Laminin 521 maintains differentiation potential of mouse and human satellite cell-derived myoblasts during long-term culture expansion
© The Author(s). 2016
Received: 11 December 2015
Accepted: 1 December 2016
Published: 13 December 2016
Large-scale expansion of myogenic progenitors is necessary to support the development of high-throughput cellular assays in vitro and to advance genetic engineering approaches necessary to develop cellular therapies for rare muscle diseases. However, optimization has not been performed in order to maintain the differentiation capacity of myogenic cells undergoing long-term cell culture. Multiple extracellular matrices have been utilized for myogenic cell studies, but it remains unclear how different matrices influence long-term myogenic activity in culture. To address this challenge, we have evaluated multiple extracellular matrices in myogenic studies over long-term expansion.
We evaluated the consequence of propagating mouse and human myogenic stem cell progenitors on various extracellular matrices to determine if they could enhance long-term myogenic potential. For the first time reported, we comprehensively examine the effect of physiologically relevant laminins, laminin 211 and laminin 521, compared to traditionally utilized ECMs (e.g., laminin 111, gelatin, and Matrigel) to assess their capacity to preserve myogenic differentiation potential.
Laminin 521 supported increased proliferation in early phases of expansion and was the only substrate facilitating high-level fusion following eight passages in mouse myoblast cell cultures. In human myoblast cell cultures, laminin 521 supported increased proliferation during expansion and superior differentiation with myotube hypertrophy. Counterintuitively however, laminin 211, the native laminin isoform in resting skeletal muscle, resulted in low proliferation and poor differentiation in mouse and human cultures. Matrigel performed excellent in short-term mouse studies but showed high amounts of variability following long-term expansion.
These results demonstrate laminin 521 is a superior substrate for both short-term and long-term myogenic cell culture applications compared to other commonly utilized substrates. Since Matrigel cannot be used for clinical applications, we propose that laminin 521 could possibly be employed in the future to provide myoblasts for cellular therapy directed clinical studies.
KeywordsSatellite cell Myoblast expansion Stem cell Laminin 521 Cell therapy
Satellite cells are the major effector cell responsible for eliciting muscle regeneration. The satellite cell name was conferred based on its identification on the periphery of the myofiber characterized by very little cytoplasm and a prominent nucleus. In this position, satellite cells remain in an inactive quiescent state characterized and regulated by the transcription factor Pax7 . Once activated in response to muscle damage, satellite cells up-regulate the transcription factor MyoD and enter the cell cycle as transit-amplifying myoblasts . Once they reach sufficient numbers, myoblasts exit the cell cycle, increase expression of myogenin, and differentiate to form multinucleated myotubes through the process of cellular fusion [3, 4]. These myotubes form the building blocks for functional, contractile muscle fibers.
In vivo, satellite cell activity is regulated by what is referred to as the satellite cell “niche,” an extracellular environment between the muscle fiber and the basal lamina. In this location, extracellular matrix adhesion proteins influence satellite cell activity in their quiescent and activated states. Laminin 211, a heterotrimeric complex composed of α2, β1, and λ1 chains, is a primary component of the niche interacting with the satellite cell via the integrin α7β1 complex . Following activation, multiple extracellular matrix (ECM) proteins are induced in the skeletal muscle including fibronectin (FN), collagen 1, and collagen 3. Fibronectin binds to satellite cells by interaction with integrin α4β1 and integrin α5β1 complexes [6, 7]. Fibronectin is up-regulated in the niche acting via Wnt7a and functions to expand the stem cell pool of satellite cells and maintain stem cell numbers [8, 9]. Laminin α5 is an additional laminin isoform localized to regenerating myofibers that is up-regulated specifically during murine skeletal muscle regeneration and in human dystrophic muscle [10, 11]. However, functional experiments have not been performed to examine the satellite cell-laminin α5 relationship in detail. Although satellite cell laminin α5-integrin studies have not been examined previously, experimental studies have revealed that laminin α5 contains the greatest number of integrin-binding sites that include those for α3β1 (2×), αVβ3, α6β1, α6β4, and α7β1 [12–16].
The expansion of satellite cells and myoblasts is a critical component for both neuromuscular drug discovery and cell therapy applications. In both of these applications, methods are required to provide large-scale expansion of myoblast cells while maintaining high differentiation capacity and diminished spontaneous differentiation. Unfortunately however, comprehensive analysis of myoblasts has not been performed after extended passaging in vitro; the majority of experimental studies have been performed on satellite cells and myoblasts that were freshly isolated due to the challenges of expansion in vitro. Moreover, our attempts to perform large expansion of satellite cell and myoblasts have been challenged due to the progressive loss of differentiation over time in culture (unpublished data). This challenge may hinder scaling of myogenic cells for multiple applications including high-throughput screening and cell therapy. One potential reason for this phenomenon may be the use of biologically irrelevant cell substrates during myoblast expansion. For instance, myoblast are often cultured on a variety of ECM substrates including laminin 111, fibronectin, gelatin, collagen I, and Matrigel (MG) [17–21]. Although laminin is commonly utilized in myogenic cell culture, the form commonly employed is laminin 111 which is composed of α1, β1, and λ1 chains; laminin structure is illustrated in Additional file 1: Figure S1. Laminin 111 is not expressed in adult skeletal muscle and differs from laminin 211, the satellite cell niche component, containing a different alpha chain . In addition, other substrates mentioned, MG, gelatin, and collagen I are not associated with the satellite cell niche. Moreover, since Matrigel is animal-derived, myogenic cells cannot be expanded on Matrigel for clinical trials or future cell therapeutic applications. Nevertheless, while short-term testing has been performed previously, long-term myoblast expansion has not been compared between cellular substrates. In this study, we present both short-term and long-term expansion analysis of primary mouse and human myoblast cultures on both commonly used substrates laminin 111 and MG, as well as previously untested but biologically relevant laminin 211 (α2, β1, λ1) and laminin 511/521 (α5, β1, λ1; α5, β2, λ1) substrates.
Isolation and culture of murine satellite cells
Primary murine satellite cells were isolated from the tibialis anterior and quadriceps muscles from 12-week-old DBA/2J male mice. Dissected muscles were minced with scalpel blades and digested in DMEM/F12 (Life Technologies, 1:1 mixture) containing 2% collagenase II (Worthington Biochemicals) and 1.2 U/ml dispase (Worthington Biochemicals) with 2.5 mM CaCL2. Digestions were incubated at 37 °C for 1 h with trituration and mixing every 15 min. The cells were filtered through 100 and 40-μM cell strainers (BD). The cells were pelleted by centrifugation for 5 min at 300×g. The cells were resuspended in FACS staining buffer (DMEM/F12/0.5% BSA/25 mM HEPES) and distributed in 200-μl aliquots into staining tubes. The cells were blocked using anti-CD16/CD32 antibody (Ebioscience) at 1:100 dilution for 10 min on ice. The cells were stained with the following antibodies on ice for 30 min: CD31-FITC (1:50, Ebioscience, 390) CD45-FITC (1:50, Ebioscience, 30-F11), PDGFRα-BV421 (1:40, BD, APA5), Sca1-BV605 (1:100, BD, D7), and integrin α7 (1:400, Ablab, R2F2). The cells were washed twice with sort buffer (HBSS/0.5% BSA/25 mM HEPES) including centrifugation for 5 min at 300 g. Compensation controls were prepared using Ultracomp beads (Ebioscience). Single only bead controls were stained in 100 μl with 2 μl of each antibody for 15 min at room temperature. The beads were washed once with sort buffer and resuspended in sort buffer. Compensation was calculated using single-stained and unstained bead controls with FACS DIVA compensation wizard. Gating was determined by using fluorescence minus one plus isotype controls. Dead cells were gated out using propidium iodide (Life Technologies).
ECM coating and culture
Laminins including laminin 111, laminin 211, laminin 332, laminin 411, laminin 421, laminin 511, and laminin 521 are human recombinant isoforms obtained from Biolamina (Sweden). Laminins are diluted at a concentration of 10 μg/ml in HBSS with calcium and magnesium and coated overnight at 4 °C. Growth factor reduced Matrigel (Corning) was diluted 1:5 with DMEM/F12 media and thin coated by covering plastic, removing excess, and drying Matrigel for 20 min at 37 °C.
For initial characterization, the cells were plated at a density of 2000 cells per well in a 96-well format in DMEM/F12/20% FBS/Primocin (Live Technologies/InvivoGen) with 10 ng/ml mouse FGF-2 (R&D). Media were replaced after 5 days and refreshed every 3 days afterwards. To induce differentiation at day 8, the media were switched to differentiation media (DM), DMEM/F12/5% HI-HS/Primocin, and maintained until day 11.
For long-term growth, the cells were plated at a density of 10,000 cells per well in a 6-well format. The cells were grown in growth media as previously described and refreshed every 3–4 days with growth media and 10 ng/ml FGF-2. Cells were split using Accutase and maintained on the same substrate for six to eight passages. To assay differentiation, the cells were split using Accutase and seeded in a 96-well format at a density of 4000 cells per well. The cells were grown in GM for 5 days, and then switched to DM for an additional 5 days. For ECM substitution experiments, the cells were thawed, expanded, and passaged twice before analysis. At the second passage, the cells were transferred to a 96-well plate containing four of the ECM substrates (laminin 111, laminin 211, laminin 521, and MG). The cells were grown and differentiated similarly to our previously mentioned long-term growth procedure.
Immunocytochemistry and imaging
Immunostaining was performed in black Corning 96-well plates. For myosin heavy chain (MHC) staining, the cells were fixed using Cytoperm/Cytofix for 15 min at room temperature. The cells were rinsed twice and then subsequently blocked using 10% HI-HS/0.1% Triton for 1 h at room temperature. The cells were stained with MHC-Alexa488 antibody at 1:100 overnight at 4 °C. The cells were rinsed four times with PBS and stained with Hoechst to identify nuclei. Images were acquired using a ×10 objective on a Cellomics ArrayScan. Analysis was performed using the Cellomics HCS Studio Version 6.5 software analyzing MHC-positive cells containing two or more nuclei. The software algorithm used was the “myotube formation” package using dynamic thresholding, three sigma or isodata, for myotube identification.
For Pax7/MyoD staining, the cells were fixed using foxp3/ki67 nuclear fixation buffer (Ebioscience) for 15 min at room temperature. The cells were rinsed twice and blocked with Block Aid (Life Technologies) for 1 h at room temperature. Pax7 (1:50, R&D) and MyoD (1:50, 5F11, Millipore) were coincubated overnight at 4 °C in Block Aid. The cells were rinsed three times, and secondary antibodies (donkey anti-mouse Alexa488, donkey anti-rat Alexa647; 1:200) were incubated for 1 h at room temperature. The cells were rinsed four times and stained with Hoechst for nuclei identification. Images were acquired using a ×20 objective on a Cellomics ArrayScan. Analysis was performed using the nuclear colocalization algorithm (Cellomics HCS Studio 6.5) analyzing proportion of Pax7 or MyoD-positive nuclei.
Integrin FACS analysis
Passage 8 mouse myoblasts were split using Accutase, collected and centrifuged at 300×g for 5 min, and resuspended in FACS staining buffer. The cells were blocked with FC block (BD biosciences) at 1:50 for 10 min on ice. Afterwards, the cells were stained with the following PE-conjugated antibodies: integrin alpha1 (BD 562115) at 1:40, integrin alpha2 (Ebioscience 12-5971-81) at 1:40, integrin alpha3 (R&D FAP2787P) at 1:10, integrin alpha4 (Ebioscience 12-0492-81) at 1:20, integrin alpha5 (BD 553930) at 1:40, integrin alpha6 (Ebioscience 12-0495-81) at 1:200, integrin alpha7 (Ablab) at 1:200, integrin alphaV (Ebioscience 12-0512-82) at 1:50, integrin beta1 (Ebioscience 12-0291-81) at 1:20, integrin beta2 (Ebioscience 12-0181-81) at 1:20, integrin beta3 (Ebioscience 12-0611-81) at 1:40, integrin beta4 (R&D FAB4054P) at 1:20, and integrin beta5 (Ebioscience 12-0497-41) at 1:20. The cells were stained for 30 min on ice followed by two washes in FACS stain buffer. The cells were resuspended in 300 μl of FACS buffer and analyzed on the FACSAria II. Gating was set according to negative unstained and isotype control Rat IgG2a K-PE (Ebioscience 12-4321-81).
Human myogenic cell isolation
Post-mortem non-diseased skeletal muscle gracillis tissue was obtained through Asterand. The muscle was trimmed of fat and connective tissue. The tissue was minced for approximately 10 min. The tissue was digested using Collagenase II (Worthington Biochemicals) and Dispase (Worthington Biochemicals), for approximately 75 min at 37 °C. Digestions were performed in gentleMACS™ Dissociators. The tissue was pulsed every 15 min. Following digestion, the cells were strained through 100-, 70-, and 30-μM cells strainers (Miltenyi), respectively. The cells are resuspended in approximately 200 μl of MACS stain buffer (Miltenyi). The cells are stained for 1 h on ice with the following antibodies: CD11b-FITC (Miltenyi Biotec, Catalog Number: 130-081-201), CD31-FITC (Miltenyi Biotec, Catalog Number: 130-092-654), CD45-FITC (Miltenyi Biotec, Catalog Number: 130-080-202), CD34-APC (BD Biosciences, Catalog Number: 560940), and CD56-PE (Miltenyi Biotec, Catalog Number: 130-090-755). Afterwards, the cells were rinsed twice and subsequently incubated with anti-FITC microbeads (Miltenyi Biotec, 130-048-701) for 30 min on ice followed by two washes. Afterwards, the cells were passed through a Miltenyi magnetic depletion column. The column binds magnetically labeled FITC+ cells (CD31, CD45, CD11b) while allowing FITC− cells to flow through. The cells move passively through the column into a collection tube. Afterwards, the cells were centrifuged, resuspended in FACS buffer, and FACS sorted (FACS ARIA II) for CD56+, CD34−, CD45−, CD31−, and CD11b− cells. Myogenic cells were grown in growth media DMEM/F12 (Gibco) supplemented with 20% FBS (Gibco)/Primocin and 10 ng/ml human FGF2 (R&D). For differentiation of human cells, cells were seeded at a density of 16,000 cells per well in a 96-well format. After 3 days, half of the media was replaced with differentiation media consisting of DMEM/F12 supplemented with 5% HS-HI (Gibco) and Primocin. Afterwards, half of the media was replaced every other day until day 11 when the cells were fixed with Cytoperm/Cytofix (BD).
Statistics for multiple comparisons were conducted using one-way ANOVA with Bonferroni correction. Significance is annotated as less than .05 (*), less than .01 (**), less than .001 (***), and less than .0001 (****). All comparisons were conducted using laminin 521 as control. Significance for myotube nuclei distribution was determined using linear regression. Statistical calculations were conducted using Graphpad Prism 6.
ECM influences myogenic potential
In order to confirm laminin 521 activity on additional background strains over long-term passaging, we cultured C57BL/6J (BL6) and C57BL/10ScNJ (BL10) primary myoblasts to eight passages and assayed for differentiation performance. Similar to previously discussed findings, laminin 521 expanded myoblasts from BL6 and BL10 strains robustly differentiated into multinucleated myotubes at a significantly higher efficiency compared to laminin 111 and laminin 211 (Fig. 3a, b). MG performance varied substantially between BL6 and BL10 expanded cells; while BL6 cells exhibited poor differentiation, BL10 cells exhibited a similar amount of MHC area per nuclei compared to laminin 521. Multi-nucleation indexes were next calculated for BL6 and Bl10 cells on each of the four substrates including laminin 111, laminin 211, laminin 521, and MG. In BL6 cultures, laminin 521 cells show a significant shift of nuclei distribution towards myotubes containing five or more nuclei while laminin 111 and MG cultures were enriched for myotubes containing one to four nuclei in MHC-positive cells (Fig. 3c). In addition, laminin 111 and MG contained a significantly higher number of undifferentiated MHC-negative cells in BL6 cultures (Fig. 3c). In BL10 cultures, laminin 521 and MG performed similarly regarding myonuclei distribution although laminin 521 contained approximately 10% increased proportion of myotubes containing 10 or more nuclei and laminin 521 contained approximately 20% reduced proportions of MHC-negative cells (Fig. 3d). Overall, these results confirm laminin 521 promotes the most optimal and consistent differentiation of long-term passaged myoblasts across background strains compared to the other substrates tested. Moreover, while MG differentiated well during BL10 expansion, both Dba and BL6 expansions lacked differentiation suggesting that laminin 521 expansion may provide more consistent differentiation following expansion.
Initial expansion on ECM dictates differentiation on other substrates
Expanded myoblasts express similar myogenic markers across matrices
FACS profiling reveals variation in integrin expression
Integrin FACS profiling on passage 8 Dba cells
Mean fluorescent intensity on passage 8 Dba cells
Laminin 521 supports enhanced human myoblast expansion and differentiation
A vast amount of research has been performed on satellite cell/myoblast biology and myogenesis over the past 50 years. This work has yielded an enormous depth of knowledge encompassing wide number of molecular regulators ranging from transcription factors, signaling molecules, and ECM proteins. Despite this progress, there remains a significant disparity in work to support cell therapy and drug discovery efforts with satellite cells and myoblasts. A number of challenges have remained unsolved; how to scale up a relatively small number of skeletal muscle precursors into hundreds of millions or billions of cells for high-throughput drug screening or stem cell engraftment. In order to perform drug discovery in relevant models derived from stem cells isolated from patients, it is critical that the cells maintain the expansion and differentiation potential of the primary cell type. In addition, while Matrigel has become a very common ECM for satellite cell and myoblast culture, it is critical to establish an alternative ECM substrate which is animal-free and non-xenogeneic for cell therapy development and clinical trials. Given that target cells for assays addressing the majority of neuromuscular disease are multinucleated and differentiated myotubes and not transit-amplifying myogenic cells, it is critical that culture conditions support the ability of the expanded primary cells to differentiate effectively into myotubes. Many of the common reagents used for basic biology research, particularly ECM substrates, have not been tested in long-term experiments with multi-passage myogenic cell cultures. Because of this disparity, it is crucial to perform culture testing over the long term; doing so will help advance both basic muscle research and accelerate drug development advancement by broadly improving myogenic cell performance. Here, we provide the first comprehensive analysis detailing the proliferation and differentiation performance of different ECM’s on both mouse and human myogenic cells in short-term and long-term expansion.
The studies reported here demonstrate that the optimal ECM substrates for large-scale myogenic expansion are those that incorporate laminin α5, a previously underappreciated isoform of the laminin family in skeletal muscle. While laminin α5 is normally expressed during regeneration in both Ctx mouse models and DMD tissue, no study has been performed regarding its utility in in vitro myogenesis studies . Here, our mouse studies show laminin 521 (α5, β2, λ1) supports superior myoblast performance in vitro by facilitating impressive proliferation and maintenance of differentiation after eight passages and approximately a 5000-fold expansion. These results indicate that laminin 521 may be a more ideal substrate for large scale-up applications, by helping to minimize the loss in differentiation potential over time. In our studies, mouse myogenic cells expanded on laminin 111 progressively lost their ability to up-regulate MHC while MG-expanded cells entered a state whereby they entered a myocyte stage expressing MHC but then fail to fuse. In addition to the mouse study presented here, we have performed an analysis of freshly isolated mdx/BL10 cells, and we observed a very similar pattern with laminin 521 outperforming laminin 111, laminin 211, and MG in differentiation (Additional file 4: Figure S4). Furthermore, we have encountered difficulty in expanding mdx/BL10 cells on laminin 111 while we have not encountered these issues on laminin 521 (data not shown). Importantly, our findings translate to human myogenic cell culture as our human cells perform optimally on laminin 521 showing superior proliferation and differentiation to all other substrates tested. Taken together, our results demonstrate laminin 521 as an optimal substrate for myoblast expansion while demonstrating translatability across several mouse backgrounds (Dba/2J, C57/BL10, C57/BL6), human cells, and disease states (mdx/BL10).
While our results strongly support laminin 521 as a superior myoblast culture matrix, additional studies will be required to further evaluate the effects of laminin 521. Particularly, additional study will need to be conducted with additional human samples to confirm that laminin 521 is broadly beneficial across multiple human myogenic progenitors. We have directly measured the amount of protein coating among the different substrates used in this study and find only a minimal difference between the different laminins and gelatin used in our study (Additional file 5: Figure S5). Matrigel resulted in an increased amount of protein compared to other substrates (Additional file 5: Figure S5). Despite these differences in coating amounts, we do not observe significant differences in cellular adherence in either freshly isolated cells (Additional file 6: Figure S6A) or long-term passaged cells (Additional file 6: Figure S6B). We have observed however that the presence of high concentrations of calcium and magnesium, as found in HBSS and PBS containing calcium and magnesium, are critical for laminin coating function. The inclusion of calcium and magnesium during laminin coating is not commonly included in many of the commercial vendor protocols, despite the critical roles of calcium and magnesium in laminin signaling.
The observed benefits of laminin 521 are likely due to the larger number of integrin binding sights on laminin α5 including unique binding sites for integrin α3, αV, and α6β4 not present in other laminin isoforms [12–15]. Laminin 521 can therefore be seen as a richer substrate capable of interacting with six integrin binding sites (α3β1 (2×), αVβ3, α6β1, α6β4, α7β1), compared to four binding sites in laminin 111 and MG (α1β1, α2β1, α6β1, α7β1) [12, 15]. The converse also may be true for laminin 211; it may fail to stimulate a high level of proliferation due to the presence of only three integrin-binding sites (α1β1, α2β1, α7β1) . Our studies have shown that a greater proportion of cells express integrin α3 on laminin 521 and MG expanded cells compared to all other substrates (Table 1). Multiple studies show that integrin α3 is a critical component during myogenic cell differentiation [23, 24]. However, our results also suggest that integrin α3-laminin α5 interaction may not be critical for differentiation since cells moved from laminin 521 to other substrates maintain the ability to differentiate, even on the minimalistic laminin 211. While our studies show MG expanded cells turn on the differentiation program through up-regulation of MHC, there is a deficit in myoblast fusion in long-term cultures. We observed an increased expression level of integrin α7 in both laminin 521 and MG cultures (Fig. 6). Since integrin α7 increases myoblast mobility and is dramatically increased during myogenic differentiation, increased expression of integrin α7 in our cultures is a possible mechanism promoting enhanced differentiation. These findings may suggest that expression of integrin α3 and high expression of integrin α7 assist cells in initiating the differentiation program but additional mechanisms are likely responsible for the difference in myotube formation between cells expanded on laminin 521 and MG. Other key differences observed included decreased expression level of integrin α5 on laminin 521 expanded cells and increased proportions of cells expressing integrin β2 and integrin β4 in MG cultures. Studies have shown expression of integrin α5 regulates myogenesis by favoring expansion and inhibiting differentiation, thus reduced integrin α5 on laminin 521 expanded cells may contribute to the maintenance of differentiation in the long term [11, 25, 26]. Overall, while our studies reveal multiple differences in the expression of integrins following long-term culture, however, there appears to be some long-lasting effects of growth on each of the ECM substrates that is poorly understood and cannot be explained merely by mapping integrin-ECM binding interactions. It will be critical in future experiments to use integrin inhibition to determine which integrins are critical for maintaining differentiation ability in myogenic cultures. While we acknowledge our integrin study is limited due to the inclusion of only the Dba mouse strain, most importantly, our findings encourage future investigation of integrin expression across backgrounds, species, and passage time points in culture. It will be crucial to expand on these findings as we expect integrin expression to vary between biological samples and conditions.
This study significantly demonstrates that various substrates are not created equal when they are employed for the expansion of myoblasts in vitro. While the performance of laminin 521 is impressive, there may be room for improvement by combining additional ECMs with laminin 521. First, it will be important to analyze the combination of laminin 521 with laminin 111 and laminin 211 to determine if there is any potential synergism. While laminin 521 fared the best in differentiation after scale-up, spontaneous differentiation was still present at a low level. Collagen VI has been described as a critical mediator of stem cell renewal in the satellite cell niche suggesting it may prevent progression into differentiation . Considering this, collagen VI in combination with laminin 521 may reduce the spontaneous differentiation during expansion, at which time collagen VI could be removed to induce differentiation. ECM stiffness is a critical regulator balancing myogenesis towards proliferation or differentiation [28–32]. Combining laminin 521 with MG or a hydrogel may provide increased performance in both proliferation and/or differentiation by providing a method to customize stiffness of the substrate to optimize both proliferation and differentiation conditions. It will be important in future studies to titrate laminin 521, which will affect both substrate stiffness and geometry of the laminin matrix, both of which may significantly affect proliferation and differentiation.
Two of the most popular myogenic matrices routinely employed in stem cell biology are MG and laminin 111; however, neither of these substrates is endogenous to the satellite cell niche. Additionally, MG is a highly variable substrate isolated from mouse EHS tumors containing over 14,000 peptides resulting in experimental variability and decreased reproducibility . In fact, even growth factor reduced MG, as used in this study, contains over 9000 peptides and significant variation as high as 50% from batch to batch has been observed which makes cell culture performance inconsistent . The laminin α1 chain contained in both MG and laminin 111 is absent from adult skeletal muscle in both resting and regenerating conditions and is only expressed in a small stage of developmental myogenesis [10, 34, 35]. This discrepancy has by and large gone unverified with the assumption that laminin 111 and laminin 211 share redundant functionality. For instance, pre-clinical animal studies have been performed injecting laminin 111 into dystrophic mice despite the lack of α1 in the muscle . However, these results are controversial since follow-up transgenic laminin 111 studies show no benefit in the mdx model . Our results show for the first time that there are significant functional differences between the laminin α1 and α2 chains. While α1 supports moderate proliferation and differentiation in our studies, α2 tends to limit proliferation and supports differentiation poorly. Considering this in combination with our results, laminin 511 or laminin 521 supplementation/overexpression may be superior therapeutically for muscular dystrophies including DMD since they contain the beneficial integrin α7 binding site similar to laminin 211 and laminin 111, but laminin 511 and laminin 521 may additionally boost the regenerative response. One caveat for potential intramuscular laminin α5 injection or overexpression is that laminin 521 is normally expressed at neuromuscular junctions while laminin 511 surrounds regenerating myofibers; thus, in vivo comparison studies between laminin 511 and laminin 521 will be crucial .
The studies presented here suggest that the laminin 521 findings may be translatable to additional technologies including cell therapy-engraftment technology and iPS skeletal muscle differentiation. In recent years, cell therapy for the muscular dystrophies has lost favor due to the inability to generate sufficient numbers of engraftment-capable myoblasts for transplantation. In the early 90s, while cell therapy was a highly promising technology due to newly developed myoblast cell culture, human myoblast trials were a failure due to the inability of the in vitro propagated cells to efficiently engraft or provide functional muscle strength recovery [38–41]. Subsequent mouse studies demonstrated that freshly isolated myogenic cells are highly efficient at engrafting, but over time, the culture engraftment potential dramatically falls . Historically, cell therapy and engraftment experiments commonly utilize gelatin, MG, or laminin 111 as expansion substrates [21, 43–46]. As demonstrated in the results presented in this report, these are not the best substrates for in vitro scale up of pre-implanted cells. Our studies suggest that laminin 521 may provide a better option for the scale-up of cells for cell therapy since we demonstrate that laminin 521 is the only substrate in our studies capable of maintaining differentiation over an extended expansion period. Studies are currently underway to analyze the engraftment potential of mouse myoblasts expanded on varying substrates including laminin 521. The differentiation of skeletal muscle differentiation from iPSCs is currently not well defined even though a number of reports have been published using protocols with varying techniques and ECM substrates [47–50]. Since laminin α2, laminin α4, and laminin α5 are expressed during muscle development, it will be important to test these substrates in iPSC studies to determine the optimal ECM induction substrate [10, 11]. Additionally, while multiple protocols exist to produce iPSC-derived myogenic cells, iPSC-generated skeletal muscle cell expansion has not been well characterized examining performance after large scale-up of the generated cells. In this avenue, laminin 521 may provide a significant advantage in maintaining iPSC myogenic cells in a differentiation-competent state while the cells are expanded into large numbers for drug screening and cell therapy applications.
Laminin 521 represents a new high performance, biologically relevant matrix for superior muscle cell performance in vitro. Laminin 521 supports generation of larger myotubes and higher amounts of nuclei per myotube compared to all other substrates with the exception of Matrigel. However, while laminin 521 matches the performance of Matrigel, it also provides more consistent and reliable differentiation over long-term culture. Particularly, laminin 521 represents an excellent substrate for cell therapy development and clinical trials due to its human origin; Matrigel on the other hand is not compatible with clinical trials or cell therapy applications in humans. Laminin 521 appears to increase differentiation potentially without altering the traditional Pax7/MyoD paradigm; it is likely that laminin 521 is providing novel cell regulation not previously appreciated. It may be beneficial to integrate laminin 521 into currently established techniques in muscle biology research to achieve synergism with other matrix proteins or technologies. In conclusion, laminin 521 extends the use of isolated primary cells and is likely to enhance many applications requiring large growth expansion such as drug discovery, genetic engineering, and cell therapy.
Fluorescent-activated cell sorting
Growth factor reduced Matrigel
Induced pluripotent stem cell
Myosin heavy chain
The project was supported by the MDA Bridge to Industry Grant (#293738 to CMP, REA, and PRA). We would like to thank Ken Wertman for scientific guidance and administrative support.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
CP conceived the study, carried out the cellular experiments, analyzed the data, and drafted the manuscript. VB, RA, and PA conceived the study, participated in its design and coordination, and helped to draft the manuscript. JP conceived the study and carried out the cellular experiments. EP carried out the immunohistological studies and helped to draft the manuscript. MP participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
All studies involving mice were conducted in accordance with the IACUC protocols approved by the University of Arizona IACUC (11-268). The human cells used in the submission were obtained through a commercial source, Asterand Bioscience. The cells were obtained with approval of the FederalWide Assurance (# IRB00003411).
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