|Year : 2017 | Volume
| Issue : 1 | Page : 16-22
Isolation, in-vitro expansion, and characterization of human muscle satellite cells from the rectus abdominis muscle
David Livingstone1, Albert A Kota2, Sanjay K Chilbule1, Karthikeyan Rajagopal1, Sukria Nayak2, Vrisha Madhuri1
1 Department of Orthopaedics, Paediatric Orthopaedics Unit, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Surgery, Unit IV, Christian Medical College, Vellore, Tamil Nadu, India
|Date of Web Publication||17-Feb-2017|
Paediatric Orthopaedics Unit, Christian Medical College, Vellore - 632 009, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Introduction: Satellite cells are a resident population of stem cells beneath the basal lamina of mature skeletal muscle fibers. Their capacity to regenerate muscle makes them a potentially ideal source for human cell therapy with respect to muscle-related diseases such as urinary and fecal incontinence, and others. In this study, we describe a protocol to isolate, expand in-vitro, and characterize human muscle satellite cells from the rectus abdominis muscle. Materials and Methods: Muscle biopsies from human donors were harvested, digested using collagenase type II, and then plated on extracellular matrix-coated plates. Results: Immunocytochemistry revealed that satellite cells on day 8 were 70–80% Pax7 positive; in contrast, cells expanded until day 12 showed 50–75% positivity for Pax7. The real-time polymerase chain reaction for day 8 culture indicated four-fold increase in Pax3 and Pax7 gene expression, four-fold increase in MyoD gene expression, and five-fold increase in Myf5 gene expression. Conclusion: These findings suggest that satellite cells can be cultured until day 8 for translational purposes. The protocol described here is modest, operational, and reproducible and involves only basic cell culture equipment.
Keywords: Cell therapy, human skeletal muscle, myoblast, satellite cells, sphincter injuries, tissue regeneration
|How to cite this article:|
Livingstone D, Kota AA, Chilbule SK, Rajagopal K, Nayak S, Madhuri V. Isolation, in-vitro expansion, and characterization of human muscle satellite cells from the rectus abdominis muscle. Paediatr Orthop Relat Sci 2017;3:16-22
|How to cite this URL:|
Livingstone D, Kota AA, Chilbule SK, Rajagopal K, Nayak S, Madhuri V. Isolation, in-vitro expansion, and characterization of human muscle satellite cells from the rectus abdominis muscle. Paediatr Orthop Relat Sci [serial online] 2017 [cited 2018 May 27];3:16-22. Available from: http://www.pors.co.in/text.asp?2017/3/1/16/200295
| Introduction|| |
A characteristic feature of working skeletal muscle is its ability to regenerate; when injured, a series of cellular reactions is triggered, resulting in the restoration of structure. Even after total disruption, skeletal muscle still has the ability to regenerate into a fully functional tissue. Satellite cells are known for their capacity for myogenic differentiation and also regarded as muscle-specific committed progenitors that are responsible for this regenerative potential and also the postnatal maintenance and growth of skeletal muscle. Mauro described satellite cells as those wedged between the plasma membrane of the myofiber and basement membrane as seen under the electron microscope. The ability of these cells to self-renew has been described, and they are also referred as muscle stem cells.,, In normal muscle regeneration, myogenic precursor cells, the descendants of activated satellite cells, undergo cell division and fuse with new or existing myofibers.,,, Severely injured muscles can be replaced with biological alternatives by tissue engineering. Muscle cell progenitors, that is, satellite cells, play a key role in such skeletal tissue engineering.,
Satellite cells were initially studied using electron microscopy, but with the advent of newer techniques of isolation, culture, characterization, and immunocytochemistry, it is now possible to identify them under the light microscope., Different protocols have been tried for isolation, in-vitro expansion, and immunostaining of myofibers and associated satellite cells from rodents. These include multiple myofiber isolation, the single myofiber explant technique, and fluorescent-activated cell sorting.
The most important characterization and immunocytochemistry (ICC) marker is the paired box transcription factor 7 (Pax7) that is actively expressed by satellite cells. In addition, Paired box transcription factor 3 (Pax3), Myogenic differentiation 1 (MyoD), Myogenic factor 5 (Myf5), Myocyte nuclear factor (MNF), c-Met, Syndecan, M-Cadherin, and Neural cell adhesion molecule (NCAM) are other markers expressed at different stages of satellite cell progression and can be used for characterization.,, Paired box protein Pax7 plays a role in the regulation of muscle precursor cell proliferation and is a marker for human muscle satellite cells. Myf5 and MyoD proteins that belong to the family of myogenic regulatory factors are the first to be expressed by activated satellite cells.
Although the potential of satellite cells has been widely studied in various animal models, similar research on human satellite cells has thus far been limited. Translational use of satellite cells to develop new skeletal muscle for clinical application has been promising and there is tremendous potential in various therapies., The use of satellite cells for treatment of muscular dystrophy has been postulated and studied in detail. Muscle-derived cells have been investigated for debilitating conditions such as urinary incontinence and fecal sphincter injuries.,, Human transplantation for sphincter injuries has previously been performed using cultured cells from muscle biopsies wherein cells with 0–9.1% staining for MyoD1, 49.5% for α-sarcomeric actin, and 32.7% for desmin were transplanted. The use of muscle-derived stem cells for the treatment of volumetric muscle loss, in tissue engineering applications, and in orthopaedic surgery is being explored at many centers. In this study, we describe our protocol for human muscle satellite cell isolation from the rectus abdominis, in-vitro expansion of the isolated satellite cells by enzymatic digestion, and characterization of cultured cells using immunocytochemistry and real-time polymerase chain reaction (RT-PCR).
| Materials and Methods|| |
This study was approved by the Institutional Review Board (IRB Min. No. 8221) and therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki, its later amendments, and the ethical guidelines of our country. The work was carried out at the Center for Stem Cell Research (a unit of inStem, Bengaluru). Four rectus abdominis muscle samples were obtained after informed consent from human donors − two men and two women 30–60 years of age − who were undergoing various reconstructive surgeries. Isolation, processing of the samples, and culture were performed under sterile conditions.
Precoating of culture flasks and coverslips
Culture flasks and coverslips were coated with extra cellular matrix (ECM; Sigma-Aldrich, St. Louis, Missouri, USA). Solubilized ECM gel was diluted 1:55 with ice-cold Dulbecco’s modified Eagle’s medium with F12 supplement (DMEM/F12; Sigma-Aldrich, St. Louis, Missouri, USA). T25 flasks were coated with 200 μL of diluted ECM (176.3 μg/mL concentration). Coverslips were coated with 40 μL of diluted ECM of same concentration and incubated overnight in a CO2 incubator at 37°C.
Satellite cell isolation
Muscle biopsies of rectus abdominis were collected in ice-cold DMEM/F12 supplemented with penicillin/streptomycin 50 units/mL (Sigma-Aldrich, St. Louis, Missouri, USA). The samples were processed within 2 h from the time of collection. They were washed in Hank’s Balanced Salt Solution (HBSS; Sigma-Aldrich, St. Louis, Missouri, USA), stripped of any visible connective tissue and fat, weighed, and minced into fine pieces of approximately 1 mm2. Enzymatic digestion of the pieces was performed in DMEM containing 3 mg/mL collagenase type II (Worthington, Lakewood, New Jersey, USA) at 37°C for 40 min with intermittent mixing. The digested samples were centrifuged at 1600 rpm and suspended in 20 mL of HBSS with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, Missouri, USA). The suspensions were successively filtered through 100 μm and 40 μm cell strainers (Corning Inc., Teterboro, New York, USA). The filtrates were centrifuged at 1600 rpm for 10 min at 25°C after which the pellets were resuspended in proliferating medium containing DMEM/F12, 10% FBS, and 50 units/mL penicillin/streptomycin.
Satellite cell culture
Immediately after isolation, cell suspensions were plated on T-25 culture flasks precoated with ECM in 1:55 dilution. On day 3, the medium was changed. All debris and unattached cells were removed during this process. Supernatants were collected and seeded on to coverslips for immunocytochemistry. Fresh DMEM/F12 along with 10% FBS was added to the flask and refreshed thrice a week. When cells reached 90–100% confluence, they were enzymatically harvested using 0.25% trypsin/EDTA (Sigma-Aldrich, St. Louis, Missouri, USA), counted with Neubauer chamber and seeded (15,000 cells/coverslip) on ECM-precoated coverslips for immunocytochemical analysis.
Coverslips were washed with phosphate-buffered saline (PBS) and then fixed with 2% paraformaldehyde for 15 min. The fixed cells were washed thrice with PBS. Cells were incubated for 30 min with 0.5% Triton X-100 in PBS and endogenous peroxidase activity inhibited by incubating with 2% H2O2 for 15 min in dark conditions. The cells were washed thrice with PBS and nonspecific-binding sites were blocked with 1% bovine serum albumin (Sigma-Aldrich, St. Louis, Missouri, USA) in PBS (blocking buffer) for 1 h at room temperature; 40 μl of primary antibody in blocking buffer was added to the coverslips in the following concentration − Pax7 (R&D Systems, Minneapolis, Minnesota, USA) 1:50 and incubated at room temperature for 1 h in humid conditions. The coverslips were washed with PBS-Tween20 (PBS-T) thrice at 5-min intervals; 40 μl of secondary antibody [Horseradish peroxidase (HRP)-conjugated immunoglobulin G 1:100 dilutions] prepared in blocking buffer was added and incubated for 1 h at room temperature in humid conditions. Secondary antibody was washed thrice with PBS-T. Forty microliter of 3,3′-diaminobenzidine was added to the coverslips and incubated for 15 min in the dark, followed by a wash with PBS (thrice with 5 min per wash). The cells were counterstained with hematoxylin and examined under a microscope. Percentage of positive cells was calculated using the formula “number of positive cells within the frame/total number of cells in that frame”. Average percentage from five images taken from five different areas of the coverslip was obtained.
RNA isolation, reverse transcription, and RT-PCR
Total RNA was extracted from muscle satellite cell using TRI reagent by following manufacturers protocol (Sigma-Aldrich, St. Louis, US). It was then reverse transcribed using reverse transcriptase commercial kit (QuantiTect® Reverse Transcription; Qiagen, Hilden, Germany) as per the manufacturer’s instructions. The obtained complementary DNA was analyzed for expression of Pax3, Pax7, Myf5, and MyoD. RT-PCR (Applied Biosystems 7500, Applied biosystems, California, US) was carried out with commercial kit (SYBR® Premix Ex Taq™ II, Takara, Shiga, Japan). The primers and the sequences obtained from qPrimerDepot database are listed in [Table 1]. All reactions were run in triplicate. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the reference gene for normalization and the real time was quantitated against the day 3 sample using the comparative threshold method.
Cryopreservation and reviving of cells was carried out in the same manner as a previously published protocol by us.
| Results|| |
Digested cells were seeded on ECM-coated plates. The cells were allowed to expand in DMEM/F12 culture and harvested when near confluence as seen in [Figure 1]a and [Figure 1]b. Samples with a mean weight of 0.802 ± 0.39 g obtained from four patients with a mean age of 49.75 ± 21.32 years yielded a cell count of 1.74 ± 0.97 × 106 when expanded for 9.25 ± 1.89 mean days [Table 2].
|Figure 1: (a) Inverted phase-contrast image of cultured human skeletal muscle satellite on different days at 10× magnification; human skeletal muscle satellite cells on day 8 with elongated satellite cell-like phenotype. (b) Human skeletal muscle satellite cells confluent culture on day 12 with changes in morphology of some cells|
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Cells in day 3 culture supernatant were seeded on to coverslips to check for the phenotype of unattached cells. The cells in the supernatant were positive for Pax7 as seen in [Figure 2], indicating that they were muscle satellite cells. The cultures expanded till confluence (day 8) analyzed by immunocytochemistry revealed 70–80% Pax7 positive cells as seen in [Figure 3]. Cells that were expanded for 12 days showed 50-75% reactivity for Pax7 as seen in [Figure 4].
|Figure 2: Light microscopic image of immunocytochemistry analysis of cells on day 3 collected from supernatant at 40× magnification; stained for Pax7 antibody (arrowhead), a satellite cell marker|
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|Figure 3: Light microscopic image of immunocytochemistry analysis of cells collected on day 8 at 40× magnification: 70–80% of the nuclei stained for Pax7 antibody(arrowhead), a satellite cell marker|
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|Figure 4: Light microscopic image of immunocytochemistry analysis of cells collected on day 12 at 40× magnification: 50–75% of the nuclei stained for Pax7 antibody(arrowhead), a satellite cell marker|
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Unadhered cells harvested on day 3 were positive for Pax7, which suggests that they still were satellite cells in the early proliferating conditions. However, when muscle satellite cells were cultured for a longer period, a change in their morphology was also observed, as seen in [Figure 1]b.
Quantification of gene expression using day 8 cultured muscle sample also confirmed the results of ICC showing that the expression of Pax3 increased 4.16-fold, 4.17-fold increase for Pax7, 5.14-fold increase for Myf5, and 4.04-fold increase for MyoD, as seen in [Figure 5] when compared with day 3 sample.
|Figure 5: Relative messenger RNA (mRNA) expression levels of genes in human skeletal muscle satellite cells analyzed by RT-PCR; error bar represents standard deviation. Increase in the levels of mRNA expression of satellite cell markers, such as Pax3 a quiescent state marker, Pax7 quiescent and proliferative state marker, Myf5 proliferative marker, and MyoD differentiated marker. RT-PCR: real-time polymerase chain reaction|
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Cryopreservation and revival
The cryopreserved cells were revived after a month and when the number of viable cells were calculated, the average viable cell yield was 91.44%, ±6.19 standard deviation.
| Discussion|| |
Posttraumatic or degenerative urinary or fecal incontinence, vocal cord injury, and facial or limb muscle damage are potential targets for stem cell therapy., This idea has been explored in many studies using different cell types. Mesoangioblasts, CD133+ cells, muscle interstitial cells, mesenchymal stem cells, endothelial progenitor cells, hematopoietic stem cells, multipotent adult progenitor cells, side population stem cells, and adipose-derived stem cells have all been previously attempted as possible triggers for skeletal muscle regeneration in many in-vitro and in-vivo experiments.
Recently, satellite cells have been recognized as a possible solution for muscle regeneration., To translate satellite cells into human clinical trials, a good manufacturing protocol needs to be established using human cells. The method of cell isolation has to follow standard operating protocols that ensure quality assurance for the final product. Various studies have described the isolation and culture of satellite cells on myofibers. Direct isolation and live muscle explant techniques are also available in animal models. However, studies on isolation of satellite cells from human subjects are scanty and mainly focused on characterization rather than clinical translation. Scott et al. isolated CD56+-positive cells from cadaveric muscle. Boldrin and Morgan have described a method of identification of human satellite cells but did not isolate them. Sharifiaghdas et al. reported the isolation and characterization of human adult muscle stem cells using explant culture. In their study, they obtained very low cell yield because of dysfunctional sorting, triple digestion, and preplating. In our study, by single digestion with collagenase, we have achieved a mean yield of 1.74 ± 0.97 × 106 cells near confluence. In our study, we found Pax7 to be a specific and reproducible marker for satellite cells. We had earlier tried markers such as CD56, CD29, CD44, and CD34 described in other studies but did not use them in this study as Pax7 was consistent and reproducibly expressed in satellite cells.
Muscle satellite cells have the ability to differentiate into myoblasts but, like most stem cells, they also show self-renewability. For clinical translation, it is important to provide a replenishable source; hence, satellite cells have an advantage over myoblast transplant. Human translation of myoblasts and muscle-derived cells has already been attempted with considerable success in some experiments. Frudinger et al. in 2010 reported the safe human translation of autologous muscle-derived myoblast transplant and objective improvement in the overall quality of life in fecal incontinence because of obstetric trauma as assessed by the Wexner incontinence score. Gerullis et al. described the injection of transurethral autologous muscle-derived cells in the urethral sphincter; 54% of their patients showed improvement in their symptoms.
Quantitative analysis of Pax7, Myf5, and MyoD has shown that cultured cells in this experiment expressed both satellite cell markers (Pax7 and Myf5) in the initial proliferative period (up to day 3). By the late proliferative period (at day 8), these cells started showing reactivity for myoblastic markers such as MyoD, myogenin, and desmin. However, expression of Pax7 and Myf5 was still retained suggesting that satellite cells, at this stage, continue to self-renew as well as generate myoblasts. Morphological analysis revealed the typical muscle satellite cell appearance of elongated spindle-shaped mononucleated cells on day 3, whereas the additional presence of myotube-like multinucleated cells was also noticed on day 12 culture. On immunocytochemistry, the cultured satellite cells on day 3 showed expression of Pax7 in the nucleus. Similar observations have been showed in past by Zammit et al. using mouse satellite cells that take a divergent fate to give rise to MyoD+ and Pax7+ cells beyond 48 h. The above observations suggest that satellite cells qualify for clinical translation as they provide differentiating cells as well as progenitor cells, and that transplantation of these cells should be carried out around day 8.
Components of the satellite cell niche are the host muscle fiber, basal lamina, and microvasculature. The anatomical location of satellite cells between the basal lamina and muscle fiber has been proved beyond doubt. Christov et al. have shown that 68% of human and 82% of mice satellite cells are located within 5 mm of neighboring capillaries or vascular endothelial cells. The muscle fiber provides mechanical, electrical, and chemical signals whereas the basal lamina acts as an anchor. These anatomical features of the satellite cell niche suggest that a combination of signals from the host muscle fiber, vasculature, and ECM govern the quiescence, activation, and proliferation of satellite cells. Coculturing and transplanting of satellite cells with endothelial cells thus has the potential to improve the outcome of human translation of satellite cells. Christov et al. have also shown that endothelial cell coculture enhances satellite cell growth through insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor BB (PDGF-BB), and vascular endothelial growth factor (VEGF). One of the limitations of using ECM is its immunogenicity because of the animal origin. Akhyari et al., in their review of extracellular matrix for myocardial tissue engineering, describe immunogenicity as a secondary issue. If cells are washed with PBS before transplantation (which removes extracellular matrix completely), the issue of immunogenicity can be solved.Another factor that could improve the outcome of satellite cell transplant is the use of scaffolds. The characteristic features of a good scaffold for muscle regeneration are biocompatibility, biodegradability, and porosity. An ideal scaffold should be similar to extracellular matrix of skeletal muscle with perfect balance of stability and elasticity. Many scaffolds have been used in vitro and in vivo for skeletal muscle regeneration in past including synthetic materials such as silicones and polymers (eg, polylactic acid, polyglycolic acid, and polycaprolactone), and their copolymers (eg, polylactide-co-glycolide) and natural materials such as collagen, fibronectin, elastin, fibrin, hyaluronan, and laminin.
Shock wave therapy has been shown to increase vascularity in and around tissues. Increase in VEGF levels in endothelial progenitor cells following shock wave therapy has been documented. Wang et al. have shown enhanced neovascularization and presence of myofibroblasts at the Achille’s tendon insertion following shock wave therapy in a canine model. It is therefore possible that coculturing of satellite cells and endothelial cells accompanied by external shock waves may provide a better niche for transplanted satellite cells.
| Conclusion|| |
This study describes a protocol for the isolation, culture, and characterization of human muscle satellite cells, which, we believe, is immediately translatable to human clinical trials. The protocol is easy, inexpensive, and reproducible and requires only basic cell culture equipment.
The authors would like to thank Dr. Noel Walter for help with revision of the manuscript, and Dr. Henrik Daa Schrøder and Dr. Jeeva Sellathurai for their valuable suggestions. The authors would also like to acknowledge the Center for Stem Cell Research (a unit of inStem, Bengaluru) for the facilities provided to carry out the bench work.
Financial support and sponsorship
This study was funded by the Institute’s fluid grant and partly by Department of Biotechnology (BT/IN/DENMARK/02/PDN/2011), Government of India.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]