Establishment of clonal myogenic cell lines from severely affected dystrophic muscles - CDK4 maintains the myogenic population - PubMed (original) (raw)

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

Guido Stadler et al. Skelet Muscle. 2011.

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.

PubMed Disclaimer

Figures

Figure 1

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.

Figure 2

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.

Figure 3

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).

Figure 4

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).

Similar articles

Cited by

References

    1. van Deutekom JC, Wijmenga C, van Tienhoven EA, Gruter AM, Hewitt JE, Padberg GW, van Ommen GJ, Hofker MH, Frants RR. FSHD associated DNA rearrangements are due to deletions of integral copies of a 3.2 kb tandemly repeated unit. Hum Mol Genet. 1993;2:2037–2042. doi: 10.1093/hmg/2.12.2037. - DOI - PubMed
    1. Winokur ST, Barrett K, Martin JH, Forrester JR, Simon M, Tawil R, Chung SA, Masny PS, Figlewicz DA. Facioscapulohumeral muscular dystrophy (FSHD) myoblasts demonstrate increased susceptibility to oxidative stress. Neuromuscul Disord. 2003;13:322–333. doi: 10.1016/S0960-8966(02)00284-5. - DOI - PubMed
    1. Gabellini D, Green MR, Tupler R. Inappropriate gene activation in FSHD: a repressor complex binds a chromosomal repeat deleted in dystrophic muscle. Cell. 2002;110:339–348. doi: 10.1016/S0092-8674(02)00826-7. - DOI - PubMed
    1. Lemmers RJ, van der Vliet PJ, Klooster R, Sacconi S, Camano P, Dauwerse JG, Snider L, Straasheijm KR, van Ommen GJ, Padberg GW, Miller DG, Tapscott SJ, Tawil R, Frants RR, van der Maarel SM. A unifying genetic model for facioscapulohumeral muscular dystrophy. Science. 2010;329:1650–1653. doi: 10.1126/science.1189044. - DOI - PMC - PubMed
    1. Dixit M, Ansseau E, Tassin A, Winokur S, Shi R, Qian H, Sauvage S, Matteotti C, van Acker AM, Leo O, Figlewicz D, Barro M, Laoudj-Chenivesse D, Belayew A, Coppee F, Chen YW. DUX4, a candidate gene of facioscapulohumeral muscular dystrophy, encodes a transcriptional activator of PITX1. Proc Natl Acad Sci USA. 2007;104:18157–18162. doi: 10.1073/pnas.0708659104. - DOI - PMC - PubMed

LinkOut - more resources