Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin Δ8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).
Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin Δ8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin Δ8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.
Skeletal muscle is a differentiated tissue with one of the most extended ability to regenerate after exercise or trauma1. This characteristic is mainly due to the presence of stem cells, called satellite cells because of their peripheral position between the basal lamina and the plasmalemma of the myofiber2. During post-natal development, satellite cells proliferate and progressively differentiate, thus contributing to skeletal muscle growth. Once in the adulthood, satellite cells enter a reversible quiescent state, and upon physiological or pathological trauma, they activate, proliferate and differentiate in order to repair the damaged muscles3. Defects in the capacity of satellite cells to properly transit through these different regenerative phases and to undergo self-renewal have been firmly linked to muscle wasting, either during physiological aging4,5,6 or in muscle degenerative diseases, such as muscular dystrophies7,8,9,10.
Two main culture approaches exist to study satellite cells ex-vivo: primary myogenic cultures from mononucleated cells, mechanically and chemically dissociated from whole muscle11,12; or culture of isolated myofibers13,14,15,16,17,18,19,20. In the first case, the process of satellite cells isolation involves the trituration of whole muscles extracted from the mouse, a chemical digestion, filtration and fluorescent activated cell sorting (FACS)21. This procedure, although effective in isolating satellite cells from a variety of models, entails several variables that expose satellite cells to stress and disrupts their physiological niche22,23. By contrast, myofiber isolation involves a gentler digestion of muscle tissue with matrix degrading enzymes and a mechanical shredding that causes reduced trauma to stem cells20. This second approach allows a much more efficient retrieval of viable satellite cells, that remain physically attached to their myofiber between the basal lamina and the sarcolemma, thus allowing analysis within their physiological niche19,20.
Many different protocols have been proposed during the past years to properly and efficiently isolate single myofibers from skeletal muscles. Already in 1986 Bischoff proposed a protocol to isolate fibers from the Flexor Digitorum Brevis13 and later, in 1995, Rosenblatt et al. modified the protocol to obtain a more efficient separation of myofibers14. Since then, many other authors proposed adjusted procedures on other muscles, such as Extensor Digitorum Longus (EDL) and Tibialis Anterior (TA)15,16,17,18,19,20, that are longer, even if more fragile, muscles14. Isolated myofibers can then be cultivated both in adhesion, to allow for the expansion of satellite cells-derived myoblasts, or in floating conditions, up to 96 hours, to follow the progeny derived from single satellite cells19 (Figure 1). Variable concentrations of serum within the culture medium are used to trigger satellite cells activation, proliferation and/or differentiation, to study their capacity to properly transit through these different phases1.
We recently described the epigenetic mechanism behind the exhaustion of the satellite stem cell pool in the mouse model of EDMD, the Lamin Δ8-11 -/- mouse7. Since these mice usually die between 4-8 weeks of age24, due to severe muscle loss, an attempt was made to capture the molecular defects underlying the early onset of the disease by focusing our analysis on post-natal muscle development. Floating single myofibers were isolated and cultured from wild type and Lamin Δ8-11 -/- mutant7 19 days-old mice. At this stage, muscle defects are already evident, but mice are still viable. However, since all the above-mentioned protocols for single myofibers extraction were optimized for skeletal muscles of adult mice, we needed to adapt them to our purposes: very small mice in term of age and size, and very fragile myofibers. Thus, we describe here our re-adaptation of the protocol proposed by the Rudnicki laboratory19 to obtain a significant number of single viable myofibers from mice during post-natal development and from severe dystrophic muscles, such as those derived from Lamin Δ8-11 -/- mice24. The final goal of this approach is to provide a standardized procedure for the study of myofibers-associated muscle stem cells in any other mouse model when the early stages of post-natal development are of interest, or in the case of mouse models carrying any specific disease that makes myofibers more susceptible to mechanical stress.
All the experimental procedures were performed under the ethical approval of the Italian Ministry of Health and the Institutional Animal Care and Use Committee (authorization n. 83/2019-PR). The animals were maintained in an authorized facility at San Raffaele Hospital, Milan, Italy (authorization n. N. 127/2012-A).
1. Muscle dissection and myofiber culture
2. Downstream applications: Myofibers crosslinking and immunofluorescence
NOTE: The myofibers-associated satellite cells can be visualized by immunofluorescence (IF) at the time of interest. Since most of the published protocols are optimized to perform IF on adult myofibers, here a detailed protocol is presented to obtain reliable results also on myofibers isolated from post-natal muscles.
We typically digest four different muscles (TA, EDL, Soleus and Gastrocnemius) to retrieve a good amount of long and viable fibers that could survive 96 h in growth-factors rich medium (Figure 4A,B). Only the most intact fibers should be transferred in culture medium, as they will survive; all the others, that are easy to discriminate and select, need to be discarded.
When myofibers are maintained in a growth-factors rich medium satellite cells derived from wild type mice start to activate and proliferate, see Figure 1. Upon 48 h of culture, in healthy condition (Lamin Δ8-11 +/+), satellite cells upregulate MyoD and undergo their first division. Activated Pax7+/MyoD+ satellite cells then proliferate and by 72 h in culture, they generate cell aggregates bound to the myofiber, that are even more visible at 96 h (Figure 5). During these divisions, some of them can repress MyoD expression, undergoing self-renewal to repopulate the stem cells pool, while those that maintain MyoD become committed to differentiation by downregulating Pax7 expression. After 96 h, satellite cells clusters contain clearly visible MyoG+ committed cells, that can differentiate into new myofibers (Figure 1 and Figure 6). Notably, with this experiment, we described a delayed dynamics of satellite cell differentiation in homozygous mutant Lamin Δ8-11 mice (-/-) as compared to their wild type counterparts (+/+), see Figure 6.
The final outcome of each single experiment let us think that the protocol developed for single myofibers isolation and culture from this model of severe muscle dystrophy ensures good quality myofibers for all further applications.
Figure 1: Graphical representation of satellite cells’ regenerative phases modelled in floating myofibers. Upon 48 h of culture in growth-factors rich medium, Pax7+ cells get activated and undergo the first division, giving rise to a doublet of Pax7+/MyoD+ cells. MyoD positive cells then proliferate and expand, giving rise, in 72 h of culture, to a cluster of several cells which are the progeny of a single satellite cell. Upon 96 h of culture Pax7+/MyoD+ cells become differentiating Pax7-/MyoG+ cells. During the expansion phase, a subset of Pax7+/MyoD+ cells downregulate MyoD expression undergoing self-renewal into quiescence. Please click here to view a larger version of this figure.
Figure 2: Preparation of bore Pasteur pipettes. (A) Longitudinal and frontal view of how the big hole bore pipette must appear. (B) Longitudinal and frontal view of how the small hole bore pipette must finally appear. Please click here to view a larger version of this figure.
Figure 3: Representative pictures of single muscle dissection. (A) Isolation of the TA muscle. Avoiding the removal of the thin layer covering the muscle protects the myofibers inside. (B) TA and EDL muscles isolated together still attached to their upper tendon at the level of the patella. (C) Division of TA and EDL after isolation by cutting them along the longitudinal axis. Please click here to view a larger version of this figure.
Figure 4: Examples of healthy and viable myofibers. Representative phase contrast images of viable myofibers in suspension. (A) The red arrow indicates a myofiber with visible sarcomere organization and a satellite cell on its side; the orange arrows indicate some pieces of broken myofibers, and some debris present in the first dish before the final selection for culture. Scale bar 100 µm. (B) More complete view of other myofibers under a smaller magnification. Scale bar 500 µm. Please click here to view a larger version of this figure.
Figure 5: Difference in the dimension of stem cell clusters in wt and mutant mice. Immunofluorescence staining of myofibers extracted from 19 days Lamin Δ8-11 mice (+/+ and -/-) after 96 h of culture. Pax7+ satellite cells are shown. The dimension of the cell cluster in most of the cases was significantly bigger in Lamin Δ8-11 +/+ than in Lamin Δ8-11 -/- in terms of number of cells. Scale bar 10 µm. Please click here to view a larger version of this figure.
Figure 6: Representative immunofluorescence experiment. Immunofluorescence experiment performed, after 96 h of culture, on myofibers extracted from 19 days Lamin Δ8-11 mice (+/+ and -/-). Pax7+/MyoG- (red) and Pax7-/MyoG+ (green) cells were observed. Images obtained with a confocal microscope. Scale bar 25 µm. Please click here to view a larger version of this figure.
Name of the solution in the text | component | percentage | suggested final volume/sample | notes | |
washing solution | DMEM high glucose | 90% | 4 mL | Keep sterile, keep at 4°C until usage | |
Horse serum (HS) | 10% | ||||
digestion solution | DMEM high glucose | 9.80% | 20 mL | Extremely harmful powder. Filter solution with 0.22µm filter and then keep sterile. Keep at 4°C until usage | |
Collagenase I | 0.20% | ||||
culture medium | DMEM high glucose | 78% | 10 mL | Keep sterile, keep at 4°C until usage | |
Fetal bovine serum (FBS) | 20% | ||||
Chicken embryo extract (CEE) | 1% | ||||
Penicillin-Streptomycin (P/S) | 1% |
Table 1: Recipes for solutions used in section 1.
Name of the solution in the text | component | percentage | suggested final volume/sample | notes | |
4% PFA | Paraformaldehyde (PFA) | 4% | 2-3 mL | Powder and then solution are extremely harmful | |
PBS | 96% | ||||
0.5% Triton X-100 | Triton X-100 | 0.50% | 2-3 mL | Triton X-100 is extremely viscous, preferentially cut the tip of the pipette to aliquot it | |
PBS | 99.50% | ||||
0.25% Tween-20 | Tween-20 | 0.25% | 10 mL | Tween-20 is extremely viscous, preferentially cut the tip of the pipette to aliquot it | |
PBS | 99.75% | ||||
0.1% Tween-20 | Tween-20 | 0.10% | 10 mL | Tween-20 is extremely viscous, preferentially cut the tip of the pipette to aliquot it | |
PBS | 99.90% | ||||
blocking solution | Fetal bovine serum (FBS) | 10% | 10 mL | Prepare fresh solution and store at 4°C for no longer than 3 weeks (always check clearness before usage) | |
PBS | 90% | ||||
DAPI | DAPI | 0.10% | 2-3 mL | Keep in the dark | |
PBS | 99.90% |
Table 2: Recipes for solutions used in section 2.
Isolation of intact single myofibers is an essential method in the field of myogenesis when the main objective is to characterize cell-autonomous regenerative capacities of muscle stem cells within their niche, in healthy and pathological conditions. However, when biochemical or genomic studies are of interest, FACS-isolated satellite cells might be the best option.
Single myofibers isolation allows to follow ex-vivo, but in the most physiological way, the dynamics of all the steps single satellite cells undergo during muscle regeneration, that are: activation, cell division (asymmetric and symmetric), differentiation and return to quiescence by self-renewal. Once myofibers are grown in floating conditions, the single satellite cells activate and expand forming a cluster of cells, all deriving from the same satellite cell. Immunofluorescence analysis for proliferation, differentiation, activation or stemness markers is then optimal to quantify the proportion between cell stages.
The key step in our protocol to obtain viable and intact myofibers can be considered the rapid but gentle muscle dissection, by tendon-to-tendon isolation, to avoid any muscle damage. Our advice is to use only sharp scissors and small sharp tweezers and to limit the entire muscle dissection procedure to ten minutes. When it is difficult to isolate very small muscles (i.e., EDL and TA), it is possible to cut them together and to later divide them by using fine scissors cutting along the longitudinal plan following the fibers. This strategy will eventually give less intact myofibers, but viability will not be compromised. The same must be performed on big muscles like Gastrocnemius to facilitate digestion. Optimization of digestion time, which needs to be empirically validated, and minimal manipulation of isolated fibers are also two crucial aspects for the positive outcome of subsequent analysis.
The advantage of the protocol reported here is that it can be applied on very small mice (in age and dimension), even when their muscles are extremely fragile. Even if not mentioned above, it is possible to follow this protocol of dissection to then culture viable myofibers for longer period using basement membrane-coated dishes18,19. It is important to consider that this situation is completely different from floating condition, where adhesion stimuli and proximity stimuli are absent.
The authors have nothing to disclose.
We thank Andrea Bianchi, the Italian Network of Laminopathies and the members of the laboratory for the support and all the constructive comments. We are grateful to Chiara Cordiglieri for the precious help at confocal microscope. The authors thank Dr. Beatrice Biferali for her help in taking pictures for figures. The work presented in here was supported by My First AIRC Grant n. 18535, AFM-Telethon n. 21030, the Italian Minister of Health n. GR-2013-02355413 and Cariplo 2017-0649 to C.L. C.M. is supported by My First AIRC grant n.18993 and AFM-Telethon n. 22489.
4′,6-diamidino-2-phenylindole | Sigma | D9542 | |
Chicken embryo extract | Seralab | CE650-DL | |
Collagenase type I | SIGMA | C0130-500MG | |
Donkey anti-Rabbit 488 antibody | Thermofisher | R37118 | to be used 1:200 |
Dulbecco's modified Eagle's medium | Gibco | 10569-010 | |
Fetal bovine serum | Corning | 35-015-CV | |
Horse serum | Gibco | 26050-088 | |
MyoG antibody | Millipore | 219998 | to be used 1:100 |
Paraformaldehyde | SIGMA | P6148 | |
Pax7 antibody | Developmental studies Hybridoma bank |
to be used 1:20 | |
Penicillin and Streptomycin | Euroclone | ECB3001D | |
Phosphate saline buffer | Euroclone | ECB4004L | |
Prolong gold antifade mountant | Thermofisher | P36930 | |
Triton X-100 | SIGMA | T8787 | |
Tween-20 | SIGMA | P1379 | |
Lab equipment | Manufacturer | ||
Bunsen burner | |||
Confocal microscope | Leica | ||
Diamond pen | bio-optica | ||
Dissection pins | |||
FACS polypropylene tubes | Falcon | ||
Falcon tubes (50 and 15 mL) | Falcon | ||
Glass coverslips | Thermofisher | ||
Glass Pasteur pipettes (22cm) | VWR | ||
Glass slides | Thermofisher | ||
Micro dissecting scissors | |||
Micropipette (1 mL and 200 µL) | Gilson | ||
Micropipette tips | Corning | ||
Petri dishes (100 and 35 mm diameter) | Thermofisher | ||
Plastic pipettes (5 and 10 mL) | VWR | ||
Rubber pipette bulbs | VWR | ||
Sharp tweezers | |||
Stereo dissection microscope with transmission illumination | Leica | ||
Tissue culture hood or lamina flow cabinet | |||
Tissue culture incubator (humidified, 37°C, 5% CO2) | |||
Water bath at 37°C | VWR |