Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.
Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily conserved structures that compose it are assembled remains under investigation. Their size and physiological features often constrain manipulation and imaging applications. The culture of immortalized cell lines is widely used, but it can only replicate the early steps of differentiation.
Here, we describe a protocol that enables easy genetic manipulation of myofibers originating from primary mouse myoblasts. After one week of differentiation, the myofibers display contractility, aligned sarcomeres and triads, as well as peripheral nuclei. The entire differentiation process can be followed by live imaging or immunofluorescence. This system combines the advantages of the existing ex vivo and in vitro protocols. The possibility of easy and efficient transfection as well as the ease of access to all differentiation stages broadens the potential applications. Myofibers can subsequently be used not only to address relevant developmental and cell biology questions, but also to reproduce muscle disease phenotypes for clinical applications.
Skeletal muscle composes up to 40% of the human body weight1. Muscle-associated disorders represent an immense health and economic burden2. How this highly complex and organized tissue is formed, maintained, and regenerated constitutes an extensive and well-established research field. Depending on the specific scientific interest, the most suited approach can range from simple myotube cultures to complex in vivo models3-6.
The goal of this protocol is to provide an in vitro system that allows for the monitoring of myogenesis through live imaging and immunofluorescence. Compared to traditional approaches, this system offers a very complete and dynamic insight into the mouse myogenic process. Cells can be followed from the myoblast stage to the mature, multinucleated myofiber displaying transversal triads and peripheral nuclei7. This maturation level can be achieved using regular cell culture equipment, without the need for complex stimulatory or mechanical apparatuses. Although some successful in vitro systems have been reported8,9, to our knowledge, this is the only protocol generating mature mouse myofibers with T-tubules transversally paired with Sarcoplasmic Reticulum (SR). Thus, this in vitro system can be used to study the molecular mechanisms of triad formation, which are still poorly understood10.
A further advantage of using this system is the availability of validated mouse-targeted resources, such as antibodies, drugs, and RNAi tools. The relatively simple protocol does not require laborious steps, highly skilled manipulation, or expensive and dedicated equipment. Matured myofibers start appearing after 5 d of culture differentiation7, displaying contractility coupled with calcium sparks (unpublished data). In one week, the different developmental stages of one of the most complex cells in the mammalian body can be studied in combination with a variety of in vitro assays.
NOTE: One mouse yields sufficient myoblasts for approximately two 35 mm dishes or two live-imaging dishes, so plan mattings, dissection, and coating (step 2.6) accordingly. Since myoblasts are isolated through sequential centrifugations and preplating, the protocol should be done in batches of 5 - 10 animals.
All procedures involving animal subjects were approved by the Animal Ethics Committee at Instituto de Medicina Molecular and University Pierre et Marie CurieÂ
1. Dissection of Neonatal Mice Hind-limb Muscles
2. Myoblast Isolation
3. Myofiber Differentiation
NOTE: After 3 d, the cells should start to fuse and form myotubes at around 70% confluency (Figure 1B).
4. Immunostaining in Glass-bottom Dishes
The extent of myofiber development is mostly determined by the purity and viability of the isolated myoblasts. The adhesion, proliferation, and fusion capacity can be used to empirically access those parameters (Figure 1 A, B). At proliferation D2, myoblasts should have adhered and should display the typical fusiform shape. Proliferation is expected to happen extensively at this stage, leading to spontaneous myotube formation the following day (Figure 1B).
Cell confluency might need slight adjustments. It should be increased if myoblasts take more than 3 d to proliferate and fuse. It should be decreased if myofibers are not allowed to grow and elongate relatively straight due to their density. Confluency typically decreases from the center to the periphery of the dish, so the best myofibers should be found towards the outer regions.
Myotubes will quickly elongate and display multiple centrally aligned nuclei (Figure 1C). By D5, some cells start acquiring striations and moving their nuclei to the periphery. The number of myofibers with mature characteristics will increase with time as well as with cell thickness (Figure 1D).
The degree of differentiation can be further observed by immunofluorescence. Myofibers fixed at differentiation D8 present transversal triads. This can be confirmed by imaging components of the T-tubules (DPHR) and the SR (triadin), which are expected to colocalize at the triads (Figure 2).
The functionality of myofibers can be addressed by live imaging. From differentiation D3 onwards, the cells display spontaneous twitching. By transfecting a calcium sensor (e.g., GCaMP6f11), it is possible to observe that the contractions are coupled with calcium peaks (Figure 3).
Using this system, we were able to identify a novel molecular pathway that is disrupted in centronuclear myopathies and myotonic dystrophies, which can therefore be a novel target for innovative molecular therapies7. We have also adapted this method to study the development of the neuromuscular junction (NMJ)12. Through the coculture with rat spinal cord explants, we have described a role for dynein in NMJ formation13.
Figure 1: Developmental Stages of the Myoblast Culture. A) At proliferation D2, myoblasts have adhered and started proliferating. B) At proliferation D3, a confluency of 60 - 80% is reached, and myoblasts start fusing spontaneously. C) At differentiation D3, myotubes containing centrally located nuclei are predominant. D) From differentiation D5 onwards (e.g., day 8), myofibers start exhibiting striations and peripheral nuclei and begin to thicken. Scale bar: 50 µm. Please click here to view a larger version of this figure.
Figure 2: Representative Confocal Image of a D8 Myofiber Immunostain. A) Immunostaining for dihydropyridine receptor (DHPR, top panel) and triadin (TRDN, middle panel). An overlay of the DHPR, TRDN, and DAPI channels shows colocalization of the triad components. B) An intensity profile of the yellow line drawn in A. C) A 3D image of volume rendering of myofibers stained for α-actinin (green) and DAPI (blue). Scale bar and grid width: 5 µm. Please click here to view a larger version of this figure.
Figure 3: Live Imaging of Calcium Levels in Myofibers with Spontaneous Twitching. A) High-speed time-lapse (20 ms frames) microscopy of a calcium spark in a twitching myofiber. Calcium was detected through the expression of GCaMP6f (Addgene plasmid #40755). B) Quantification of the fluorescence intensity over time for the calcium sensor in panel A. Please click here to view a larger version of this figure.
The use of this protocol for the cultivation of primary myoblasts gives rise to a special niche that greatly nurtures the development of myofibers. This is partially due to other cell types that are also present in very small numbers. A balance between myoblast concentration and culture purity must be achieved. A good cell culture also depends on the quality of the products used for the medium formulation. All products derived from animal sources should be thoroughly tested. In our experience, the digestion conditions should also be monitored.
As usual for primary cultures, experimental variability can be higher than in studies with isolated fibers or immortalized myoblasts. This variability can be diminished by the standardization of medium and digestion components, mice age and size, and the time points for culture manipulation and results collection. Nevertheless, the advantage of scrutinizing in real time the intricate mechanisms necessary for myofiber development greatly surpasses the variability drawback.
This protocol confers the advantages of in vitro approaches without compromising cell differentiation. Myofibers mature until triads are formed and contractions are coupled to calcium sparks. These functional outputs can be accessed in different experimental conditions. Furthermore, there can be many technical variations made to the protocol. Myoblasts can be harvested from neonatal mice with mutations of interest relating to muscle development. Cells can be lysed for biochemical analysis at different differentiation time points. Calcium indicators can be added to the culture to follow its dynamics. Optogenetic constructs can be used to enforce certain signaling pathways or to induce specific local responses. Finally, the myofibers can be cocultured with other cells types to study their interactions.
This work was supported by the European Research Council (ERG) and EMBO installation (ERG) and by a PhD fellowship from the Fundação para a Ciência e Tecnologia (MP).
Name | Company | Catalog Number | Comments |
Dispase II | Gibco | 17105041 | |
Collagenase type V | Sigma-Aldrich-Aldrich | C9263 | |
IMDM, Glutamax supplemented | Gibco | 31980022 | |
Matrigel Growth reduced factor | Corning | 354230 | protein concentration of the lot should be around 10mg/ml and endotoxin result should be <1.5 |
Chicken Embryo Extract | MP biomedical | 2850145 | it is also possible to prepare in the lab (Danoviz ME, Yablonka-Reuveni Z. Methods Mol Biol (2012)) |
Recombinant rat agrin | R&D systems | 550-AG-100 | |
Penicillin-Streptomycin | Thermo Fisher Scientific | 15140122 | |
Horse Serum | GE Healthcare | 11581831 | |
Lipofectamine RNAiMAX | Invitrogen | 13778-150 | used to transfect siRNA |
Lipofectamine LTX | Invitrogen | 15338-100 | used to transfect DNA |
Lipofectamine 2000 | Invitrogen | 11668030 | used to transfect siRNA plus DNA |
DPBS | Gibco | 14190094Â | |
Fetal Bovine Serum | Eurobio | CVFSVF0001 | |
Cell strainer | Corning | 21008-949 | |
Fluorodishes | World precision instruments | FD35-100 | dishes used to cultivate cells for live imaging |
Triton X-100 | Sigma-Aldrich | T9284 | |
16% PFA (Paraformaldehyde) | Science Services GmbH | E15710 | |
Goat Serum | Sigma-Aldrich | G9023 | |
BSA (Bovine Serum Albumine) | Sigma-Aldrich | A7906 | |
Saponine | Sigma-Aldrich | 47036 | |
DAPI | Sigma-Aldrich | D9542 | use at 200ng/ul |
Fluoromount-G | SouthernBiotech | 0100-01Â | |
Name | Company | Catalog Number | Comments |
Solutions and Media | |||
Digestion Mix | in DPBS 5 mg/ml collagenase 3.5 mg/ml dispase sterile filtered, can be stored in working aliquotes for 2 weeks at -20 °C | ||
Dissection Medium | in IMDM Glutamax supplemented 10% FBS 1% Penicillin-Streptomycin sterile filtered | ||
Growth Medium | in IMDM Glutamax supplemented 20% FBS 1% Chicken Embryo Extract 1% Penicillin-Streptomycin1% Penicillin-Streptomycin sterile filtered | ||
Differentiation Medium | in IMDM Glutamax supplemented 2% Horse Serum 1% Penicillin-Streptomycin sterile filtered | ||
Blocking Solution | in DPBS 10% Goat Serum 5% BSA add 0.1% saponine when incubating with primary and secondary antibodies |
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