Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs and their progeny — myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.


Introduction
Disease-and age-related progressive loss of skeletal muscle mass and function results in frailty, decline in strength and decrease in quality of life of aged people. Skeletal muscle accounts for approximately 40% body mass 1 . During ageing and disease, progressive atrophy of individual myofibers and reduction of muscle quality due to the infiltration of fat and fibrosis occurs 1,2,3,4,5,6 . Recently, it has been proposed that species-specific differences in ageing of skeletal muscle occur, specifically that muscle fiber loss occurring in rodents, may not occur in humans 7 . Nevertheless, the remaining muscle fibers of aged mammals are characterized by increased susceptibility to damage and impaired regeneration 8 . Adult muscle repair and maintenance is mediated by satellite cells 9,10 . Upon muscle injury and other relevant cues, satellite cells become activated and proliferate. A subset of the cells returns to the quiescent state and the remainder progresses into myoblasts (Myogenic Progenitor Cells -MPCs). These contribute to repair of the existing myofiber 11 . The functionality of satellite cells determines the success of muscle regeneration and the changes in satellite cell availability with ageing have been demonstrated 12,13,14,15 . Moreover, satellite cells from the muscle of old humans and rodents show a transcriptional profile switch and reduced regenerative potential 16,17,18,19 . Satellite cells of muscle from old mice and humans have also been shown to undergo senescence resulting in their reduced functionality 20 .
The most established cell line enabling the study of muscle homeostasis is murine C2C12 cell line 21 . A significant amount of studies have also used murine primary myoblasts 22 . These cultures have led to a significant understanding of murine and vertebrate myogenesis as well as muscle regeneration, myotube/myofiber atrophy, and hypertrophy processes occurring during muscle disease and ageing 23,24,25,26 . More recently, several groups have described using human primary myoblasts to study myogenesis and muscle ageing. However, there is lack of consensus with regards to differences between primary myoblasts isolated from the muscle of adult and aged humans 27,28,29,30,31 . Despite differences characterized in the systemic and local environment occurring during development, ageing and disease 4. Culture and Passaging of Cells 1. Change the media every 2 -3 d and split the cells as soon as groups of cells are visible under the microscope (100X total magnification, example in Figure 2, cells from aged people, passage 0, after 7 d) in order to avoid spontaneous differentiation. 2. At the first passage (P1), change the media to high-glucose DMEM complemented with 20% FBS, 10% HS, 1% P/S, 1% α-glutamine. Avoid using FGF-b from this point, as FGF is a potent mitogen and important factor at the beginning of the culture, but it can promote fibroblast overgrowth if used longer in culture. 3. For cell passaging: 1. Remove the media and wash the cells twice with DPBS. 2. Add a minimum volume of 0.25% EDTA-trypsin to cover the surface of the cells. Rock gently to ensure all the cells are covered by the detachment solution, incubate for 10 seconds at room temperature, and remove it. 3. Incubate the cells in a humidified 37 °C, 5% CO 2 incubator for 3 -5 min. Tap gently and check under the bright light microscope (100X total magnification) that the cells are rounded but not completely detached from the surface. If no change is observed in the cells, incubate for 5 more min.

Transfections Protocol
1. Seed 50,000 cells/well in a 12-well plate for MF 20, senescence and viability assays. Culture cells on cover slips or in a dish coated with laminin. For Ki67 staining, seed 25,000 cells/well in a 12-well plate. 2. For transfections, follow the manufacturer's procedure using 5 µL of transfection reagent, 100 nM of control microRNA mimic or inhibitor, 100 nM of microRNA mimic or 100 nM of microRNA inhibitor per well, with a total volume of 1 mL. 3. Change to Differentiation Medium 6 h after transfection (high-glucose DMEM complemented with 2% HS, 1% P/S, 1% α-glutamine). For proliferation experiments, stain the cells 2 d after transfection; for senescence, 7 d after transfection; and for MF20 staining, seven days after transfection.

Immunostaining of the Cells
The representative results shown here and published data from our laboratory 22 aim to support our isolation and culture protocol and demonstrate the different techniques that can be used for functional studies of human primary myoblasts. Myoblast proliferation can be studied using Ki67 immunostaining and viability using staining for cell viability assay (Figure 3). For differentiation, the culture media should be changed to differentiation media. Myotubes should form in 5 -7 d and be myosin heavy chain positive (Figure 3). Note that the myotube formation may be less efficient when myoblasts are isolated from the muscle of aged people (Figure 3). Senescence (SA-β-galactosidase) staining can also be performed in order to establish the percentage of senescent cells in the culture (Figure 3). We observed that with longer cultures (Figure 3, passage 4), more myoblasts isolated from the muscle of aged people show senescence.
For functional studies, gene and microRNA expression can be manipulated using lipophilic transfection reagents-mediated delivery of expression vectors, siRNAs, microRNA mimics and antimiRs (Figure 4). This allows for 40 -70% transfection efficiency with gene/microRNA levels being up-or down-regulated within a physiological range (Figure 4; 22 ).

Discussion
Here, we present a simple, robust, inexpensive, reproducible and efficient method of isolating muscle progenitor cells/primary myoblasts from adult and aged humans from extensor digitorium brevis, tibialis anterior or abductor halluces muscles. This protocol aims to allow studies using human primary myoblasts from adult and aged humans, especially when more sophisticated methods, such as FACS-or MACS-sorting, are not possible or not practical.
The isolation method presented in this manuscript takes approximately 2 hours. During muscle isolation, muscle was washed in 70% ethanol in order to avoid contamination. Prior to enzymatic dissociation of the muscle, it is important to cut the muscle into small but visible pieces, and avoid cell damage from too much mincing. The digestion results in the dissociation of myofibers and the release of satellite cells and myogenic precursor cells. In our case for ~20 mg of skeletal muscle, one 60 mm (20 cm 2 ) Petri dish was the most appropriate surface area for harvesting the cells. Cells plated onto a larger surface showed reduced proliferation, whereas cells plated onto a smaller surface showed increased cell death and agglutination.
Upon isolation, the cells were cultured and expanded on laminin-covered plates. The use of non-coated surfaces tended to decrease the success of the isolation. For this reason, cells can be preferably harvested on a pre-coated surface directly after isolation. Fibroblasts-enriched cultures will predominate rather than myoblasts-derived cells if the cells are harvested on a non-coated surface directly after isolation. Apart from laminin, the use of other cell attachment solutions such as Matrigel and collagen-based reagents can be used. Coating solutions may include growth factors and other compounds that will promote cell growth, but these could alter the cell behavior and therefore the experimental results. In our experience, 10 µg/mL laminin is the optimum concentration and appropriate coating reagent for satellite cells and myoblast attachment and proliferation as it lacks any growth factor or other complements. Moreover, laminin is naturally present in the basal lamina, directly linked to the sarcolemma, which plays a key function in satellite cell attachment and migration through the skeletal muscle fiber.
The supplements of the culture media may also have a detrimental influence on the behavior of the primary myoblast. For example growth factor groups, such as FGFs or IGFs, have pleiotropic effects on primary myoblast cultures, with FGF-2 controlling both mitogenic and programmed cell death response 31 . It is therefore necessary to rigorously control the culture conditions, especially because the differences in the behavior of primary myoblasts isolated from muscle of adult and aged people are very likely to be due to the purity of the cultures and the likelihood of fibroblasts overrunning the myoblasts in culture during long-term cultures 35 . We have used 1-hour pre-plating of the cells during the first splitting onto a non-coated surface in order to decrease the contamination of the cultures with fibroblasts.
The method we describe is appropriate for isolating myogenic progenitor cells from the muscles of both adult and aged humans. The isolated cell consists of a representative myogenic population of cells as indicated by a high percentage of myogenic cells (MyoD expression and myogenic properties visualized by MF20 immunostaining in Figures 1 and 2) and can be used as an in vitro model for functional studies of processes associated with muscle homeostasis.
Previous studies have characterized the isolation and differences in properties, or the lack thereof, of human primary myoblasts from adult and aged people 6,20,27,28,29,30,31,35,36,37,38 . The existence of geriatric and/or non-functional human MPCs has been demonstrated 6,20,22 . However, no difference in the behavior of freshly isolated human MPCs has also been shown 27 . Our protocol allows for the isolation of primary myoblasts that at least partially retain their phenotype, such as reduced proliferative potential or senescence of primary myoblasts isolated from muscle of aged people and permits the use of these cells for functional studies of molecular mechanisms of muscle homeostasis during ageing 22 . The primary myoblasts isolated using the method described here can be used not only for myogenic differentiation studies but also to investigate intracellular changes, such as changes in gene expression occurring in human myogenic precursor cells during ageing. However, changes that occur in cells during prolonged ex vivo culture need to be considered when analyzing phenotypic and genotypic changes occurring during ageing. We recommend using freshly isolated cells for this purpose.
Moreover, the primary myoblast culture method described here allows for expansion and relatively long-term culture of human primary myoblasts, allowing for robust in vitro functional studies. We have previously shown that myogenic progenitor cells isolated using our method can be used for both expression profiling and functional studies of processes associated with muscle ageing 22 . This method is also applicable to the muscles of adult and old rodents and allows for isolation of an enriched culture of myoblasts that can be used for profiling genetic and epigenetic changes during ageing and functional studies 22 . The limitations of this method include the use of, to some degree, mixed population of cells rather than a pure population of satellite cells, which can be obtained using more sophisticated published methods 6,28,29,39,40,41,42,43 .
We present a simplified, affordable, and reproducible protocol for the isolation of primary myoblasts cells from adult and aged humans. In our experience, the available, more sophisticated methods of isolation and culture of human primary myoblasts (such as MACS-or FACS-sorted satellite cells) are ideal for some types of studies, such as profiling transcriptomic or proteomic changes in the cells. However, these methods are expensive, require at least some level of expertise, and may prove difficult due to the low proliferative rate of pure primary myoblast cultures and fibroblasts overgrowing myoblasts.
We present a reproducible protocol that permits the simple isolation and culture of human primary myoblasts for use in functional studies. Additionally, we propose the use of laminin 42 and the limited use of bFGF as key factors for a successful culture 44 . We also propose avoiding the stress generated by centrifugation when splitting the cells and one pre-plating step at the first passage 45 . To summarize, we have optimized an efficient protocol for isolation and culture of primary myoblasts/MPCs from the muscles of adult and aged humans that is also applicable to the muscles of rodents and enables expression and functional studies of muscle homeostasis.

Disclosures
There is nothing to disclose.