemocytometer. For expansion, line using the calcium phosphate technique [36], and cells were seeded at ~2000 to 3000 cells/cm in growth the virus-containing supernatant was used to infect the medium. For freezing, an equal volume of ice-cold 2 × amphotropic packaging cell line PA317 [37] to obtain freezing medium (20% dimethysulfoxide, 50% FBS, 30% stable virus-producing cell lines after selection with 0.5 growth medium) was added to cells and mixed well, mg/ml G418 or hygromycin (EMD Biosciences, San then cells were incubated in the liquid nitrogen vapor Diego, CA, USA). All infections were performed in the phase for at least 1 hour before storage at -140°C. presence of 2 μg/ml polybrene (Sigma-Aldrich). To standardize conditions for growth of primary and For infection of myoblasts, 5 × 10 cells were seeded immortalized cells, primary myoblasts were subsequently in six-well dishes 24 hours before infection. Selection cultured in a 4:1 mixture of Dulbecco modified Eagle was started 48 hours after infection using 300 mg/l medium (DMEM) and Medium 199 (Hyclone), supple- hygromycin and 400 mg/l G418 for 1 and 2 weeks, mented with 15% FBS (Hyclone), 0.02 M HEPES buffer, respectively. Depending on growth rates and infection 1.4 mg/l vitamin B12 (both Sigma-Aldrich, St. Louis, efficiency, cells were passaged during the selection per- MO, USA), 0.03 mg/l ZnSO (Fisher Scientific, Fair iod before becoming confluent. Lawn,NJ, USA),0.055 mg/l dexamethasone(Sigma- Aldrich), 2.5 μg/l hepatocyte growth factor (Chemicon Isolation of myogenic immortalized clones International, Temecula, CA, USA) and 10 μg/l basic Immortalized populations were seeded at low densities, fibroblast growth factor (BioPioneer), on dishes coated (50, 100, 200 or 400 cells per 100 mm dish). About 10 with 0.1% pigskin gelatin (Sigma-Aldrich) in a 2-5% oxy- days after seeding, clones were isolated using cloning gen environment [34]. Hepatocyte and fibroblast growth rings and seeded into 48-well dishes, followed by six factors were added to a volume of medium that was well and 100 mm dishes. The exact time point for clone used within 1 week. Cells were routinely grown in 100 isolation was chosen to maximize the cell number and mm tissue culture dishes (BD Falcon, Franklin Lakes, minimize cell density of individual clones, to facilitate Stadler et al. Skeletal Muscle 2011, 1:12 Page 9 of 10 http://www.skeletalmusclejournal.com/content/1/1/12 growth after passage but avoid fusion of myoblasts, were collected on microscope slides (Fisherbrand Super- respectively. We estimate that clones contained 100 to frost Plus; Fisher Scientific), air-dried, and fixed with 4% 1000 cells at the time of isolation. All clones were ana- formaldehyde in PBS for 15 minutes at room tempera- lyzed for CD56 expression by flow cytometry (see ture. Slides were washed three times with PBS and below) and fusion potential in differentiation medium. stained with hematoxylin and eosin. Sections were The percentage of cell nuclei in multinucleated and/or imaged under a microscope (AxioImager M2, Zeiss) and MF20-positive cells was determined for 10 random images captured with a camera (Digital microscopy fields after 3 or 7 days in differentiation medium. camera Axio Cam, ICC3, Zeiss). Terminal restriction fragment assay Acknowledgements Terminal restriction fragment assay was performed as We thank Kendal Hanger and Leigh Warsing for technical support. We also previously described [38]. thank the two subjects for their generous participation in this research. This work was performed within the framework of the Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center for FSHD, supported by Flow cytometry National Institutes of Health grant 5U54HD060848. GS is an Ellison/AFAR Cells were detached with trypsin, counted, and sedimen- Postdoctoral Fellow in Aging Research and was supported by an Erwin Schroedinger grant (project number J2737-B12) from the Austrian Science ted by centrifugation, then 1.5 × 10 cells were resus- Fund. pended in 0.2 ml DMEM with 10% calf serum (Cosmic Calf Serum; Hyclone) containing 5% supernatant from Author details Department of Cell Biology, UT Southwestern Medical Center at Dallas, the hybridoma line 5.1H11 [39], which recognizes CD56 Dallas, TX, USA. Boston Biomedical Research Institute, Watertown, MA, USA. (kindly provided by Helen Blau). As a control, primary 3 Center for Genetic Muscle Disorders, The Kennedy Krieger Institute, Johns antibody was omitted. Cells were incubated for 30 min at Hopkins School of Medicine, Baltimore, MD, USA. room temperature, washed twice with 10% calf serum Authors’ contributions (Cosmic Calf Serum; Hyclone) in PBS, and incubated KW, CPE and WEW designed this study. GS, JC, KW and JR performed the with secondary Alexa488-conjugated goat anti-mouse experiments and analyzed the results. GS, JC, KW, JWS and WEW wrote the manuscript. All authors read and approved the final manuscript. antibody (Invitrogen, Eugene, OR, USA) for 30 min at room temperature. After washing with PBS, cells were Competing interests resuspended in 0.2 ml PBS and analyzed on a flow cyt- The authors declare that they have no competing interests. ometer (FACSCalibur; Becton Dickinson). A second flow Received: 19 November 2010 Accepted: 8 March 2011 cytometer (FACSAria; Becton Dickinson) was used for Published: 8 March 2011 sorting, with 5 × 10 cells stained in upscaled volumes. 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Cite this article as: Stadler et al.: Establishment of clonal myogenic cell 19. Scherer WF, Syverton JT, Gey GO: Studies on the propagation in vitro of lines from severely affected dystrophic muscles - CDK4 maintains the poliomyelitis viruses. IV. Viral multiplication in a stable strain of human myogenic population. Skeletal Muscle 2011 1:12. malignant epithelial cells (strain HeLa) derived from an epidermoid carcinoma of the cervix. J Exp Med 1953, 97:695-710. 20. Todaro GJ, Wolman SR, Green H: Rapid Transformation of Human Fibroblasts with Low Growth Potential into Established Cell Lines by Sv40. J Cell Physiol 1963, 62:257-265. 21. Morales CP, Holt SE, Ouellette M, Kaur KJ, Yan Y, Wilson KS, White MA, Wright WE, Shay JW: Absence of cancer-associated changes in human fibroblasts immortalized with telomerase. Nat Genet 1999, 21:115-118. 22. 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Smith LL, Coller HA, Roberts JM: Telomerase modulates expression of growth-controlling genes and enhances cell proliferation. Nat Cell Biol • Convenient online submission 2003, 5:474-479. 26. Park JI, Venteicher AS, Hong JY, Choi J, Jun S, Shkreli M, Chang W, Meng Z, • Thorough peer review Cheung P, Ji H, McLaughlin M, Veenstra TD, Nusse R, McCrea PD, • No space constraints or color figure charges Artandi SE: Telomerase modulates Wnt signalling by association with • Immediate publication on acceptance target gene chromatin. Nature 2009, 460:66-72. 27. Tsivitse S: Notch and Wnt signaling, physiological stimuli and postnatal • Inclusion in PubMed, CAS, Scopus and Google Scholar myogenesis. Int J Biol Sci 2010, 6:268-281. • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Establishment of clonal myogenic cell lines from severely affected dystrophic muscles - CDK4 maintains the myogenic population

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Springer Journals
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Copyright © 2011 by Stadler et al; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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2044-5040
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10.1186/2044-5040-1-12
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21798090
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Abstract

Background: A hallmark of muscular dystrophies is the replacement of muscle by connective tissue. Muscle biopsies from patients severely affected with facioscapulohumeral muscular dystrophy (FSHD) may contain few myogenic cells. Because the chromosomal contraction at 4q35 linked to FSHD is thought to cause a defect within myogenic cells, it is important to study this particular cell type, rather than the fibroblasts and adipocytes of the endomysial fibrosis, to understand the mechanism leading to myopathy. Results: We present a protocol to establish clonal myogenic cell lines from even severely dystrophic muscle that has been replaced mostly by fat, using overexpression of CDK4 and the catalytic component of telomerase (human telomerase reverse transcriptase; hTERT), and a subsequent cloning step. hTERT is necessary to compensate for telomere loss during in vitro cultivation, while CDK4 prevents a telomere-independent growth arrest affecting CD56 + myogenic cells, but not their CD56- counterpart, in vitro. Conclusions: These immortal cell lines are valuable tools to reproducibly study the effect of the FSHD mutation within myoblasts isolated from muscles that have been severely affected by the disease, without the confounding influence of variable amounts of contaminating connective-tissue cells. Background (FSHD), but the technique is also applicable to other Most muscular dystrophies result from a defect within muscular dystrophies. myogenic cells that leads to progressive muscle weak- FSHD has been linked to deletions of D4Z4 tandem ness and wasting, and in severe cases, to the replace- repeats at chromosome 4q [1], but it is still unclear how ment of muscle fibers by connective tissue and/or fat. this deletion causes disease. Primary skeletal-muscle cul- At advanced stages, skeletal muscle is replaced by tures have been used in attempts to model the disease, fibroblasts and adipocytes. Although these cell types and several FSHD-specific phenotypes, such as increased probably promote disease in later stages, it is generally vacuolization and sensitivity to oxidative stress [2] have believed that the root cause leading to most muscular been described. In addition, there have been reports dystrophiesisadefect originating in myogenic cells. describing incorrect expression of various FSHD candi- We therefore developed a protocol to enrich, immorta- date genes in muscles and myogenic cells from patients lizeand isolateraremuscleprogenitorcells from with FSHD, including FSHD region gene (FRG)1 and severely affected dystrophic muscles to obtain clonal the double homeobox (DUX)4 gene [3-6]. However, in myogenic cell lines. In this report, the protocol is many instances, these findings have not been consistent described for cells isolated from skeletal muscle of between different laboratories, perhaps as a result of people with facioscapulohumeral muscular dystrophy variables such as muscle type, disease progression, cul- ture purity, in vitro culture conditions or replicative age * Correspondence: Woodring.Wright@UTSouthwestern.edu of the cells. In particular, because of their low replicative Department of Cell Biology, UT Southwestern Medical Center at Dallas, potential, it is difficult to use the same purified primary Dallas, TX, USA cultures for multiple large-scale experiments in different Full list of author information is available at the end of the article © 2011 Stadler et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Stadler et al. Skeletal Muscle 2011, 1:12 Page 2 of 10 http://www.skeletalmusclejournal.com/content/1/1/12 laboratories to reproduce the findings with the same cells and validated this by several observations. First, material. Immortal cell lines provide a solution to this CD56+ and CD56- cells were distinct and stable popula- problem, and are a useful and unlimited resource for tions (Figure 1C), and skeletal-muscle cells did not the research community. switch between a CD56+ and CD56- phenotype. Second, To immortalize FSHD muscle-derived myogenic CD56+, but not CD56- cells, expressed the intermediate cells, we used the same strategy as previously filament protein, desmin, another marker of skeletal- described for normal skeletal-muscle cells; that is, muscle cells (Figure 1D). Third, purified CD56+ but not ectopic expression of human telomerase reverse tran- CD56- populations fused into multinucleated myotubes when changed to differentiation medium (Figure 1E). scriptase (hTERT) to overcome replicative senescence, and of CDK4 to block the growth arrest due to cell- culture stress (stress or aberrant signaling-induced CDK4 maintains the myogenic population senescence (stasis)) [7]. Replicative senescence is an To test the hypothesis that stasis of CD56+ cells is irreversible growth arrest triggered by a DNA damage responsible for their overgrowth by CD56- cells in vitro, signal from critically short telomeres [8,9]. Telomere we infected primary 01Abic cells with a retrovirus con- shortening occurs during each cell division, because of taining CDK4 at the fifth population doubling (PD5), the end replication problem [10], end processing [11] when the fraction of CD56+ cells was about 20%. and oxidative damage [12], and can be prevented by Whereas the percentage of CD56+ cells quickly dropped addition of telomeric repeats by the reverse transcrip- towards zero in uninfected cells (Figure 2A, blue line tase telomerase [13,14]. Overexpression of hTERT, the with yellow circles), CDK4 overexpression maintained catalytic subunit of telomerase, has been shown to the CD56+ cells at roughly 20% for an additional 50 prevent senescence of a variety of human somatic days, corresponding to about 25 PDs (Figure 2A, blue cells that do not express this gene endogenously line with blue squares). The effect of CDK4 on mainte- [15-17]. However, inadequate in vitro culture condi- nance of the CD56+ population was also dramatic in tions can cause accumulating stress to certain cell the primary cells cultured from the 01U biceps (desig- types, which may lead to stasis, a premature growth nated 01Ubic) (Figure 2A, red lines). At early PDs, this arrest independent of telomere length. We have cell strain consisted of a seemingly pure myogenic popu- shown that overexpression of CDK4 is able to bypass lation, which nonetheless was overgrown by initially rare stasis in many cell types, including myoblasts, while CD56- cells (Figure 2A, red line with yellow circles) at maintaining normal phenotypes and cell cycle control between 30 and 50 days of culture. CDK4 overexpres- [7,18]. We show in this paper that overexpression of sion prevented this overgrowth, and maintained the CDK4 delays or even prevents overgrowth of myo- CD56+ population for the entire culture period (Figure genic CD56+ cells by their non-myogenic CD56- 2A, red line with red squares). These experiments sug- counterparts, and therefore facilitates the isolation of gest that CDK4 overexpression confers a selective clonal myogenic cell lines from severely affected dys- growth advantage on CD56+ cells compared with their trophic muscles. CD56- counterparts. This hypothesis was confirmed by infection of CD56 sorted 01Abic cells with CDK4 Results (Figure 2B). Overexpression of CDK4 led to a doubling Primary cultures from severely affected FSHD muscles of the replicative lifespan of CD56+ cells, from about 40 primarily consist of non-myogenic cells to 80 PDs. In the absence of CDK4, the growth rate of During progression of FSHD, the muscle fibers of cer- the CD56+ cells began to slow down as early as PD20, tain skeletal muscles are progressively replaced by con- contributing to the overgrowth by CD56- cells long nective tissue. The biceps is one of the muscles before the CD56+ cells actually had their growth preferentially affected by FSHD (Figure 1A). Despite arrested. By contrast, CDK4 had only a minor effect on good strength in the biopsied biceps, muscle fibers from the lifespan of CD56- cells. Telomeres of CD56- cells the biceps of a 42-year-old man with FSHD (subject were shorter than those of their CD56+ counterparts at 01A) were found to be in close association with fibrotic growth arrest in the absence of CDK4, consistent with tissue and surrounded by large pockets of adipose cells. the interpretation that CD56- cells arrested because of By contrast, the biceps muscle from his 46-year-old replicative aging, whereas CD56+ cells arrested because healthy brother (subject 01U, inset) exhibited no endo- of stasis before reaching the telomere-based replicative mysial fibrosis, and was virtually free of adipocytes. The limits. CDK4 overexpression led to further telomere percentage of myogenic cells in the primary cell cultures shortening during the increased lifespan of the CD56+ from the biceps muscle of 01A (designated 01Abic) was population, reaching lengths similar to those of the senescent CD56- population when they finally became only 20%, and decreased rapidly during in vitro cultiva- senescent (not shown). tion (Figure 1B). We used CD56 to identify myogenic Stadler et al. Skeletal Muscle 2011, 1:12 Page 3 of 10 http://www.skeletalmusclejournal.com/content/1/1/12 Figure 1 Replacement of FSHD muscle by non-myogenic cells. (A) Transverse section of the biceps muscle from subject 01A with facioscapulohumeral muscular dystrophy (FSHD), showing extensive fibrosis and large pockets of adipocytes; muscle fibers displayed variable diameter. By contrast, biceps muscle from his brother (subject 01U, inset) had a relatively uniform array of myogenic fibers with no endomysial fibrosis or fatty infiltration. Hematoxylin and eosin, original magnification 20 ×. (B) Percentage of CD56+ cells in primary cell cultures isolated from FSHD biceps muscle shown in (A) during in vitro cultivation. PD = population doubling. (C) Growth curves of sorted CD56+ (red squares) and CD56- (green squares) populations from the primary 01A biceps cell culture. The percentage of CD56+ cells in sorted, CD56+ and CD56- cultures over time are also displayed (circles outlined in red (CD56+) or green (CD56-)). (D) Desmin immunostaining (green) of sorted CD56+ and CD56- cells. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (blue). (E) Sorted CD56+ and CD56- cells after 3 days in differentiation medium. Stadler et al. Skeletal Muscle 2011, 1:12 Page 4 of 10 http://www.skeletalmusclejournal.com/content/1/1/12 Figure 2 CDK4 maintains the myogenic population. (A) Percentage of CD56+ cells in primary cultures from biceps muscle of a subject with facioscapulohumeral muscular dystrophy (FSHD) (01Abic) and his unaffected brother (01Ubic) with and without overexpression of CDK4. (B) Growth curves of CD56 sorted 01Abic cells with and without overexpression of CDK4. Myogenic clones uniformly expressing CD56 can be which survived the subsequent expansion to the ~2 × isolated after immortalization of primary cultures 10 cells necessary for cryopreservation and analysis for consisting mainly of CD56- cells myogenicity (CD56 expression and fusion competence). Even though pure myogenic populations can be isolated Of 15 randomly analyzed clones, four (27%) were myo- from mixed primary cultures using antibody based genic. The higher percentage of myogenic clones than methods, such as fluorescence or magnetic-activated cell CD56+ cells in the immortal population (27% vs. 14%) sorting(FACS or MACS), thosecells haveavery is consistent with the observed growth advantage for restricted replicative lifespan, and therefore can be used CD56+ over CD56- cells at clonal density (data not for only a limited number of experiments. We thus shown). Growth curves of three immortal myogenic established the following protocol to isolate immortal clones are shown in Figure 3C, in comparison with the clonal myogenic cell lines from severely affected dys- CD56 sorted primary culture, with and without CDK4. trophic muscle. Primary cultures (in this case, 01Abic, Immortalized myoblasts had a doubling time of about containing only 20% CD56+ cells) were consecutively 24 to 30 hours, which we found was only slightly higher infected with retroviruses for overexpression of CDK4 than that of primary cultures at early PDs (~20 to 26 and hTERT, and then uniformly CD56-expressing hours). All clones underwent >200 PDs without any sign clones were isolated (Figure 3A). In our hands, differ- of growth retardation. Whereas telomere restriction ences in viral titers, multiplicity of infection (MOI) and fragment analysis showed that telomeres were extended virus type have not had significant effects on rate of by ectopic hTERT for most myoblast clones (Figure 3B, immortalization, and we have obtained similar results immortal clone number 6 as a representative example), using low MOI retroviruses or high MOI lentiviruses. some clones maintained telomere lengths at levels simi- For the experiments published here, about 1% of the lar to that of the primary culture (Figure 3B, compare cells survived selection and therefore had been trans- immortal clone number 2 with the primary CD56+ duced, corresponding to an MOI of ~0.01. As CDK4 population). The different telomere dynamics may be expression maintains the myogenic population, the due to a variety of factors, including hTERT integration immortal population had an increased and stable per- site-specific hTERT transcription, or differences in fac- centage of CD56+ cells after drug selection compared tors affecting telomerase enzyme assembly, recruitment with the parental population at the same time point or activity at telomeres. (14% vs. 2%), and no sorting step before infection was All immortal clones stably expressed CD56 and des- necessary to isolate myogenic clones, which could be min, and retained their ability to fuse into myosin- carried out easily and with little effort. Healthy-looking expressing, multinucleated myotubes when moved to and well-separated clones were picked, roughly 90% of differentiation medium (Figure 4, 01Abic immortal Stadler et al. Skeletal Muscle 2011, 1:12 Page 5 of 10 http://www.skeletalmusclejournal.com/content/1/1/12 Figure 3 Isolation of immortal CD56+ clones. (A) FACS histograms of primary 01Abic cells at PD7 (left) and two clones at PD35, one CD56+ and the other CD56-, after immortalization with CDK4 and human telomerase reverse transcriptase (hTERT) (right). Red lines represent cells treated with anti-CD56 antibody; green lines represent controls where the primary antibody was omitted, and hence they correspond to nonspecific background fluorescence. The percentage of CD56+ cells (fluorescence greater than background) is indicated. (B) Telomere restriction fragment analysis gel showing telomere length dynamics of the CD56+ population and two immortalized CD56+ clones at different PDs. (C) Growth curves of primary 01Abic cells sorted for CD56 (CD56+ population, red squares), cells infected with CDK4 (CD56+ population CDK4, red diamonds) and three CD56+ immortalized clones (blue lines). clone number 6 is shown as a representative example); Discussion however, the differentiation kinetics were found to slow Since the description of the first cancer cell line [19], down with time in culture. Immortal clones at early PDs infinitely growing human cell lines have been invaluable (Figure 4, PD35) readily formed very large myotubes tools in biology. In contrast to most cancer cells that after only 3 days in differentiation medium; ~70% of are immortal, human somatic cells have a finite lifespan nuclei were present in multinucleated and/or myosin- and have to be ‘immortalized’ to obtain unlimited prolif- positive cells, detected by immunostaining with MF20 erative capacity. Historically, this was initially achieved antibody. By contrast, after >100 PDs (Figure 4, PD120 by overexpression of viral oncogenes such as simian and PD234), fusion was delayed and resulting myotubes virus(SV)40[20], whichisalwaysassociatedwithat were thinner, with <10% of nuclei present in MF20- least some degree of neoplastic transformation. By con- positive cells on day 3, and approximately 50% (PD120) trast, hTERT overexpression not only immortalizes a and 30% (PD234) appearing in multinucleated/MF20- variety of human primary cultures, but has been positive cells at day 7. reported to do this while also maintaining the cellular Stadler et al. Skeletal Muscle 2011, 1:12 Page 6 of 10 http://www.skeletalmusclejournal.com/content/1/1/12 PD 35 PD 120 PD 234 100% 100% CD56 100% CD56 CD56 CD56 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 fluorescence intensity fluorescence intensity fluorescence intensity desmin differentiation (brightfield) differentiation (myosin heavy chain in green, DAPI in blue) Figure 4 Characterization of an immortal CD56+ clone during in vitro propagation. Three time points are shown: population doubling (PD) 35, 120 and 234 (see growth curve in Figure 3C). Top row: flow cytometry histograms of CD56 stained cells (red lines) and control cells without the primary antibody (green lines, representing background fluorescence). Uniform expression of CD56 was maintained throughout >200 PDs of in vitro cultivation (percentage of CD56+ cells is indicated). Second row: cells stained for expression of the muscle-specific intermediate-filament protein, desmin (green) and counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). Third and fourth rows: phase (third row) and fluorescence (fourth row) images of cells in differentiation medium after 3 days (PD 35) or 7 days (PD 120 and PD234) and stained for myosin heavy chain (MF20, green) and DNA (DAPI, blue). phenotype [15,21,22]. However, this latter notion has recombinase-mediated excision of the hTERT cassette been challenged by the observation of putative non-telo- (Stadler and Wright, unpublished data). The slower dif- meric activities of hTERT [23-25], with strong evidence ferentiation kinetics and formation of thinner myotubes seen in high passage hTERT cells (Figure 4) were not for its potential to modulate the Wnt pathway in mice [26]. In most myoblast clones, expression of hTERT for due to a direct effect of ectopic hTERT, as this pheno- just a few PDs is sufficient to elongate telomeres by sev- type was not seen with lower passage cells, in which eral kilobases (Figure 3C, clone number 6). Although hTERT had already been expressed for >20 PDs. We the importance of the Wnt pathway in skeletal-muscle suggest that this change in differentiation kinetics is differentiation is well documented [27], we have not instead due to prolonged in vitro cultivation and its found any obvious phenotypic change between the associated potential for epigenetic drift and constant hTERT-expressing cells described here and cell models selection for proliferation. Hence, it is crucial to take that only transiently overexpress hTERT before Cre the replicative history of a cell into account, even for counts 0 40 80 counts 0 40 80 counts 0 40 80 Stadler et al. Skeletal Muscle 2011, 1:12 Page 7 of 10 http://www.skeletalmusclejournal.com/content/1/1/12 immortal cell lines, and to perform parallel analyses only is myostatin, which has been shown to be expressed by on cell lines at roughly similar PD levels. In situations both skeletal-muscle fibroblasts and myoblasts [31], and where this is not practical, it may be necessary to con- to inhibit proliferation of myoblasts [32]. Our immorta- firm that myogenic potential and differentiation kinetics lized CD56+ and CD56- clones from skeletal muscle have not changed with additional culturing. These are provide a model system to study the relationship also important variables to consider when comparing between them and to help elucidate the intercellular crosstalk and regulatory pathways between different cell different cell lines and cell clones with one another. types. Although there may be insertion-specific variation, our Although we cannot exclude the possibility that pri- findings link changes in the differentiation ability of a cell line with the time spent by that cell line in culture. mary cultures from skeletal muscle contain non-myo- The conditions for immortalization of human myo- genic CD56+ cells, such as infiltrating immune cells blasts were initially established using primary cultures [33], all CD56+ immortal clones analyzed to date in our from the pectoralis major muscle[7].Inadditionto laboratory (>20) were desmin-positive and fusion- cell-culture supplements, such as dexamethasone and competent. hepatocyte growth factor, ectopic CDK4 was necessary to prevent telomere-independent growth arrest (stasis). Conclusions Fetal lung fibroblasts require the spin-trap compound n- In summary, we describe a simple and efficient protocol tert-butyl hydroxylamine in addition to low oxygen to to establish immortal myogenic cell lines from dys- avoid stasis [28], but this substance was extremely toxic trophicmusclethatconsistsmainlyofnon-muscletis- to myoblasts (unpublished observations). This might sue. Additionally, we provide evidence that stasis of reflect the consequences of blocking nitric oxide signal- myoblasts and not their CD56- counterpart is responsi- ing in myoblasts [29]. ble for overgrowth of the former by the latter in primary In fetal fibroblasts and epithelial cells, stasis is often cultures, which can be prevented by ectopic CDK4. To associated with an increase in p16 [18,28], and therefore our knowledge, this is the first report of immortal myo- ectopic expression of CDK4 was chosen to titer out the genic human cell lines harboring the FSHD mutation, upregulation of this cell cycle inhibitor. Whereas others and will be useful in investigating this enigmatic disease. have shown that p16 is increased in growth-arresting myoblasts [30], we found that p16 levels are not Methods increased in human myoblasts during stasis [7]. Differ- The study was approved by The Johns Hopkins Medi- ential regulation of cell cycle inhibitors in vitro may be cine Institutional Review Board. explained by different culture conditions, or by develop- mental differences of fetal versus adult myoblasts (used Human subjects and tissue collection in the former and latter studies, respectively), as has Themaleproband (subject 01A) firstexhibited weak- been shown for fibroblasts [28]. We are currently inves- ness in the legs as a teenager. At time of enrollment, he tigating which factors prevent adult myoblasts from was 42 years old, and exhibited mild facial weakness, being immortalized by hTERT in the absence of CDK4 scapular winging and asymmetric weakness in the limbs, and are hence responsible for stasis. consistent with the FSHD phenotype. Molecular diagno- In contrast to CD56+ myoblasts, CD56- cells did not sis of FSHD was confirmed by the University of Iowa exhibit stasis under our culture conditions, which were Diagnostic Laboratories, which identified a HindIII 4qA originally optimized for myoblasts. This is supported by allele of 26 kb in length. An open muscle biopsy was the following observations: First, CDK4 overexpression taken from the biceps muscle (01Abic), which had its in CD56- cells did not lead to a significant increase in strength rated as 4+ out of 5 on the modified Medical their proliferative potential. Second, CD56- cells could Research Council scale. Tissue was also obtained from be immortalized by hTERT alone in conditions of low the biceps muscle (01Ubic) of the proband’s 46-year-old oxygen (data not shown), suggesting that CD56+ cells brother (01U), who had full muscle strength and no dis- are more vulnerable to ‘stress’ (which triggers stasis) ease allele found on gene testing. Approximately 300 mg compared with CD56- cells, and therefore CD56- cells of tissue was reserved for histological and biochemical may have a growth advantage in ‘stressful’ situations, assays, and ~500 to 700 mg tissue was used for primary such as during pathological regeneration of muscle in cell isolation. vivo. Additionally, it seems that at low passage, sorted CD56+ cells have growth rates similar to those of Primary cell isolation and cell culture CD56-, whereas the former are rapidly overgrown when The biceps muscle tissue was minced into fragments of unsorted, arguing that CD56- cells might inhibit growth <5 mm , and stored at 4°C in a Ham’s F10 medium con- of CD56+ cells. One candidate for this inhibitory activity taining 20% fetal bovine serum (FBS; Hyclone, Thermo Stadler et al. Skeletal Muscle 2011, 1:12 Page 8 of 10 http://www.skeletalmusclejournal.com/content/1/1/12 Scientific, Logan, UT, USA), 2% chick embryo extract NJ, USA) covered by 10 ml medium, with a weekly and 2.5 ng/ml basic fibroblast growth factor (Biopioneer, medium change if cells were not passaged before that San Diego, CA, USA), supplemented with 1% antibiotic/ (usually one passage every 5 to 7 days). Cells were pas- antimycotic (Cellgro, Manassas, VA, USA) until pro- saged at 50 to 90% confluency using 0.05% trypsin/ cessed for cell isolation (within approximately 24 hours EDTA (Gibco), and cell numbers determined (Z1 Coul- of biopsy) (viable cells have been successfully isolated ter Particle Counter; Beckman Coulter, Miami, FL, from tissues stored in this manner up to 6 days after USA). PD was calculated using the formula: PD = ln biopsy). Tissue was cleaned and minced in Hanks’ [(final number of cells)/(initial number of cells)]/In(2). For differentiation, 1.2 × 10 balanced salt solution (HBSS, catalogue number 14175; myoblasts were seeded in Gibco, Grand Island, NY, USA) and dissociated in 5 ml six-well dishes, then 24 hours later, cells were washed of enzyme solution (1 mg/ml collagenase IV, 2.4 U/ml twice with phosphate-buffered saline (PBS) and fed with dispase (both Worthington, Lakewood, NJ, USA) and differentiation medium (DMEM and Medium 199 in a 2.5 mmol/l CaCl in HBSS] for 45 minutes at 37°C, tri- ratio of 4:1, supplemented either with 2% horse serum turating every 15 minutes with a 5 ml serological pipette (Gibco) or with 0.02 mol/l HEPES plus 10 mg/l insulin (moving it up and down ~10 to 15 times), and filtered and 100 mg/l apo-transferrin (both Sigma-Aldrich)). through 100 μmand 40 μm cell sieves (BD Biosciences, Results obtained were similar with both differentiation Bedford, MA, USA). Cells were pelleted by spinning in a media, although a delay of ~1 day was observed with centrifuge at 1000 g for 5 minutes, and resuspended in horse serum relative to insulin/transferrin. 2mlgrowthmedium(Ham’s F-10 supplemented with 20% FBS (Hyclone), 0.5% chick embryo extract, 1.2 Retroviral infection mmol/l CaCl and 1% antibiotic/antimycotic (Cellgro), The construction of the vectors used for immortaliza- prepared as fresh stock at least every 7 days) and tion (CDK4-pBabe-neo and hTERT-pBabe-Hygro), has counted with a hemocytometer. The 01Abic cells were been described previously [7,18]. In brief, mouse CDK4 seeded in 2 ml growth medium on a 35 mm gelatin- (kindly provided by Charles J. Sherr) and hTERT coated dish, and the 01Ubic cells were seeded on a 60 cDNAs were inserted into pBabe vectors [35] containing mm dish in 5 ml growth medium. Cells were cultured neomycin- and hygromycin-resistance genes, respec- undisturbed for 48 hours, after which they were given tively. Additionally, loxP sites were placed internal to fresh growth medium daily for 2 to 4 days. When the long terminal repeats of the hTERT expression vec- approximately 50 to 70% confluent, cells were treated tor, to allow for excision of the entire expression cas- with trypsin (TrypLE Express/Gibco) for 5 minutes at sette by Cre recombinase. These vectors were 37°C, neutralized with an equal volume of growth med- transfected into the Phoenix ecotropic packaging cell ium, and counted with a hemocytometer. For expansion, line using the calcium phosphate technique [36], and cells were seeded at ~2000 to 3000 cells/cm in growth the virus-containing supernatant was used to infect the medium. For freezing, an equal volume of ice-cold 2 × amphotropic packaging cell line PA317 [37] to obtain freezing medium (20% dimethysulfoxide, 50% FBS, 30% stable virus-producing cell lines after selection with 0.5 growth medium) was added to cells and mixed well, mg/ml G418 or hygromycin (EMD Biosciences, San then cells were incubated in the liquid nitrogen vapor Diego, CA, USA). All infections were performed in the phase for at least 1 hour before storage at -140°C. presence of 2 μg/ml polybrene (Sigma-Aldrich). To standardize conditions for growth of primary and For infection of myoblasts, 5 × 10 cells were seeded immortalized cells, primary myoblasts were subsequently in six-well dishes 24 hours before infection. Selection cultured in a 4:1 mixture of Dulbecco modified Eagle was started 48 hours after infection using 300 mg/l medium (DMEM) and Medium 199 (Hyclone), supple- hygromycin and 400 mg/l G418 for 1 and 2 weeks, mented with 15% FBS (Hyclone), 0.02 M HEPES buffer, respectively. Depending on growth rates and infection 1.4 mg/l vitamin B12 (both Sigma-Aldrich, St. Louis, efficiency, cells were passaged during the selection per- MO, USA), 0.03 mg/l ZnSO (Fisher Scientific, Fair iod before becoming confluent. Lawn,NJ, USA),0.055 mg/l dexamethasone(Sigma- Aldrich), 2.5 μg/l hepatocyte growth factor (Chemicon Isolation of myogenic immortalized clones International, Temecula, CA, USA) and 10 μg/l basic Immortalized populations were seeded at low densities, fibroblast growth factor (BioPioneer), on dishes coated (50, 100, 200 or 400 cells per 100 mm dish). About 10 with 0.1% pigskin gelatin (Sigma-Aldrich) in a 2-5% oxy- days after seeding, clones were isolated using cloning gen environment [34]. Hepatocyte and fibroblast growth rings and seeded into 48-well dishes, followed by six factors were added to a volume of medium that was well and 100 mm dishes. The exact time point for clone used within 1 week. Cells were routinely grown in 100 isolation was chosen to maximize the cell number and mm tissue culture dishes (BD Falcon, Franklin Lakes, minimize cell density of individual clones, to facilitate Stadler et al. Skeletal Muscle 2011, 1:12 Page 9 of 10 http://www.skeletalmusclejournal.com/content/1/1/12 growth after passage but avoid fusion of myoblasts, were collected on microscope slides (Fisherbrand Super- respectively. We estimate that clones contained 100 to frost Plus; Fisher Scientific), air-dried, and fixed with 4% 1000 cells at the time of isolation. All clones were ana- formaldehyde in PBS for 15 minutes at room tempera- lyzed for CD56 expression by flow cytometry (see ture. Slides were washed three times with PBS and below) and fusion potential in differentiation medium. stained with hematoxylin and eosin. Sections were The percentage of cell nuclei in multinucleated and/or imaged under a microscope (AxioImager M2, Zeiss) and MF20-positive cells was determined for 10 random images captured with a camera (Digital microscopy fields after 3 or 7 days in differentiation medium. camera Axio Cam, ICC3, Zeiss). Terminal restriction fragment assay Acknowledgements Terminal restriction fragment assay was performed as We thank Kendal Hanger and Leigh Warsing for technical support. We also previously described [38]. thank the two subjects for their generous participation in this research. This work was performed within the framework of the Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center for FSHD, supported by Flow cytometry National Institutes of Health grant 5U54HD060848. GS is an Ellison/AFAR Cells were detached with trypsin, counted, and sedimen- Postdoctoral Fellow in Aging Research and was supported by an Erwin Schroedinger grant (project number J2737-B12) from the Austrian Science ted by centrifugation, then 1.5 × 10 cells were resus- Fund. pended in 0.2 ml DMEM with 10% calf serum (Cosmic Calf Serum; Hyclone) containing 5% supernatant from Author details Department of Cell Biology, UT Southwestern Medical Center at Dallas, the hybridoma line 5.1H11 [39], which recognizes CD56 Dallas, TX, USA. Boston Biomedical Research Institute, Watertown, MA, USA. (kindly provided by Helen Blau). As a control, primary 3 Center for Genetic Muscle Disorders, The Kennedy Krieger Institute, Johns antibody was omitted. Cells were incubated for 30 min at Hopkins School of Medicine, Baltimore, MD, USA. room temperature, washed twice with 10% calf serum Authors’ contributions (Cosmic Calf Serum; Hyclone) in PBS, and incubated KW, CPE and WEW designed this study. GS, JC, KW and JR performed the with secondary Alexa488-conjugated goat anti-mouse experiments and analyzed the results. GS, JC, KW, JWS and WEW wrote the manuscript. All authors read and approved the final manuscript. antibody (Invitrogen, Eugene, OR, USA) for 30 min at room temperature. After washing with PBS, cells were Competing interests resuspended in 0.2 ml PBS and analyzed on a flow cyt- The authors declare that they have no competing interests. ometer (FACSCalibur; Becton Dickinson). A second flow Received: 19 November 2010 Accepted: 8 March 2011 cytometer (FACSAria; Becton Dickinson) was used for Published: 8 March 2011 sorting, with 5 × 10 cells stained in upscaled volumes. 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