Analyses of the differentiation potential of satellite cells from myoD-/-, mdx, and PMP22 C22 mice

Abstract

Background: Sporadic and sometimes contradictory studies have indicated changes in satellite cell behaviour associated with the progressive nature of human Duchenne muscular dystrophy (DMD). Satellite cell proliferation and number are reportedly altered in DMD and the mdx mouse model. We recently found that satellite cells in MSVski transgenic mice, a muscle hypertrophy model showing progressive muscle degeneration, display a severe ageing-related differentiation defect in vitro. We tested the hypothesis that similar changes contribute to the gradual loss of muscle function with age in mdx and PMP22 mice, a model of human motor and sensory neuropathy type 1A (HMSN1A).

Methods: Single extensor digitorum longus muscle fibres were cultured from mdx and PMP22 mice and age- and genetic background-matched controls. Mice at several ages were compared with regard to the differentiation of satellite cells, assayed as the proportion of desmin-expressing cells that accumulated sarcomeric myosin heavy chain.

Results: Satellite cells of 2 month, 6 month, and 12 month old mdx mice were capable of differentiating to a similar extent to age-matched wild type control animals in an in vitro proliferation/differentiation model. Strikingly, differentiation efficiency in individual 6 month and 12 month old mdx animals varies to a much higher extent than in age-matched controls, younger mdx animals, or PMP22 mice. In contrast, differentiation of myoblasts from all myoD null mice assayed was severely impaired in this assay system. The defect in satellite cell differentiation that occurs in some mdx animals arises from a delay in differentiation that is not overcome by IGF-1 treatment at any phase of cultivation.

Conclusion: Overall, a defect in satellite cell differentiation above that arising through normal ageing does not occur in mdx or PMP22 mouse models of human disease. Nonetheless, the impaired differentiation of satellite cells from some mdx animals suggests that additional factors, environmental or epigenetic, may lead to deteriorating muscle repair through poor differentiation of satellite cells in genetically predisposed individuals.

Figure 1

A method to quantitate satellite cell differentiation. Single fibres from EDL muscles of 2 month old mice were isolated and cultured for five days, switched to differentiation medium for two days and the fraction of desmin-positive (desmin⁺) cells expressing myosin heavy chain determined by immunofluorescence microscopy. A: Satellite cell cultures from myoD heterozygous (myoD^+/-) and homozygous null (myoD^-/-) animals. Essentially all cells (blue nuclei: DAPI) express desmin (red: Cy3) in both genotypes, but note the lack of multinucleate myotubes and cells expressing MyHC (green: FITC) in the myoD^-/-. B-D: Quantification of satellite cell-derived myoblast growth and differentiation by genotype. Error bar = SD. Numbers above columns indicate number of wells counted (1 fibre/well) from 4 animals of myoD^+/+, myoD^+/-, and myoD^-/- genotype, respectively. B: Differentiation of myoD^-/- myoblasts is poor compared to littermates. C: Increase in cell number after the switch from plating to proliferation medium is independent from the genotype of the animals. myoD^+/+, myoD^+/- and myoD^-/- cells proliferate to the same extent. D: The fraction of differentiated myogenic cells is unaffected by either the number of myogenic desmin⁺ cells or the number of non-myogenic desmin^- cells in each well. In all genotypes (myoD^+/+, myoD^+/-, and myoD^-/-) all fibres yielded myogenic cells.

Figure 2

Differentiation of satellite cells of the HMSN1A mouse model PMP22 C22 at three ages. A. Variation in soleus muscle cryosections reacted for slow MyHC reveals variable pathology of individual PMP22 C22 mice. Note fibre atrophy (arrowheads) and large, hypertrophied fibres in the middle compared to the right panel. B. Satellite cells from single fibre culture of PMP22 C22 and littermate controls were analysed for cell yield, proliferation and terminal differentiation. No significant differences were observed.

Figure 3

Differentiation of satellite cells of control and mdx mice of different ages in vitro. A. Box and whisker plot: The box extends from the 25th percentile to the 75th percentile between all animals of one age, with a horizontal line at the median (50th percentile). Whiskers show the range of the data. B. Mean differentiation for each individual animal shows heterogeneity between mdx animals. Individuals showing poor differentiation (8 in total) are significantly more common in mdx (X², P = 0.0076). C. Satellite cell cultures from single fibres of 12 month old control C57BL/10ScSn and mdx animals showing good differentiation in each case. Blue nuclei: DAPI; desmin: red, Cy3; MyHC: green, FITC in both genotypes.

Figure 4

Prolongation of differentiation rescues the satellite cell differentiation defect. Satellite cells of 6 months old mdx or control animals were plated and allowed to proliferate as described in Materials and Methods and then cultivated either for 2 days (2d diff) or for 5 days (5d diff) in differentiation medium. Prolongation of the differentiation period to 5 days enhances differentiation efficiency (* P < 0.0028) of affected mdx satellite cells to the level observed in controls.

Figure 5

IGF-1 treatment fails to rescue differentiation of satellite cells in culture. Satellite cells from control or mdx animals at the age of 6 months were treated with IGF-1 (100 ng/ml medium) during either proliferation (P+IGF), differentiation (D+IGF), both (P+IGF D+IGF), or neither (P+D+, control). A. Differentiation was unaltered by IGF-1 addition. B. Non-myogenic cells are more abundant in mdx than control single fibre cultures.