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Skeletal muscle cells derived from mouse skin cultures
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Skeletal muscle cells derived from mouse skin cultures Hideki Ohnota a, b, *, 1, Hiromi Nakazawa a, 1, Morimichi Hayashi c, Yuji Okuhara c, Takayuki Honda a, Alessandra d’Azzo d, Yoshiki Sekijima e a
Department of Drug Discovery Science, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, Japan Research Division, R&D, Kissei Pharmaceutical Co. Ltd., 4365-1 Kashiwabara, Hotaka, Azumino, Nagano, 399-8304, Japan c Safety Research Laboratory, R&D, Kissei Pharmaceutical Co. Ltd., 2320-1 Maki, Hotaka, Azumino, Nagano, 399-8304, Japan d Department of Genetics, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA e Department of Medicine (Neurology and Rheumatology), Shinshu University School of Medicine, Matsumoto, 390-8621, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 November 2019 Accepted 15 December 2019 Available online xxx
We have established a novel, simple, and highly reproducible method to generate skeletal muscle cells from mouse skin. Small pieces of skin from the back of mice were cultured in extracellular materialcoated dishes in typical culture medium for about 3 weeks. Myotubes formed after about a week, grew into twitching myotubes, and became twitching myotube clumps after 3 weeks. Skeletal muscle cells are formed spontaneously with no induction. Myotubes were immunologically positive for myosin heavy chains, MyoD, and myogenin. Ultrastructural analysis revealed the presence of the sarcomere structure. Furthermore, PAX7þ/MyoD- muscle stem cells proliferated around these myotubes, and MyoDþ/myogeninþ/MHC- cells were also observed. Moreover, we investigated the formation of skeletal muscle cells from the sialidosis mouse skin, and showed that it is decreased compared to that of the wild type. Our method to generate skeletal muscle cells from skin is thought to be useful for the investigation of muscle cell development and muscle-related disorders. © 2020 Elsevier Inc. All rights reserved.
Keywords: Panniculus carnosus PAX7 Myotube Twitching Sialidosis Skin
1. Introduction Skin-derived precursors (SKPs), which differentiate into neurons, glia, smooth muscle cells, and adipocytes [1], are reported to develop into skeletal muscle progenitors in vitro, and to differentiate into skeletal muscle cells in vivo [2]. SKPs were obtained as it follows. Skin from the abdomen and back of mice was cut into pieces, digested with trypsin, dissociated by pipetting, and grown in a sphere culture system in presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Then, muscle progenitor cells were induced by treatment with 5-azacytidine, followed by cultivation in a differentiation medium containing 2% fetal bovine serum (FBS), hydrocortisone, insulin-like growth factor 1 (IGF-1), and bFGF. These progenitors were injected into cardiotoxin-injured tibialis anterior muscle of mice, and it has been demonstrated that they differentiate into muscle cells in vivo. Wakabayashi et al. reported that murine dermal Sca-1- cells develop into skeletal muscle cells through cell aggregation cultures.
* Corresponding author. Department of Drug Discovery Science, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, Japan. E-mail address: [email protected] (H. Ohnota). 1 Hideki Ohnota and Hiromi Nakazawa contributed equally to this work.
They isolated Sca-1- cells from the dermis, after epidermis and subcutaneous tissue removal, by trypsin digestion and FACS sorting. These cells grew into myotubes by aggregation cultures in presence of growth factors, and, when transplanted, integrated into host muscles in vivo [3]. García-Parra et al. engineered murine muscles from dermal precursors. They dissected skin fragments from the back of mice, digested them with collagenase, and performed sphere culture with various growth factors to generate twitching myotubes [4]. In addition, they identified and characterized dermal panniculus carnosus (PC) muscle stem cells, which are Myf5þ/PAX7þ and differentiate into myotubes by similar culture methods [5]. These results demonstrated that skeletal muscle stem cells, also called satellite cells, are present in the skin from the back of mice. In this study, we developed a new method to generate skeletal muscle cells from mouse skin fragments. In most animals, the PC muscle functions in skin twitching and contraction, while in humans, it is present in the skin of some upper body regions, as a remnant of evolution [6]. It also plays important roles for the investigation of human diseases such as amyotrophic lateral sclerosis, and muscular dystrophies, including facioscapulohumeral muscular dystrophy (FSHD). Innervations from the lateral thoracic nerve to the distal cutaneous maximus muscle degenerate earlier than those of the proximal one in SOD1 mouse
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Please cite this article as: H. Ohnota et al., Skeletal muscle cells derived from mouse skin cultures, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.067
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Abbreviations SKPs EGF bFGF IGF-1 PC FSHD NEU1 FBS PBS PBST TEM MyoG SR MHC
Skin-derived precursors epidermal growth factor basic fibroblast growth factor insulin-like growth factor 1 panniculus carnosus facioscapulohumeral muscular dystrophy lysosomal neuraminidase fetal bovine serum phosphate buffered saline 0.3% Triton X-100 in PBS transmission electron microscopy myogenin sarcoplasmic reticulum myosin heavy chain
model, and these results highlighted the significance of axonal length in pathological conditions [7]. FSHD exhibits focal myopathy, which mainly affects the shoulder and facial muscles [8]. Caruso et al. reported that protocadherin FAT1-disrupted mice show selective defects in the scapular muscles and abnormally shaped shoulder muscles, cutaneous maximus abnormalities during development, and significant volume and thickness reduction of the rhomboid superficialis and rhomboid profundus [9]. They also showed that FAT-1 was misregulated in FSHD patients mainly due to genomic changes [9]. Therefore, our method, which enables easy formation of skeletal muscle cell cultures from skin pieces, will be very useful to investigate various diseases of skeletal muscles. Sialidosis is an inherited rare lysosomal storage disease that is caused by the lack or low activity of lysosomal neuraminidase (NEU1) [10]. Skeletal deformities and muscle hypotonia were reported in sialidosis patients, and Neu1-disrupted mice [11] showed skeletal muscle pathologic alterations, with expanded connective tissue and infiltration of fibroblast-like cells [12]. It was also reported that skeletal muscle regeneration is impaired in these mice [13]. In addition, it was reported that Neu1-/- fibroblasts secrete large amounts of exosomes, and induce fibrosis of skeletal muscles [14]. Therefore, we used our skeletal muscle culture method to examine the formation of skeletal muscle cells from the skin of Neu1-/- mice. 2. Materials and methods 2.1. Animals Male and female FVB/NJ mice (Neu1-/þ [9]) were obtained from Dr. A. d’Azzo (St. Jude Children’s Research Hospital, Memphis, TN, USA), and mated. Offspring were genotyped, and wild type (Neu1þ/ þ ) and homozygote mutant (Neu1-/-) mice were used in this study. They were housed in controlled conditions (12 h day-night cycle, 22e26 C, 60% humidity), with free access to food and water. All experimental procedures were carried out in accordance with the Regulations for Animal Experimentation of the Shinshu University. The animal protocol was approved by the Committee for Animal Experiments of the Shinshu University (Approval number 300029). 2.2. Mouse skin culture Five to 12-week-old male or female mice were euthanized, and a portion of the back skin (about 1 cm2) was excised after clipping the hairs. Each skin fragment was placed in a 10 cm plastic dish
containing 10 mL high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich Japan, Tokyo), supplemented with 10% immobilized FBS (Hyclone, GE healthcare, Chicago, IL, USA), MEM NEAA (Thermo Fisher Scientific, Waltham, MA, USA), 2 mM glutamine (Nacalai Tesque, Kyoto, Japan), penicillin (100 units/mL, ThermoFishrer Scientific), and streptomycin (100 mg/mL,Thermo Fisher Scientific). The skin was cut into small pieces of about 1 mm2 using surgical knives. Three to four skin pieces were placed in a 3.5 cm dish (AGC Techno Glass, Shizuoka, Japan) with the PC downside, and cultured in the medium described above at 37 C in an atmosphere supplemented with 5% CO2, 100% humidity for about 3 weeks. Culture medium was replaced every 2 or 3 days. 2.3. Phase-contrast and fluorescence microscope observations Phase-contrast microscope observations were performed under a routine inverted microscope (Primovert, ZEISS, Jena, Germany). Images and movies were obtained using the Axiocam ERc 5s (ZEISS). Sequential pictures were taken every 15 min using the Axio Observer and Axio Vision 4.8 (ZEISS). Immunofluorescence analysis was also performed using the system. 2.4. Fluorescent immunostaining Cultured skeletal muscle cells were fixed with 4% paraformaldehyde (Nacalai Tesque) for 10 min at room temperature, washed three times with PBS, blocked and permeabilized with 0.3% Triton X-100 (Nacalai Tesque) in PBS (Takara-bio, Shiga, Japan, PBST) containing 5% normal goat serum (Immunobioscience, Mukilteo, WA, USA) for 1 h at room temperature, and incubated with primary antibodies in PBST at 4 C overnight. The following day, after washing with PBS, secondary antibodies in PBST were applied and incubated at room temperature for 1 h. Then, 3.3 mM DAPI (Thermo Fisher Scientific) in PBS were added into the dish and incubated for 5 min at room temperature to stain the nuclei. Primary antibodies were rabbit anti-myosin heavy chain (MHC) antibody (ab124205, 1: 500, Abcam, Cambridge, UK), mouse anti-MHC antibody (clone A4.1025, 1:100, Merck KGaA, Darmstadt, Germany), mouse anti-PAX7 antibody (1: 100, DSHB, Iowa, IO, USA), mouse anti-myogenin (MyoG) antibody (ab1835, x200, Abcam), mouse anti-MyoD antibody (sc-377460, 1: 400, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-MyoD1 antibody (18943-1-AP, 1:100, Proteintech, Tokyo, Japan), and rabbit anti-MyoG antibody (orb6492, Biorbyt, Cambridge, UK). Control antibodies for primary antibodies were normal rabbit IgG (1: 5000, Fujifilm Wako Chemicals, Osaka, Japan), normal mouse IgG1 (1: 400, sc-3877, Santa Cruz Biotechnology), normal mouse IgG 2a (1: 400, sc-3883, Santa Cruz Biotechnology), and normal mouse IgM (1: 500, M-079-3, MBL, Tokyo, Japan). Secondary antibodies were goat anti-rabbit IgG (1: 500, ab150077, Alexa Fluor 488, Abcam), goat anti-mouse IgG (1:500, ab150113, Alexa Fluor 488, Abcam), goat anti-rabbit IgG (1: 500, ab150080, Alexa Fluor 594, Abcam), and goat anti-mouse IgG (1: 500, ab150116, Alexa Fluor 594, Abcam). 2.5. Transmission electron microscopy (TEM) Cultured myotubes were fixed with 2.5% glutaraldehyde and 5% osmium tetroxide, embedded in epoxy resin, and cut into ultrathin sections. These sections were electron-stained with uranyl acetate and lead citrate, subjected to carbon shadowing, and observed by TEM (JEM1400; JEOL, Tokyo, Japan). 2.6. Skeletal muscle cell formation assessment Skin pieces from female Neu1þ/þ (wild) or Neu1-/- mice were
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cultured for about 3 weeks, as described above. Culture dishes were observed under a phase-contrast microscope every 2 or 3 days. Each skin piece that generated skeletal muscle cells was counted as one positive piece. The migration of fibroblastic cells was also evaluated, and the ratio of skin pieces with myotubes among those with fibroblastic cells was estimated. 2.7. Statistical analysis Data are presented as mean ± SEM. A Statistical analysis for the incidence rate of myotube formation was performed through the Brunner-Munzel test using BellCurve for Excel (Social Survey Research Information, Tokyo, Japan). 3. Results 3.1. Formation of twitching skeletal muscle cells from Neu1þ/þ mice skin pieces
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https://doi.org/10.1016/j.bbrc.2019.12.067 3.2. Immunohistological analyses When well-developed myotubes were stained with anti-MHC antibody, the striated pattern of myotubes was observed in correlation with MyoGþ/MyoDþ in their nuclei (Fig. 2, A upper left and middle). In addition, myoblast-like cells with a small spindle shape or round cells, which were positive for both MHC and MyoG or MyoD, were also observed (Fig. 2A lower left and middle). In our analyses, most MHCþ cells (including myotubes) were double positive for MyoG and MyoD. The majority of MyoDþ single cells were also MyoGþ, and some cells were MyoD-/MyoGþ (Supplemental Fig. 1). In addition, many PAX7þ cells proliferated around the myotubes. They were MyoD- and MHC-, and no cells were double positive for MyoD or MHC with PAX7 (Fig. 2A upper and lower right). 3.3. Ultrastructural analysis
Typical myotube formation was observed as it follows. Keratinocytes and fibroblasts migrated from the skin piece between the 3rd and 5th day of culture. Between the 6th and 10th day, bright rod-like myotubes emerged (Fig. 1A upper left). Then, they extended by fusing with other myoblast-like cells or myotubes, and formed large groups of myotubes (Fig. 1A upper right, lower left). Spontaneous twitches of these cells were observed (Supplemental Video 1). These myotubes developed into large clumps of myotubes (Fig. 1A lower right). They seemed to overlap several times, and were twitching synchronously (Supplemental Video 2). Supplementary video related to this article can be found at https://doi.org/10.1016/j.bbrc.2019.12.067 We also observed the formation of myotubes by live-imaging every 15 min. Myotubes were formed through repeated stretching and shrinking (Fig. 1B, Supplemental Videos 3 and 4). During the early stage of myotube formation (Fig. 1B upper, Supplemental Video 3), relatively short myotubes elongated and suddenly shortened to become round masses or myotubes with a swelling in the central cell region. And then, they grew again into extended myotubes. We sometimes observed myotubes rupture (Supplemental Video 3), and swelled cell twitching (Supplemental Video 5). In the late stage (Fig. 1B lower, Supplemental Video 4), myotubes elongated and shrunk suddenly, become round, and elongated again repeatedly. Supplementary video related to this article can be found at
We examined the ultrastructure of cultured myotubes. Myofibril assemblies with sarcomere formation were confirmed (Fig. 2B). Z lines, A bands, and M lines in sarcomeres were observed. Relatively short myotubes also formed sarcomeres (Fig. 2B right). Sarcoplasmic reticulums (SRs) and mitochondria were between myofibrils. SR T-tubule triad-like structures were also observed (Supplemental Fig. 2A), and immature actin filaments were detected (Supplemental Fig. 2B). 3.4. Skeletal muscle formation from sialidosis model mouse skin Myotube formations from Neu1-/- mouse skin pieces were compared to that of wild type mice (Fig. 3). The incidence of myotube formation from Neu1-/- mouse skin was significantly lower than that of wild mouse skin (Fig. 3B: 21 ± 9% vs. 73 ± 11%), while the fibroblast migration rates for the two types of skin pieces were similar (Fig. 3A: 92 ± 5% vs. 101 ± 1%). Myotubes derived from Neu1-/- mouse skin did not seem to differ from those formed from wild mouse skin in their shape, twitching, and immunological staining (data not shown). 4. Discussion We have established a novel method to generate skeletal muscle
Fig. 1. Generations of myotubes from skin cultures. (A) A 6 week-old male wild mouse skin was cultured and myotube formations were observed over time. Myotubes on 10th, 13th, 15th, and 22nd day. Myotubes on15th day and the 22nd day were twitching (Supplemental Video 1 and 2). (B) The early stage of myotubes(Upper panel) and the late stage myotubes (Lower panel), both of which were observed for about 5 days every 15 min (Supplemental Video 3 and 4).
Please cite this article as: H. Ohnota et al., Skeletal muscle cells derived from mouse skin cultures, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.067
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Fig. 2. (A) Immunofluorescence staining of myotubes derived from 5 week old male wild mice skin. Myosin heavy chain(MHC), myogenin (MyoG), and MyoD were stained. Nuclei were also stained with DAPI. The Phase-contrast images were modulated for clarity. (B) TEM image of cultured myotubes derived from 12 week old male wild mice skin. Sarcomere formations surrounded with mitochondria and SRs were observed.
Fig. 3. Fibroblasts migration incidents per skin pieces of wild and Neu1-/-mice were calculated(A) and myotube formation rates per skin pieces with fibroblasts(B) were shown. **: P < 0.01 vs wild mice by Brunner-Munzel test.
cells from mouse skin fragment cultures. Our method is highly unique in that we did not use either of the traditional conditions, such as trypsin or collagenase digestion, growth factors like EGF or bFGF, aggregate or sphere culture system, or low nutrient medium to induce cell differentiation [2e4]. In our culture conditions,
proliferation and differentiation of myogenic precursors were thought to be induced by injury with surgical knives, while growth factors might have been supplied by the surrounding fibroblasts. Many reports concerning co-cultures of myogenic progenitors with other cells residing in their stem cell niche have been published.
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Among these, some examples are fibroblasts [14], endothelial cells [15], neural cells [16], and fibro/adipogenic progenitors [17]. They revealed a higher degree of muscle cell differentiation and myotube maturation, vascularized tissue development, and neuromuscular junction formation. However, our culture method provides host myogenic precursors with host myogenic niche cells. This is an ideal condition to elucidate skeletal muscle development under physiological and pathological conditions. We believe our culture method is a useful tool for the investigation of skeletal muscles and the related disorders. During myotube formations in our culture system, we observed elongated myotubes that contracted suddenly, became swollen, and extended again. This stretch and shrink process was sometimes repeated in the same myotube. In addition, we observed myotubes that split into two thick spindle-shaped myotubes (Supplemental Video 3), which then stretched and continued to grow. In some cases, shrunken myotubes became spherical, then, elongated again (Supplemental Video 4). To our knowledge, these phenomena have not been reported so far, and their physiological meaning, if any, is not known. However, they are highly reproducible. The contraction of a cultured myotube due to unknown causes and resulting in cell death is common. The possibility that these observations are artifacts in our culture system cannot be excluded, as thick spindleshaped myotube formation as an intermediary during myotube formation has not been so far identified in vivo. However, we are very interested in the stretch and shrink process, and further examinations will be mandatory. In addition, the tearing and regeneration of myotubes is surprising. We recognized at least two myotubes cleaved into two cells in the Supplemental Video 3, which are located in the upper middle and center of the image. When myotubes are split by strain, calcium influx and necrosis occur generally [18]. The cleaving observed in our culture seemed similar to that induced by overstrains, and the contraction of fragmented cells was observed, possibly due to calcium influx. Then, these cleaved cells did not die, and, instead, regenerated and
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elongated as myotubes. This movie presents the sequential images taken every 15 minutes, and the magnification was fifty times. Therefore, more detailed examinations with additional analytical methods will be necessary to elucidate what happened in this situation. Myoblasts or myotubes are known to fuse with one another, and the molecular mechanisms underlying this process have been clarified [19]. Therefore, a speculation is that immature myotubes could close the cleavage in the plasma membrane by fusing. The muscle cell development was known to be associated with stage-specific nuclear factors. The satellite cells or myogenic precursors are positive for PAX7, then MyoD is induced for myoblast commitment, while myogenin emerges in the final differential stage [20]. In our culture system, PAX7þ stem cells or precursors proliferated around the myotubes, and uninucleated PAX7-/ MyoDþ/myogeninþ/MHC- or PAX7-/MyoDþ/MyoGþ/MHCþ cells were also observed. Most myotubes were double positive for MyoD and MyoG. PAX7þ/MyoDþ cells were not observed. In addition, MyoDþ/MyoG- cell were not determined, and MyoD-/MyoGþ cells were rare. These are thought to be characteristic to our myotube formation system. Myotubes showed characteristic striated pattern by immunofluorescence staining and the sarcomere ultrastructure was observed under a transmission electron microscope. Moreover, these myotubes sometimes overlapped, and formed large twitching clumps. We understand that these myotubes are similar to other ones produced in vitro. However, immature sarcomeres with streaming Z lines were also observed, and the SR T-tubule triad was rare. In addition, nuclei did not move to the edge of myotubes. Therefore, a complete reproduction of skeletal muscle formation in vivo was not reached. It will be interesting to vary the culture conditions (e.g. adding various growth factors) to replicate the physiological development of skeletal muscles. The speculated skeletal muscle formation in our culture is depicted in Fig. 4. Initially, PAX7þ cells proliferate, and become PAX7-/MyoDþ/MyoGþ myoblasts. Then, cells express MHC and fuse
Fig. 4. Speculated myotube developmental process in our culture. Initially, PAX7þ cells emerge, then they become PAX7-/MyoDþ/MyoGþ myoblasts without MHC expression. Next, MHCþ cells appear, whose shape is round or spindle. Then cells are fused and sarcomeres formed. In the process, stretches and shrinking are observed, and shrunken cells have vacuole-like structure in the swollen part (Supplemental Videos 3 and 4). Finally they become twitching clamps of myotubes. Dotted line indicates sarcomeres. MyoG, Myogenin; MHC, Myosin Heavy Chain.
Please cite this article as: H. Ohnota et al., Skeletal muscle cells derived from mouse skin cultures, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.067
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to form myotubes with multiple nuclei, and myofibers. Myotubes exhibited repeated stretching and shrinking during their growth process. They suddenly shrunk, and became round clumps or corpulent bodies, then elongated again. Finally, they become a twitching clump of myotubes. In addition, it is not clear what type of stem cell from the skin piece culture contributed to the formation of myotubes. Three candidates could be skin-derived multipotent precursors [1], dermis-derived Sca 1- cells [3], or satellite cells in PC [5]. This subject will require further investigations in the future. Neu1-/- sialidosis model mice were known to show muscle degeneration with expanded connective tissues [12e14]. In the present study, we investigated the myotube formation rate using our skin piece culture system, and revealed that skeletal muscle generation from Neu1-/- mouse skin in vitro was impaired, while fibroblast-like cell migration was not altered. In our culture system, Neu1-/- fibroblastic cells are co-cultured with myogenic precursors, and exosome hypersecretion, which is an underlying cause for this disease, could be closely associated. Therefore, our results are compatible with the previous report, and show that the system could be employed to understand the mechanisms underlying muscle disorders and for the treatment discovery. Once the mouse model for skeletal muscle disorder is obtained, affected skeletal muscles can be easily generated by skin piece culture. In addition, we consider the possibility to grow skeletal muscle cells from the skin of animals other than rodents. In conclusion, we have established a novel and simple skeletal muscle culture method, and observed some novel phenomena during myotubes formations. We believe these findings lead to the novel knowledge about skeletal muscle generation, and our skin culture method will be applied in various fields, including developmental biology, medical science, and pharmacology. Declaration of competing interest (1) This work was supported by a grant of Kissei Pharmaceutical CO., LTD. (2) Hideki Ohnota, Morimichi Hayashi, and Yuji Okuhara receive their salaries from Kissei Pharmaceutical CO., LTD. (3) A patent filing on this work is scheduled on December 4. (4) No other author has reported a potential conflict of interest relevant to this article. Acknowledgements This work was supported by a grant of Kissei Pharmaceutical Co. Ltd. The authors thank Ms. Suzuki, Ms. Yamada, and Ms. Ishikawa for their excellent technical supports and specimen preparations. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.067. References -Heider, A. Sadikot, [1] J.G. Toma, M. Akhavan, K.J. Fernandes, F. Barnabe D.R. Kaplan, F.D. Miller, Isolation of multipotent adult stem cells from the dermis of mammalian skin, Nat. Cell Biol. 3 (2001) 778e784. https://doi: 10. 1038/ncb0901-778.
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Okano, Design of prevascularized three-dimensional cell-dense tissues using a cell sheet stacking manipulation technology, Biomaterials 31 (2010) 1646e1654. https://doi: 10.1016/j.biomaterials.2009.11.036. [16] A.S. Smith, S.L. Passey, N.R. Martin, D.J. Player, V. Mudera, L. Greensmith, M.P. Lewis, Creating interactions between tissue-engineered skeletal muscle and the peripheral nervous system, Cells Tissues Organs 202 (2016) 143e158. https://doi: 10.1159/000443634. [17] A.W. Joe, L. Yi, A. Natarajan, F. Le Grand, L. So, J. Wang, M.A. Rudnicki, F.M. Rossi, Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis, Nat. Cell Biol. 12 (2010) 153e163. https://doi: 10.1038/ ncb2015. [18] J. Souza, C. Gottfried, Muscle injury: review of experimental models, J. Electromyogr. Kinesiol. 23 (2016) 1253e1260. https://doi: 10.1016/j.jelekin. 2013.07.009. [19] S.M. Hindi, M.M. Tajrishi, A. Kumar, Signaling mechanisms in mammalian myoblast fusion, Sci. 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Skeletal muscle cells derived from mouse skin cultures Hideki Ohnota a, b, *, 1, Hiromi Nakazawa a, 1, Morimichi Hayashi c, Yuji Okuhara c, Takayuki Honda a, Alessandra d’Azzo d, Yoshiki Sekijima e a
Department of Drug Discovery Science, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, Japan Research Division, R&D, Kissei Pharmaceutical Co. Ltd., 4365-1 Kashiwabara, Hotaka, Azumino, Nagano, 399-8304, Japan c Safety Research Laboratory, R&D, Kissei Pharmaceutical Co. Ltd., 2320-1 Maki, Hotaka, Azumino, Nagano, 399-8304, Japan d Department of Genetics, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA e Department of Medicine (Neurology and Rheumatology), Shinshu University School of Medicine, Matsumoto, 390-8621, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 November 2019 Accepted 15 December 2019 Available online xxx
We have established a novel, simple, and highly reproducible method to generate skeletal muscle cells from mouse skin. Small pieces of skin from the back of mice were cultured in extracellular materialcoated dishes in typical culture medium for about 3 weeks. Myotubes formed after about a week, grew into twitching myotubes, and became twitching myotube clumps after 3 weeks. Skeletal muscle cells are formed spontaneously with no induction. Myotubes were immunologically positive for myosin heavy chains, MyoD, and myogenin. Ultrastructural analysis revealed the presence of the sarcomere structure. Furthermore, PAX7þ/MyoD- muscle stem cells proliferated around these myotubes, and MyoDþ/myogeninþ/MHC- cells were also observed. Moreover, we investigated the formation of skeletal muscle cells from the sialidosis mouse skin, and showed that it is decreased compared to that of the wild type. Our method to generate skeletal muscle cells from skin is thought to be useful for the investigation of muscle cell development and muscle-related disorders. © 2020 Elsevier Inc. All rights reserved.
Keywords: Panniculus carnosus PAX7 Myotube Twitching Sialidosis Skin
1. Introduction Skin-derived precursors (SKPs), which differentiate into neurons, glia, smooth muscle cells, and adipocytes [1], are reported to develop into skeletal muscle progenitors in vitro, and to differentiate into skeletal muscle cells in vivo [2]. SKPs were obtained as it follows. Skin from the abdomen and back of mice was cut into pieces, digested with trypsin, dissociated by pipetting, and grown in a sphere culture system in presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Then, muscle progenitor cells were induced by treatment with 5-azacytidine, followed by cultivation in a differentiation medium containing 2% fetal bovine serum (FBS), hydrocortisone, insulin-like growth factor 1 (IGF-1), and bFGF. These progenitors were injected into cardiotoxin-injured tibialis anterior muscle of mice, and it has been demonstrated that they differentiate into muscle cells in vivo. Wakabayashi et al. reported that murine dermal Sca-1- cells develop into skeletal muscle cells through cell aggregation cultures.
* Corresponding author. Department of Drug Discovery Science, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, Japan. E-mail address: [email protected] (H. Ohnota). 1 Hideki Ohnota and Hiromi Nakazawa contributed equally to this work.
They isolated Sca-1- cells from the dermis, after epidermis and subcutaneous tissue removal, by trypsin digestion and FACS sorting. These cells grew into myotubes by aggregation cultures in presence of growth factors, and, when transplanted, integrated into host muscles in vivo [3]. García-Parra et al. engineered murine muscles from dermal precursors. They dissected skin fragments from the back of mice, digested them with collagenase, and performed sphere culture with various growth factors to generate twitching myotubes [4]. In addition, they identified and characterized dermal panniculus carnosus (PC) muscle stem cells, which are Myf5þ/PAX7þ and differentiate into myotubes by similar culture methods [5]. These results demonstrated that skeletal muscle stem cells, also called satellite cells, are present in the skin from the back of mice. In this study, we developed a new method to generate skeletal muscle cells from mouse skin fragments. In most animals, the PC muscle functions in skin twitching and contraction, while in humans, it is present in the skin of some upper body regions, as a remnant of evolution [6]. It also plays important roles for the investigation of human diseases such as amyotrophic lateral sclerosis, and muscular dystrophies, including facioscapulohumeral muscular dystrophy (FSHD). Innervations from the lateral thoracic nerve to the distal cutaneous maximus muscle degenerate earlier than those of the proximal one in SOD1 mouse
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Please cite this article as: H. Ohnota et al., Skeletal muscle cells derived from mouse skin cultures, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.067
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Abbreviations SKPs EGF bFGF IGF-1 PC FSHD NEU1 FBS PBS PBST TEM MyoG SR MHC
Skin-derived precursors epidermal growth factor basic fibroblast growth factor insulin-like growth factor 1 panniculus carnosus facioscapulohumeral muscular dystrophy lysosomal neuraminidase fetal bovine serum phosphate buffered saline 0.3% Triton X-100 in PBS transmission electron microscopy myogenin sarcoplasmic reticulum myosin heavy chain
model, and these results highlighted the significance of axonal length in pathological conditions [7]. FSHD exhibits focal myopathy, which mainly affects the shoulder and facial muscles [8]. Caruso et al. reported that protocadherin FAT1-disrupted mice show selective defects in the scapular muscles and abnormally shaped shoulder muscles, cutaneous maximus abnormalities during development, and significant volume and thickness reduction of the rhomboid superficialis and rhomboid profundus [9]. They also showed that FAT-1 was misregulated in FSHD patients mainly due to genomic changes [9]. Therefore, our method, which enables easy formation of skeletal muscle cell cultures from skin pieces, will be very useful to investigate various diseases of skeletal muscles. Sialidosis is an inherited rare lysosomal storage disease that is caused by the lack or low activity of lysosomal neuraminidase (NEU1) [10]. Skeletal deformities and muscle hypotonia were reported in sialidosis patients, and Neu1-disrupted mice [11] showed skeletal muscle pathologic alterations, with expanded connective tissue and infiltration of fibroblast-like cells [12]. It was also reported that skeletal muscle regeneration is impaired in these mice [13]. In addition, it was reported that Neu1-/- fibroblasts secrete large amounts of exosomes, and induce fibrosis of skeletal muscles [14]. Therefore, we used our skeletal muscle culture method to examine the formation of skeletal muscle cells from the skin of Neu1-/- mice. 2. Materials and methods 2.1. Animals Male and female FVB/NJ mice (Neu1-/þ [9]) were obtained from Dr. A. d’Azzo (St. Jude Children’s Research Hospital, Memphis, TN, USA), and mated. Offspring were genotyped, and wild type (Neu1þ/ þ ) and homozygote mutant (Neu1-/-) mice were used in this study. They were housed in controlled conditions (12 h day-night cycle, 22e26 C, 60% humidity), with free access to food and water. All experimental procedures were carried out in accordance with the Regulations for Animal Experimentation of the Shinshu University. The animal protocol was approved by the Committee for Animal Experiments of the Shinshu University (Approval number 300029). 2.2. Mouse skin culture Five to 12-week-old male or female mice were euthanized, and a portion of the back skin (about 1 cm2) was excised after clipping the hairs. Each skin fragment was placed in a 10 cm plastic dish
containing 10 mL high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich Japan, Tokyo), supplemented with 10% immobilized FBS (Hyclone, GE healthcare, Chicago, IL, USA), MEM NEAA (Thermo Fisher Scientific, Waltham, MA, USA), 2 mM glutamine (Nacalai Tesque, Kyoto, Japan), penicillin (100 units/mL, ThermoFishrer Scientific), and streptomycin (100 mg/mL,Thermo Fisher Scientific). The skin was cut into small pieces of about 1 mm2 using surgical knives. Three to four skin pieces were placed in a 3.5 cm dish (AGC Techno Glass, Shizuoka, Japan) with the PC downside, and cultured in the medium described above at 37 C in an atmosphere supplemented with 5% CO2, 100% humidity for about 3 weeks. Culture medium was replaced every 2 or 3 days. 2.3. Phase-contrast and fluorescence microscope observations Phase-contrast microscope observations were performed under a routine inverted microscope (Primovert, ZEISS, Jena, Germany). Images and movies were obtained using the Axiocam ERc 5s (ZEISS). Sequential pictures were taken every 15 min using the Axio Observer and Axio Vision 4.8 (ZEISS). Immunofluorescence analysis was also performed using the system. 2.4. Fluorescent immunostaining Cultured skeletal muscle cells were fixed with 4% paraformaldehyde (Nacalai Tesque) for 10 min at room temperature, washed three times with PBS, blocked and permeabilized with 0.3% Triton X-100 (Nacalai Tesque) in PBS (Takara-bio, Shiga, Japan, PBST) containing 5% normal goat serum (Immunobioscience, Mukilteo, WA, USA) for 1 h at room temperature, and incubated with primary antibodies in PBST at 4 C overnight. The following day, after washing with PBS, secondary antibodies in PBST were applied and incubated at room temperature for 1 h. Then, 3.3 mM DAPI (Thermo Fisher Scientific) in PBS were added into the dish and incubated for 5 min at room temperature to stain the nuclei. Primary antibodies were rabbit anti-myosin heavy chain (MHC) antibody (ab124205, 1: 500, Abcam, Cambridge, UK), mouse anti-MHC antibody (clone A4.1025, 1:100, Merck KGaA, Darmstadt, Germany), mouse anti-PAX7 antibody (1: 100, DSHB, Iowa, IO, USA), mouse anti-myogenin (MyoG) antibody (ab1835, x200, Abcam), mouse anti-MyoD antibody (sc-377460, 1: 400, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-MyoD1 antibody (18943-1-AP, 1:100, Proteintech, Tokyo, Japan), and rabbit anti-MyoG antibody (orb6492, Biorbyt, Cambridge, UK). Control antibodies for primary antibodies were normal rabbit IgG (1: 5000, Fujifilm Wako Chemicals, Osaka, Japan), normal mouse IgG1 (1: 400, sc-3877, Santa Cruz Biotechnology), normal mouse IgG 2a (1: 400, sc-3883, Santa Cruz Biotechnology), and normal mouse IgM (1: 500, M-079-3, MBL, Tokyo, Japan). Secondary antibodies were goat anti-rabbit IgG (1: 500, ab150077, Alexa Fluor 488, Abcam), goat anti-mouse IgG (1:500, ab150113, Alexa Fluor 488, Abcam), goat anti-rabbit IgG (1: 500, ab150080, Alexa Fluor 594, Abcam), and goat anti-mouse IgG (1: 500, ab150116, Alexa Fluor 594, Abcam). 2.5. Transmission electron microscopy (TEM) Cultured myotubes were fixed with 2.5% glutaraldehyde and 5% osmium tetroxide, embedded in epoxy resin, and cut into ultrathin sections. These sections were electron-stained with uranyl acetate and lead citrate, subjected to carbon shadowing, and observed by TEM (JEM1400; JEOL, Tokyo, Japan). 2.6. Skeletal muscle cell formation assessment Skin pieces from female Neu1þ/þ (wild) or Neu1-/- mice were
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cultured for about 3 weeks, as described above. Culture dishes were observed under a phase-contrast microscope every 2 or 3 days. Each skin piece that generated skeletal muscle cells was counted as one positive piece. The migration of fibroblastic cells was also evaluated, and the ratio of skin pieces with myotubes among those with fibroblastic cells was estimated. 2.7. Statistical analysis Data are presented as mean ± SEM. A Statistical analysis for the incidence rate of myotube formation was performed through the Brunner-Munzel test using BellCurve for Excel (Social Survey Research Information, Tokyo, Japan). 3. Results 3.1. Formation of twitching skeletal muscle cells from Neu1þ/þ mice skin pieces
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https://doi.org/10.1016/j.bbrc.2019.12.067 3.2. Immunohistological analyses When well-developed myotubes were stained with anti-MHC antibody, the striated pattern of myotubes was observed in correlation with MyoGþ/MyoDþ in their nuclei (Fig. 2, A upper left and middle). In addition, myoblast-like cells with a small spindle shape or round cells, which were positive for both MHC and MyoG or MyoD, were also observed (Fig. 2A lower left and middle). In our analyses, most MHCþ cells (including myotubes) were double positive for MyoG and MyoD. The majority of MyoDþ single cells were also MyoGþ, and some cells were MyoD-/MyoGþ (Supplemental Fig. 1). In addition, many PAX7þ cells proliferated around the myotubes. They were MyoD- and MHC-, and no cells were double positive for MyoD or MHC with PAX7 (Fig. 2A upper and lower right). 3.3. Ultrastructural analysis
Typical myotube formation was observed as it follows. Keratinocytes and fibroblasts migrated from the skin piece between the 3rd and 5th day of culture. Between the 6th and 10th day, bright rod-like myotubes emerged (Fig. 1A upper left). Then, they extended by fusing with other myoblast-like cells or myotubes, and formed large groups of myotubes (Fig. 1A upper right, lower left). Spontaneous twitches of these cells were observed (Supplemental Video 1). These myotubes developed into large clumps of myotubes (Fig. 1A lower right). They seemed to overlap several times, and were twitching synchronously (Supplemental Video 2). Supplementary video related to this article can be found at https://doi.org/10.1016/j.bbrc.2019.12.067 We also observed the formation of myotubes by live-imaging every 15 min. Myotubes were formed through repeated stretching and shrinking (Fig. 1B, Supplemental Videos 3 and 4). During the early stage of myotube formation (Fig. 1B upper, Supplemental Video 3), relatively short myotubes elongated and suddenly shortened to become round masses or myotubes with a swelling in the central cell region. And then, they grew again into extended myotubes. We sometimes observed myotubes rupture (Supplemental Video 3), and swelled cell twitching (Supplemental Video 5). In the late stage (Fig. 1B lower, Supplemental Video 4), myotubes elongated and shrunk suddenly, become round, and elongated again repeatedly. Supplementary video related to this article can be found at
We examined the ultrastructure of cultured myotubes. Myofibril assemblies with sarcomere formation were confirmed (Fig. 2B). Z lines, A bands, and M lines in sarcomeres were observed. Relatively short myotubes also formed sarcomeres (Fig. 2B right). Sarcoplasmic reticulums (SRs) and mitochondria were between myofibrils. SR T-tubule triad-like structures were also observed (Supplemental Fig. 2A), and immature actin filaments were detected (Supplemental Fig. 2B). 3.4. Skeletal muscle formation from sialidosis model mouse skin Myotube formations from Neu1-/- mouse skin pieces were compared to that of wild type mice (Fig. 3). The incidence of myotube formation from Neu1-/- mouse skin was significantly lower than that of wild mouse skin (Fig. 3B: 21 ± 9% vs. 73 ± 11%), while the fibroblast migration rates for the two types of skin pieces were similar (Fig. 3A: 92 ± 5% vs. 101 ± 1%). Myotubes derived from Neu1-/- mouse skin did not seem to differ from those formed from wild mouse skin in their shape, twitching, and immunological staining (data not shown). 4. Discussion We have established a novel method to generate skeletal muscle
Fig. 1. Generations of myotubes from skin cultures. (A) A 6 week-old male wild mouse skin was cultured and myotube formations were observed over time. Myotubes on 10th, 13th, 15th, and 22nd day. Myotubes on15th day and the 22nd day were twitching (Supplemental Video 1 and 2). (B) The early stage of myotubes(Upper panel) and the late stage myotubes (Lower panel), both of which were observed for about 5 days every 15 min (Supplemental Video 3 and 4).
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Fig. 2. (A) Immunofluorescence staining of myotubes derived from 5 week old male wild mice skin. Myosin heavy chain(MHC), myogenin (MyoG), and MyoD were stained. Nuclei were also stained with DAPI. The Phase-contrast images were modulated for clarity. (B) TEM image of cultured myotubes derived from 12 week old male wild mice skin. Sarcomere formations surrounded with mitochondria and SRs were observed.
Fig. 3. Fibroblasts migration incidents per skin pieces of wild and Neu1-/-mice were calculated(A) and myotube formation rates per skin pieces with fibroblasts(B) were shown. **: P < 0.01 vs wild mice by Brunner-Munzel test.
cells from mouse skin fragment cultures. Our method is highly unique in that we did not use either of the traditional conditions, such as trypsin or collagenase digestion, growth factors like EGF or bFGF, aggregate or sphere culture system, or low nutrient medium to induce cell differentiation [2e4]. In our culture conditions,
proliferation and differentiation of myogenic precursors were thought to be induced by injury with surgical knives, while growth factors might have been supplied by the surrounding fibroblasts. Many reports concerning co-cultures of myogenic progenitors with other cells residing in their stem cell niche have been published.
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Among these, some examples are fibroblasts [14], endothelial cells [15], neural cells [16], and fibro/adipogenic progenitors [17]. They revealed a higher degree of muscle cell differentiation and myotube maturation, vascularized tissue development, and neuromuscular junction formation. However, our culture method provides host myogenic precursors with host myogenic niche cells. This is an ideal condition to elucidate skeletal muscle development under physiological and pathological conditions. We believe our culture method is a useful tool for the investigation of skeletal muscles and the related disorders. During myotube formations in our culture system, we observed elongated myotubes that contracted suddenly, became swollen, and extended again. This stretch and shrink process was sometimes repeated in the same myotube. In addition, we observed myotubes that split into two thick spindle-shaped myotubes (Supplemental Video 3), which then stretched and continued to grow. In some cases, shrunken myotubes became spherical, then, elongated again (Supplemental Video 4). To our knowledge, these phenomena have not been reported so far, and their physiological meaning, if any, is not known. However, they are highly reproducible. The contraction of a cultured myotube due to unknown causes and resulting in cell death is common. The possibility that these observations are artifacts in our culture system cannot be excluded, as thick spindleshaped myotube formation as an intermediary during myotube formation has not been so far identified in vivo. However, we are very interested in the stretch and shrink process, and further examinations will be mandatory. In addition, the tearing and regeneration of myotubes is surprising. We recognized at least two myotubes cleaved into two cells in the Supplemental Video 3, which are located in the upper middle and center of the image. When myotubes are split by strain, calcium influx and necrosis occur generally [18]. The cleaving observed in our culture seemed similar to that induced by overstrains, and the contraction of fragmented cells was observed, possibly due to calcium influx. Then, these cleaved cells did not die, and, instead, regenerated and
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elongated as myotubes. This movie presents the sequential images taken every 15 minutes, and the magnification was fifty times. Therefore, more detailed examinations with additional analytical methods will be necessary to elucidate what happened in this situation. Myoblasts or myotubes are known to fuse with one another, and the molecular mechanisms underlying this process have been clarified [19]. Therefore, a speculation is that immature myotubes could close the cleavage in the plasma membrane by fusing. The muscle cell development was known to be associated with stage-specific nuclear factors. The satellite cells or myogenic precursors are positive for PAX7, then MyoD is induced for myoblast commitment, while myogenin emerges in the final differential stage [20]. In our culture system, PAX7þ stem cells or precursors proliferated around the myotubes, and uninucleated PAX7-/ MyoDþ/myogeninþ/MHC- or PAX7-/MyoDþ/MyoGþ/MHCþ cells were also observed. Most myotubes were double positive for MyoD and MyoG. PAX7þ/MyoDþ cells were not observed. In addition, MyoDþ/MyoG- cell were not determined, and MyoD-/MyoGþ cells were rare. These are thought to be characteristic to our myotube formation system. Myotubes showed characteristic striated pattern by immunofluorescence staining and the sarcomere ultrastructure was observed under a transmission electron microscope. Moreover, these myotubes sometimes overlapped, and formed large twitching clumps. We understand that these myotubes are similar to other ones produced in vitro. However, immature sarcomeres with streaming Z lines were also observed, and the SR T-tubule triad was rare. In addition, nuclei did not move to the edge of myotubes. Therefore, a complete reproduction of skeletal muscle formation in vivo was not reached. It will be interesting to vary the culture conditions (e.g. adding various growth factors) to replicate the physiological development of skeletal muscles. The speculated skeletal muscle formation in our culture is depicted in Fig. 4. Initially, PAX7þ cells proliferate, and become PAX7-/MyoDþ/MyoGþ myoblasts. Then, cells express MHC and fuse
Fig. 4. Speculated myotube developmental process in our culture. Initially, PAX7þ cells emerge, then they become PAX7-/MyoDþ/MyoGþ myoblasts without MHC expression. Next, MHCþ cells appear, whose shape is round or spindle. Then cells are fused and sarcomeres formed. In the process, stretches and shrinking are observed, and shrunken cells have vacuole-like structure in the swollen part (Supplemental Videos 3 and 4). Finally they become twitching clamps of myotubes. Dotted line indicates sarcomeres. MyoG, Myogenin; MHC, Myosin Heavy Chain.
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to form myotubes with multiple nuclei, and myofibers. Myotubes exhibited repeated stretching and shrinking during their growth process. They suddenly shrunk, and became round clumps or corpulent bodies, then elongated again. Finally, they become a twitching clump of myotubes. In addition, it is not clear what type of stem cell from the skin piece culture contributed to the formation of myotubes. Three candidates could be skin-derived multipotent precursors [1], dermis-derived Sca 1- cells [3], or satellite cells in PC [5]. This subject will require further investigations in the future. Neu1-/- sialidosis model mice were known to show muscle degeneration with expanded connective tissues [12e14]. In the present study, we investigated the myotube formation rate using our skin piece culture system, and revealed that skeletal muscle generation from Neu1-/- mouse skin in vitro was impaired, while fibroblast-like cell migration was not altered. In our culture system, Neu1-/- fibroblastic cells are co-cultured with myogenic precursors, and exosome hypersecretion, which is an underlying cause for this disease, could be closely associated. Therefore, our results are compatible with the previous report, and show that the system could be employed to understand the mechanisms underlying muscle disorders and for the treatment discovery. Once the mouse model for skeletal muscle disorder is obtained, affected skeletal muscles can be easily generated by skin piece culture. In addition, we consider the possibility to grow skeletal muscle cells from the skin of animals other than rodents. In conclusion, we have established a novel and simple skeletal muscle culture method, and observed some novel phenomena during myotubes formations. We believe these findings lead to the novel knowledge about skeletal muscle generation, and our skin culture method will be applied in various fields, including developmental biology, medical science, and pharmacology. Declaration of competing interest (1) This work was supported by a grant of Kissei Pharmaceutical CO., LTD. (2) Hideki Ohnota, Morimichi Hayashi, and Yuji Okuhara receive their salaries from Kissei Pharmaceutical CO., LTD. (3) A patent filing on this work is scheduled on December 4. (4) No other author has reported a potential conflict of interest relevant to this article. Acknowledgements This work was supported by a grant of Kissei Pharmaceutical Co. Ltd. The authors thank Ms. Suzuki, Ms. Yamada, and Ms. Ishikawa for their excellent technical supports and specimen preparations. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.067. References -Heider, A. Sadikot, [1] J.G. Toma, M. Akhavan, K.J. Fernandes, F. Barnabe D.R. Kaplan, F.D. Miller, Isolation of multipotent adult stem cells from the dermis of mammalian skin, Nat. Cell Biol. 3 (2001) 778e784. https://doi: 10. 1038/ncb0901-778.
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Please cite this article as: H. Ohnota et al., Skeletal muscle cells derived from mouse skin cultures, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.067