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Laminin-α2 related congenital muscular dystrophy (LAMA2-CMD) is a fatal muscle disease caused by mutations in the LAMA2 gene. Laminin-α2 is critical for the formation of laminin-211 and -221 heterotrimers in the muscle basal lamina. LAMA2-CMD patients exhibit hypotonia from birth and progressive muscle loss that results in developmental delay, confinement to a wheelchair, respiratory insufficiency and premature death. There is currently no cure or effective treatment for LAMA2-CMD. Several studies have shown laminin-111 can serve as an effective protein-replacement therapy for LAMA2-CMD. Studies have demonstrated early treatment with laminin-111 protein results in an increase in life expectancy and improvements in muscle pathology and function. Since LAMA2-CMD patients are often diagnosed after advanced disease, it is unclear if laminin-111 protein therapy at an advanced stage of the disease can have beneficial outcomes. In this study, we tested the efficacy of laminin-111 protein therapy after disease onset in a mouse model of LAMA2-CMD. Our results showed laminin-111 treatment after muscle disease onset increased life expectancy, promoted muscle growth and increased muscle stiffness. Together these studies indicate laminin-111 protein therapy either early or late in the disease process could serve as an effective protein replacement therapy for LAMA2-CMD.

Volumetric muscle loss (VML) is characterized by a critical loss of muscle tissue that is accompanied by severe functional impairment and often long-term disability. Clinical therapies currently used in the treatment of VML are ineffective at regenerating lost muscle and restoring function. In this study, we used a novel hydrogel composed of fibrinogen and laminin-111, to promote regeneration and recovery of VML-traumatized muscle. Our previous study showed that these hydrogels exhibit a fibrous structure and Young's modulus of ∼2 kPa, while supporting C(2)C(12) myoblast proliferation, myogenic marker expression, and proregenerative growth factor secretion in vitro. In a murine model of VML injury, the implantation of these hydrogels showed significant improvements in muscle weights and heightened infiltration of endothelial, hematopoietic, and immune cells at 2 weeks postinjury compared to the untreated muscles. At 4 weeks postinjury, the hydrogel-treated muscle showed increased myogenic activity, acetylcholine receptor clustering, and induction of the anti-inflammatory M2-like macrophage phenotype. However, improvements in muscle weights and force production were not observed at 4 weeks. An adjunct therapy such as physical rehabilitation or codelivery of stem cells may be required for the effective treatment of VML. Overall, these results will inform and guide the development of a successful tissue engineering strategy for the regeneration of skeletal muscle following trauma. IMPACT STATEMENT: Extremity injuries make up the most common survivable injuries in vehicular accidents and modern military conflicts. A majority of these injuries involve volumetric muscle loss (VML). The potential for donor site morbidity may limit the clinical use of autologous muscle grafts for VML injuries. Treatments that can improve the regeneration of functional muscle tissue are critically needed to improve limb salvage and reduce the rate of delayed amputations. The development of a laminin-111-enriched fibrin hydrogel will offer a potentially transformative and “off-the-shelf” clinically relevant therapy for functional skeletal muscle regeneration.

Volumetric muscle loss (VML) is the surgical or traumatic loss of skeletal muscle which can cause loss of limb function or permanent disability. VML injuries overwhelms the endogenous regenerative capacity of skeletal muscle and results in poor functional healing outcomes. Currently, there are no approved tissue engineering treatments for VML injuries. In this study, fibrin hydrogels enriched with laminin-111 (50-450 µg/ml) were used for the treatment of VML of the tibialis anterior muscle in a rat model. Treatment with fibrin hydrogel containing 450 µg/ml of laminin-111 (FBN450) improved muscle regeneration following VML injury. FBN450 hydrogel treatment increased the relative proportion of contractile to fibrotic tissue as indicated by the myosin: collagen ratio on day 28 post-VML injury. FBN450 hydrogels also enhanced myogenic protein expression and increased the quantity of small to medium size myofibers (500-2000 µm2) as well as innervated myofibers. Improved contractile tissue deposition due to FBN450 hydrogel treatment resulted in a significant improvement (~60%) in torque production at day 28 post-injury. Taken together, these results suggest that the acellular FBN450 hydrogels provide a promising therapeutic strategy for VML that is worthy of further investigation.

Prostate cancer is a life-threatening condition worldwide. As the tumor progresses, smooth muscle cells (SMCs) become atrophic/dedifferentiated, within a series of stromal changes named stromal reaction. Here, we tested whether a laminin 111-rich extracellular matrix (Lr-ECM) could affect SMCs phenotype and differentiation status. Using time-lapse microscopy, image analyses, quantitative real-time reverse transcription polymerase chain reaction, immunohistochemistry and immunoblotting, and transmission electron microscopy, we showed that SMCs acquires a migratory behavior with a decreased expression of differentiation markers and relocation of focal adhesion kinase. SMCs set homotypic cell junctions and were active in autophagy/phagocytosis. Analysis of the migratory behavior showed that SMCs polarized and migrated toward each other, recognizing long-distance signals such as matrix tensioning. However, half of the cell population were immotile, irrespective of the nearest neighbor distance, suggesting they do not engage in productive interactions, possibly as a result of back-to-back positioning. In conclusion, the Lr-ECM, mimics the effects of the proliferating and infiltrating tumor epithelium, causing SMCs phenotypical change similar to that observed in the stromal reaction, in addition to a hitherto undescribed, stereotyped pattern of cell motility resulting from cell polarization.

Neurons require adhesive scaffolds for their growth and differentiation. Laminins are a major cell adhesive component of basement membranes and have various biological activities in the peripheral and central nervous systems. Here, we evaluated the biological activities of 5 peptides derived from laminin-111 as a scaffold for mouse neuroblastoma Neuro2a cells and rat neural stem/progenitor cells (NPCs). The 5 peptides showed Neuro2a cell attachment activity similar to that of poly-d-lysine. However, when NPCs were cultured on the peptides, 2 syndecan-binding peptides, AG73 (RKRLQVQLSIRT, mouse laminin α1 chain 2719-2730) and C16 (KAFDITYVRLKF, laminin γ1 chain 139-150), demonstrated significantly higher cell attachment and neurite extension activities than other peptides including integrin-binding ones. Long-term cell culture experiments showed that both AG73 and C16 supported the growth of neurons and astrocytes that had differentiated from NPCs. Furthermore, C16 markedly promoted the expression of neuronal markers such as synaptosomal-associated protein-25 and syntaxin 1A. These results indicate that AG73 and C16 are useful for NPC cultures and that C16 can be applied to specialized research on synapses in differentiated neurons. These peptides have the potential for use as valuable biomaterials for NPC research.

Background Laminin-α2-related congenital muscular dystrophy (LAMA2-CMD) is a devastating genetic disease caused by mutations in the LAMA2 gene. These mutations result in progressive muscle wasting and inflammation leading to delayed milestones, and reduced lifespan in affected patients. There is currently no cure or treatment for LAMA2-CMD. Preclinical studies have demonstrated that mouse laminin-111 can serve as an effective protein replacement therapy in a mouse model of LAMA2-CMD. Methods In this study, we generated a novel immunocompromised dyW mouse model of LAMA2-CMD to study the role the immune system plays in muscle disease progression. We used this immune-deficient dyW mouse model to test the therapeutic benefits of recombinant human laminin-111 and laminin-211 protein therapy on laminin-α2-deficient muscle disease progression. Results We show that immunodeficient laminin-α2 null mice demonstrate subtle differences in muscle regeneration compared to immunocompetent animals during early disease stages but overall exhibit a comparable muscle disease progression. We found human laminin-111 and laminin-211 could serve as effective protein replacement strategies with mice showing improvements in muscle pathology and function. We observed that human laminin-111 and laminin-211 exhibit differences on satellite and myoblast cell populations and differentially affect muscle repair. Conclusions This study describes the generation of a novel immunodeficient mouse model that allows investigation of the role the immune system plays in LAMA2-CMD. This model can be used to assess the therapeutic potential of heterologous therapies that would elicit an immune response. Using this model, we show that recombinant human laminin-111 can serve as effective protein replacement therapy for the treatment of LAMA2-CMD.

Laminin (LM)-111 supplementation has improved muscle regeneration in several models of disease and injury. This study investigated a novel hydrogel composed of fibrinogen and LM-111. Increasing LM-111 concentration (50-450 μg/mL) in fibrin hydrogels resulted in highly fibrous scaffolds with progressively thinner interlaced fibers. Rheological testing showed that all hydrogels had viscoelastic behavior and the Young's modulus ranged from 2-6KPa. C2C12 myobalsts showed a significant increase in VEGF production and decrease in IL-6 production on LM-111 enriched fibrin hydrogels as compared to pure fibrin hydrogels on day 4. Western blotting results showed a significant increase in MyoD and desmin protein quantity but a significant decrease in myogenin protein quantity in myoblasts cultured on the LM-111 (450 μg/mL) enriched fibrin hydrogel. Combined application of electromechanical stimulation significantly enhanced the production of VEGF and IGF-1 from myoblast seeded fibrin-LM-111 hydrogels. Taken together, these observations offer an important first step toward optimizing a tissue engineered constructs for skeletal muscle regeneration.

Laminin-111 (Ln-1), an extracellular matrix (ECM) glycoprotein found in the basement membrane of mammary gland epithelia, is essential for lactation. In mammary epithelial cells (MECs), dystroglycan (Dg) is believed to be necessary for polymerization of laminin-111 into networks., thus we asked whether correct polymerization could compensate for Dg loss. Artificially polymerized laminin-111 and the laminin-glycoprotein mix Matrigel, both formed branching, spread networks with fractal dimensions from 1.7 to 1.8, whereas laminin-111 in neutral buffers formed small aggregates without fractal properties (a fractal dimension of 2). In Dg knockout cells, either polymerized laminin-111 or Matrigel readily attached to the cell surface, whereas aggregated laminin-111 did not. In contrast, polymerized and aggregated laminin-111 bound similarly to Dg knock-ins. Both polymerized laminin-111 and Matrigel promoted cell rounding, clustering, formation of tight junctions, and expression of milk proteins, whereas aggregated Ln-1 did not attach to cells or promote functional differentiation. These findings support that the microstructure of Ln-1 networks in the basement membrane regulates mammary epithelial cell function.

Duchenne muscular dystrophy (DMD) is a devastating X-linked disease affecting ~1 in 5000 males. DMD patients exhibit progressive muscle degeneration and weakness, leading to loss of ambulation and premature death from cardiopulmonary failure. We previously reported that mouse Laminin-111 (msLam-111) protein could reduce muscle pathology and improve muscle function in the mdx mouse model for DMD. In this study, we examined the ability of msLam-111 to prevent muscle disease progression in the golden retriever muscular dystrophy (GRMD) dog model of DMD. The msLam-111 protein was injected into the cranial tibial muscle compartment of GRMD dogs and muscle strength and pathology were assessed. The results showed that msLam-111 treatment increased muscle fiber regeneration and repair with improved muscle strength and reduced muscle fibrosis in the GRMD model. Together, these findings support the idea that Laminin-111 could serve as a novel protein therapy for the treatment of DMD.

Endothelial basement membranes constitute barriers to extravasating leukocytes during inflammation, a process where laminin isoforms define sites of leukocyte exit; however, how this occurs is poorly understood. In addition to a direct effect on leukocyte transmigration, we show that laminin 511 affects endothelial barrier function by stabilizing VE-cadherin at junctions and downregulating expression of CD99L2, correlating with reduced neutrophil extravasation. Binding of endothelial cells to laminin 511, but not laminin 411 or non-endothelial laminin 111, enhanced transendothelial cell electrical resistance (TEER) and inhibited neutrophil transmigration. Data suggest that endothelial adhesion to laminin 511 via β1 and β3 integrins mediates RhoA-induced VE-cadherin localization to cell-cell borders, and while CD99L2 downregulation requires integrin β1, it is RhoA-independent. Our data demonstrate that molecular information provided by basement membrane laminin 511 affects leukocyte extravasation both directly and indirectly by modulating endothelial barrier properties.

This study detected the distribution of laminin during embryonic formation by immunofluorescence. To determine the possible function of laminin on developmental ability of in vitro fertilized embryos, the presumptive zygotes were divided and transferred to CR1aa medium supplemented with different concentrations (0 μg/ml, 5 μg/ml, 10 μg/ml and 20 μg/ml) of laminin. To explore the association with sperm-oocyte fusion, oocytes and/or sperm were pre-incubated with laminin or anti-β1 antibody before insemination. Laminin was absent in mature oocytes and could be detected first at the 8-cell stage and then displayed an increasing tendency. Adding 10 μg/ml laminin to the culture medium improved embryonic development including cleavage rate, blastocyst rate, total cell numbers in the blastocyst and cell numbers in the inner cell mass. Laminin inhibited sperm-oocyte fusion when incubated with oocytes and/or sperm before in vitro fertilization, and only integrin-β1 of sperm was involved in sperm-oocyte binding. Inhibition may be caused by blocking β1, but why laminin inhibits fertilization is still unknown. The results suggest that laminin plays an important role during embryonic formation and has a negative function in sperm-oocyte fusion, but improves embryonic development. However, only integrin-β1 is involved in sperm-oocyte binding.

Recurrent laryngeal nerve (RLN) injury causes vocal fold paralysis from which functional recovery is typically absent due to nonselective reinnervation. This study investigates expression of axon guidance cues and their modulators relative to the chronology of reinnervation by examining the expression of glial-derived neurotrophic factor (GDNF), netrin 1, and laminin 111 (LAMA1) in nonpooled laryngeal muscles. This study is the first to describe the post-RLN injury expression pattern of LAMA1, a target of particular interest as it has been shown to switch netrin 1-mediated growth cone attraction to repulsion.Animal experiment (rat model).Basic science laboratory.The right RLNs of 64 female Sprague-Dawley rats were transected, with sacrifice at 1, 3, 7, 21, 28, and 56 days postinjury (DPI). Single-animal messenger RNA was isolated from the ipsilateral posterior cricoarytenoid (PCA), lateral thyroarytenoid (LTA), and medial thyroarytenoid (MTA) for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. Immunostaining for LAMA1 expression was performed in the same muscles.LAMA1 was elevated in the PCA at 3 to 56 DPI, LTA at 7 DPI, and MTA at 14 and 28 DPI. This correlates with the chronology of laryngeal reinnervation. Using a new protocol, single-animal muscle qRT-PCR possible and expression results for GDNF and netrin 1 were similar to previous pooled investigations.Reliable qRT-PCR is possible with single rat laryngeal muscles. The expression of netrin 1 and LAMA1 is chronologically coordinated with muscle innervation in the LTA and MTA. This suggests that LAMA1 may influence netrin 1 to repel axons and delay LTA and MTA reinnervation.

Skeletal muscle has a remarkable regenerative capability following mild physical or chemical insult. However, following a critical loss of muscle tissue, the regeneration process is impaired due to the inadequate myogenic activity of muscle resident stem cells (i.e., satellite cells). Laminin (LM) is a heterotrimeric structural protein in the satellite cell niche that is crucial for maintaining its function. In this study, we created hydrogels composed of poly (ethylene glycol) (PEG) and LM-111 to provide an elastic substrate for satellite cell proliferation at the site of injury. The PEG-LM111 conjugates were mixed with 5% and 10% (w/v) pure PEG-diacrylate (PEGDA) and photopolymerized to form 5% and 10% PEGLM gels. Pure 5% and 10% PEGDA gels were used as controls. The modulus of both hydrogels containing 10% (w/v) PEGDA was significantly higher than the hydrogels containing 5% (w/v) PEGDA. The 5% PEGLM hydrogels showed significantly higher swelling in aqueous medium suggesting a more porous structure. C2C12 myoblasts cultured on the softer 5% PEGLM hydrogels showed a flat and spread-out morphology when compared to the rounded, multicell clusters formed on the 5% PEGDA, 10% PEGDA, and 10% PEGLM hydrogels. The 5% PEGLM hydrogels also promoted a significant increase in both vascular endothelial growth factor and interleukin-6 (IL-6) production from the myoblasts. Additionally, the expression of MyoD was significantly higher while that of myogenin and α-actinin trended higher on the 5% PEGLM hydrogels compared to 5% PEGDA on day 5. Our data suggests that the introduction of LM-111 into compliant PEG hydrogels promoted myoblast adhesion, survival, pro-regenerative growth factor production, and myogenic activity.

Epithelial progenitor cells are dependent upon a complex 3D niche to promote their proliferation and differentiation during development, which can be recapitulated in organoids. The specific requirements of the niche remain unclear for many cell types, including the proacinar cells that give rise to secretory acinar epithelial cells that produce saliva. Here, using ex vivo cultures of E16 primary mouse submandibular salivary gland epithelial cell clusters, we investigated the requirement for mesenchymal cells and other factors in producing salivary organoids in culture. Native E16 salivary mesenchyme, but not NIH3T3 cells or mesenchymal cell conditioned medium, supported robust protein expression of the progenitor marker Kit and the acinar/proacinar marker AQP5, with a requirement for FGF2 expression by the mesenchyme. Enriched salivary epithelial clusters that were grown in laminin-enriched basement membrane extract or laminin-111 together with exogenous FGF2, but not with EGF, underwent morphogenesis to form organoids that displayed robust expression of AQP5 in terminal buds. Knockdown of FGF2 in the mesenchyme or depletion of mesenchyme cells from the organoids significantly reduced AQP5 levels even in the presence of FGF2, suggesting a requirement for autocrine FGF2 signaling in the mesenchyme cells for AQP5 expression. We conclude that basement membrane proteins and mesenchyme cells function as niche factors in salivary organoids.

Cell adhesive biomaterials have been used for various cells in culture, especially for primary cultures of neurons. Here we examined laminin-111 and its active peptides conjugated to chitosan matrices (ChtMs) for primary culture of rat cortical neurons. Laminin-111 on poly-d-lysine substrate promoted neuronal cell attachment and differentiation. The biological activity of six active laminin-111-derived peptides was examined using a peptide-ChtM construct. When the syndecan-binding peptides, AG73 (RKRLQVQLSIRT, mouse laminin α1 chain 2719-2730) and C16 (KAFDITYVRLKF, laminin γ1 chain 139-150), were conjugated to chitosan, AG73-ChtM and C16-ChtM showed potent neuronal cell attachment activity and promoted axon extension by primary cultured rat cortical neurons. However, the remaining peptides, including integrin-binding peptides, did not show activity when conjugated to ChtM. AG73-ChtM and C16-ChtM also supported neuron survival for at least 4 weeks in serum-free medium without a glia feeder layer. These data suggest that AG73-ChtM and C16-ChtM are useful for primary cultures of central nervous system neurons and have a potential for use as functional biomaterials for tissue engineering in the central nervous system.

Hyposalivation reduces the patient quality of life, as saliva is important for maintaining oral health. Current treatments for hyposalivation are limited to medications such as the muscarinic receptor agonists, pilocarpine and cevimeline. However, these therapies only provide temporary relief. Therefore, alternative therapies are essential to restore salivary gland function. An option is to use bioengineered scaffolds to promote functional salivary gland regeneration. Previous studies demonstrated that the laminin-111 protein is critical for intact salivary gland cell cluster formation and organization. However, laminin-111 protein as a whole is not suitable for clinical applications as some protein domains may contribute to unwanted side effects such as degradation, tumorigenesis and immune responses. Conversely, the use of synthetic laminin-111 peptides makes it possible to minimize the immune reactivity or pathogen transfer. In addition, it is relatively simple and inexpensive as compared to animal-derived proteins. Therefore, the goal of this study was to demonstrate whether a 20 day treatment with laminin-111-derived peptide conjugated fibrin hydrogel promotes tissue regeneration in submandibular glands of a wound healing mouse model. In this study, laminin-111-derived peptide conjugated fibrin hydrogel significantly accelerated formation of salivary gland tissue. The regenerated gland tissues displayed not only structural but also functional restoration.

Minced muscle autografting mediates de novo myofiber regeneration and promotes partial recovery of neuromuscular strength after volumetric muscle loss injury (VML). A major limitation of this approach is the availability of sufficient donor tissue for the treatment of relatively large VMLs without inducing donor site morbidity. This study evaluated a laminin-111 supplemented hyaluronic acid based hydrogel (HA+LMN) as a putative myoconductive scaffolding to be co-delivered with minced muscle grafts. In a rat tibialis anterior muscle VML model, delivery of a reduced dose of minced muscle graft (50% of VML defect) within HA+LMN resulted in a 42% improvement of peak tetanic torque production over unrepaired VML affected limbs. However, the improvement in strength was not improved compared to a 50% minced graft-only control group. Moreover, histological analysis revealed that the improvement in in vivo functional capacity mediated by minced grafts in HA+LMN was not accompanied by a particularly robust graft mediated regenerative response as determined through donor cell tracking of the GFP+ grafting material. Characterization of the spatial distribution and density of macrophage and satellite cell populations indicated that the combination therapy damps the heightened macrophage response while re-establishing satellite content 14 days after VML to a level consistent with an endogenously healing ischemia-reperfusion induced muscle injury. Moreover, regional analysis revealed that the combination therapy increased satellite cell density mostly in the remaining musculature, as opposed to the defect area. Based on the results, the following salient conclusions were drawn: 1) functional recovery mediated by the combination therapy is likely due to a superposition of de novo muscle fiber regeneration and augmented repair of muscle fibers within the remaining musculature, and 2) The capacity for VML therapies to augment regeneration and repair within the remaining musculature may have significant clinical impact and warrants further exploration.

The isolation of islets by collagenase digestion can cause damage and impact the efficiency of islet engraftment and function. In this study, we assessed the basement membranes (BMs) of mouse pancreatic islets as a molecular biomarker for islet integrity, damage after isolation, and islet repair in vitro as well as in the absence or presence of an immune response after transplantation. Immunofluorescence staining of BM matrix proteins and the endothelial cell marker platelet endothelial cell adhesion molecule-1 (PECAM-1) was performed on pancreatic islets in situ, isolated islets, islets cultured for 4 days, and islet grafts at 3–10 days posttransplantation. Flow cytometry was used to investigate the expression of BM matrix proteins in isolated islet β-cells. The islet BM, consisting of collagen type IV and components of Engelbreth–Holm–Swarm (EHS) tumor laminin 111, laminin α2, nidogen-2, and perlecan in pancreatic islets in situ, was completely lost during islet isolation. It was not reestablished during culture for 4 days. Peri- and intraislet BM restoration was identified after islet isotransplantation and coincided with the migration pattern of PECAM-1+ vascular endothelial cells (VECs). After islet allotransplantation, the restoration of VEC-derived peri-islet BMs was initiated but did not lead to the formation of the intraislet vasculature. Instead, an abnormally enlarged peri-islet vasculature developed, coinciding with islet allograft rejection. The islet BM is a sensitive biomarker of islet damage resulting from enzymatic isolation and of islet repair after transplantation. After transplantation, remodeling of both peri- and intraislet BMs restores β-cell–matrix attachment, a recognized requirement for β-cell survival, for isografts but not for allografts. Preventing isolation-induced islet BM damage would be expected to preserve the intrinsic barrier function of islet BMs, thereby influencing both the effector mechanisms required for allograft rejection and the antirejection strategies needed for allograft survival.

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a severe and fatal muscle-wasting disease with no cure. MDC1A patients and the dy(W-/-) mouse model exhibit severe muscle weakness, demyelinating neuropathy, failed muscle regeneration and premature death. We have recently shown that laminin-111, a form of laminin found in embryonic skeletal muscle, can substitute for the loss of laminin-211/221 and prevent muscle disease progression in the dy(W-/-) mouse model. What is unclear from these studies is whether laminin-111 can restore failed regeneration to laminin-α2-deficient muscle. To investigate the potential of laminin-111 protein therapy to improve muscle regeneration, laminin-111 or phosphate-buffered saline-treated laminin-α2-deficient muscle was damaged with cardiotoxin and muscle regeneration quantified. Our results show laminin-111 treatment promoted an increase in myofiber size and number, and an increased expression of α7β1 integrin, Pax7, myogenin and embryonic myosin heavy chain, indicating a restoration of the muscle regenerative program. Together, our results show laminin-111 restores muscle regeneration to laminin-α2-deficient muscle and further supports laminin-111 protein as a therapy for the treatment of MDC1A.

Laminin-111 is a large trimeric basement membrane glycoprotein with many active sites. In particular, four peptides active in tumor malignancy studies have been identified in laminin-111 using a systematic peptide screening method followed by various assays. Two of the peptides (IKVAV and AG73) are found on the α1 chain, one (YIGSR) of the β1 chain and one (C16) on the γ1 chain. The four peptides have distinct activities and receptors. Since three of the peptides (IKVAV, AG73 and C16) strongly promote tumor growth, this may explain the potent effects laminin-111 has on malignant cells. The peptide, YIGSR, decreases tumor growth and experimental metastasis via a 32/67 kD receptor while IKVAV increases tumor growth, angiogenesis and protease activity via integrin receptors. AG73 increases tumor growth and metastases via syndecan receptors. C16 increases tumor growth and angiogenesis via integrins. Identification of such sites on laminin-111 will have use in defining strategies to develop therapeutics for cancer.

Previous studies showed that mouse submandibular gland cells form three-dimensional structures when grown on Laminin-111 gels. The use of Laminin-111 for tissue bioengineering is complicated due to its lack of purity. By contrast, the use of synthetic peptides derived from Laminin-111 is beneficial due to their high purity and easy manipulation. Two Laminin-111 peptides have been identified for salivary cells: the A99 peptide corresponding to the α1 chain from Laminin-111 and the YIGSR peptide corresponding to the β1 chain from Laminin-111, which are important for cell adhesion and migration. We created three-dimensional salivary cell clusters using a modified fibrin hydrogel matrix containing immobilized Laminin-111 peptides. Results indicate that the YIGSR peptide improved morphology and lumen formation in rat parotid Par-C10 cells as compared to cells grown on unmodified fibrin hydrogel. Moreover, a combination of both peptides not only allowed the formation of functional three-dimensional salivary cell clusters but also increased attachment and number of cell clusters. In summary, we demonstrated that fibrin hydrogel decorated with Laminin-111 peptides supports attachment and differentiation of salivary gland cell clusters with mature lumens.

Gap junctions within extracellular matrix (ECM)-defined boundaries ensure synchronous activity between cells destined to become functional mediators that regulate cell behavior. However, the role of ECM in connexin (Cx) function in mouse embryonic stem cells (mESCs) has not been elucidated. Therefore, we examined the role of laminin-111 in the control of Cx43 functions and related signal pathways in mESCs. ECM components (laminin-111, fibronectin, and collagen I) increased Cx43 phosphorylation and decreased Lucifer yellow (Ly) diffusion. In addition, laminin-111 increased the proliferation index through reduction of gap junctional intercellular communication (GJIC), which was confirmed by 18α-glycyrrhetinic acid (18α-GA). Laminin-111 increased phosphorylation of focal adhesion kinase (FAK)/Src and protein kinase C (PKC), which were inhibited by integrin β1 antibody (Ab) and laminin receptor-1 (LR-1) Ab, respectively. In addition, inhibition of both FAK/Src and PKC blocked Cx43 phosphorylation. Laminin-111 increased the Ras homolog gene family, member A (RhoA) activation, which was blocked by FAK/Src and PKC inhibitors, suggesting the existence of parallel pathways that merge at RhoA. Inhibition of RhoA reversed the laminin-111-induced increase of Cx43 phosphorylation and reduction of GJIC. Laminin-111 also stimulated the dissociation of Cx43/ZO-1 complex followed by disruption of Cx43/drebrin and Cx43/F-actin complexes, which were reversed by C3 (RhoA inhibitor). ZO-1 small interfering (si) RNA significantly decreased Ly diffusion. Moreover, laminin-111 decreased Cx43 labeling at the intercellular junction, whereas pretreatment with degradation inhibitors (lysosomal protease inhibitor, chloroquine; proteasome inhibitor, lactacystin) increased Cx43 expression, reversely. In conclusion, laminin-111 stimulated mESC proliferation through a reduction of GJIC via RhoA-mediated Cx43 phosphorylation and Cx43/ZO-1/drebrin complex instability-mediated Cx43 degradation.

Functional differentiation is orchestrated by precise growth-regulatory controls conveyed by the tissue microenvironment. Cues from laminin 111 (LN1) lower transcription and suppress mammary epithelial cell growth in culture, but how LN1 induces quiescence is unknown. Recent literature points to involvement of nuclear β-actin in transcriptional regulation. Here, we show that quiescence induced by growth factor withdrawal, or LN1 addition, rapidly decreases nuclear β-actin. LN1, but not other extracellular matrix (ECM) molecules, decreases the levels of nuclear β-actin and destabilizes RNA polymerase (RNA Pol) II and III binding to transcription sites, leading to a dramatic drop in transcription and DNA synthesis. Constitutive overexpression of globular β-actin in the nucleus reverses the effect of LN1 on transcription and RNA Pol II association and prevents the cells from becoming quiescent in the presence of LN1. The physiological relevance of our findings was verified by identifying a clear spatial separation of LN1 and β-actin in developing mammary end buds. These data indicate a novel role for nuclear β-actin in growth arrest of epithelial cells and underscore the importance of the integrity of the basement membrane in homeostasis.

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease caused by mutations in the gene encoding dystrophin. Loss of dystrophin results in reduced sarcolemmal integrity and increased susceptibility to muscle damage. The α 7 β 1 -integrin is a laminin-binding protein up-regulated in the skeletal muscle of DMD patients and in the mdx mouse model. Transgenic overexpression of the α 7 -integrin alleviates muscle disease in dystrophic mice, making this gene a target for pharmacological intervention. Studies suggest laminin may regulate α 7 -integrin expression. To test this hypothesis, mouse and human myoblasts were treated with laminin and assayed for α 7 -integrin expression. We show that laminin-111 (α 1 , β 1 , γ 1 ), which is expressed during embryonic development but absent in normal or dystrophic skeletal muscle, increased α 7 -integrin expression in mouse and DMD patient myoblasts. Injection of laminin-111 protein into the mdx mouse model of DMD increased expression of α 7 -integrin, stabilized the sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscle from exercised-induced damage. These findings demonstrate that laminin-111 is a highly potent therapeutic agent for the mdx mouse model of DMD and represents a paradigm for the systemic delivery of extracellular matrix proteins as therapies for genetic diseases.

Mutations in the alpha7 integrin gene cause congenital myopathy characterized by delayed developmental milestones and impaired mobility. Previous studies in dystrophic mice suggest the alpha7beta1 integrin may be critical for muscle repair. To investigate the role that alpha7beta1 integrin plays in muscle regeneration, cardiotoxin was used to induce damage in the tibialis anterior muscle of alpha7 integrin-null mice. Unlike wild-type muscle, which responded rapidly to repair damaged myofibers, alpha7 integrin-deficient muscle exhibited defective regeneration. Analysis of Pax7 and MyoD expression revealed a profound delay in satellite cell activation after cardiotoxin treatment in alpha7 integrin-null animals when compared with wild type. We have recently demonstrated that the muscle of alpha7 integrin-null mice exhibits reduced laminin-alpha2 expression. To test the hypothesis that loss of laminin contributes to the defective muscle regeneration phenotype observed in alpha7 integrin-null mice, mouse laminin-111 (alpha1, beta1, gamma1) protein was injected into the tibialis anterior muscle 3 days before cardiotoxin-induced injury. The injected laminin-111 protein infiltrated the entire muscle and restored myogenic repair and muscle regeneration in alpha7 integrin-null muscle to wild-type levels. Our data demonstrate a critical role for a laminin-rich microenvironment in muscle repair and suggest laminin- 111 protein may serve as an unexpected and novel therapeutic agent for patients with congenital myopathies.

Laminins are a major component of basement membranes. Each laminin molecule is a heterotrimeric glycoprotein composed of one alpha, one beta, and one gamma chain. Fifteen laminin isoforms exist, assembled from various combinations of 5alpha, 3beta, and 3gamma chains. The embryonic lung has abundant laminin isoforms. Increasing evidence suggests that different laminin isoforms have unique functions in lung development. Studies of embryonic lung explants and organotypic co-cultures show that laminin alpha1 and laminin 111 are important for epithelial branching morphogenesis and that laminin alpha2 and laminin 211 have a role in smooth muscle cell differentiation. In vivo studies of laminin alpha5-deficient mice indicate that this laminin chain, found in laminins 511 and 521, is essential for normal lobar septation in early lung development and normal alveolization and distal epithelial cell differentiation and maturation in late lung development. However, not all of the laminin chains present in the developing lung appear to be necessary for normal lung development since laminin alpha4 null mice do not have obvious lung abnormalities and laminin gamma2 null mice have only minimal changes in lung development. The mechanisms responsible for the lung phenotypes in mice with laminin mutations are unknown, but it is clear that multiple laminin isoforms are crucial for lung development and that different laminin isoforms exhibit specific, non-overlapping functions.

A simplification of the laminin nomenclature is presented. Laminins are multidomain heterotrimers composed of alpha, beta and gamma chains. Previously, laminin trimers were numbered with Arabic numerals in the order discovered, that is laminins-1 to -5. We introduce a new identification system for a trimer using three Arabic numerals, based on the alpha, beta and gamma chain numbers. For example, the laminin with the chain composition alpha5beta1gamma1 is termed laminin-511, and not laminin-10. The current practice is also to mix two overlapping domain and module nomenclatures. Instead of the older Roman numeral nomenclature and mixed nomenclature, all modules are now called domains. Some domains are renamed or renumbered. Laminin epidermal growth factor-like (LE) domains are renumbered starting at the N-termini, to be consistent with general protein nomenclature. Domain IVb of alpha chains is named laminin 4a (L4a), domain IVa of alpha chains is named L4b, domain IV of gamma chains is named L4, and domain IV of beta chains is named laminin four (LF). The two coiled-coil domains I and II are now considered one laminin coiled-coil domain (LCC). The interruption in the coiled-coil of beta chains is named laminin beta-knob (Lbeta) domain. The chain origin of a domain is specified by the chain nomenclature, such as alpha1L4a. The abbreviation LM is suggested for laminin. Otherwise, the nomenclature remains unaltered.

The dynamic interplay between the extracellular matrix and embryonic stem cells (ESCs) constitutes one of the key steps in understanding stem cell differentiation in vitro. Here we report a biologically-active laminin-111 fragment generated by matrix metalloproteinase 2 (MMP2) processing, which is highly up-regulated during differentiation. We show that the β1-chain-derived fragment interacts via α3β1-integrins, thereby triggering the down-regulation of MMP2 in mouse and human ESCs. Additionally, the expression of MMP9 and E-cadherin is up-regulated in mouse ESCs--key players in the epithelial-to-mesenchymal transition. We also demonstrate that the fragment acts through the α3β1-integrin/extracellular matrix metalloproteinase inducer complex. This study reveals a previously unidentified role of laminin-111 in early stem cell differentiation that goes far beyond basement membrane assembly and a mechanism by which an MMP2-cleaved laminin fragment regulates the expression of E-cadherin, MMP2, and MMP9.

We have identified a number of cell-adhesive peptides from laminins, a major component of basement membranes. Cell-adhesive peptides derived from basement membrane proteins are potential candidates for incorporating cell-binding activities into scaffold materials for tissue engineering. Our goal is development of a chemically synthetic basement membrane using laminin-derived cell-adhesive peptides and polymeric materials. In this study, we used hyaluronic acid (HA) as a scaffold material and laminin-derived cell-adhesive peptides, A99 (AGTFALRGDNPQG, binds to integrin αvβ3), AG73 (RKRLQVQLSIRT, binds to syndecans), and an A99/AG73 mixture (molar ratio = 9:1) conjugated to two-dimensional (2D) HA matrices. As a result, it was found that the 2D A99/AG73-HA matrices have strong biological functions, such as cell attachment, cell spreading, and neurite outgrowth, similar to that of basement membrane extract (BME)-coated plates. Next, we developed three-dimensional (3D) peptide-HA matrices using the A99/AG73 mixture. The 3D A99/AG73-HA matrices promoted cell spreading and improved cell viability and collagen gene expression. Further, PC12 neurite extension was observed in the 3D A99/AG73-HA matrices. These biological activities of the 3D A99/AG73-HA matrices were similar to those of the 3D BME matrices. These results suggest that the peptide-HA matrices are useful as 2D and 3D matrices and can be applied for tissue engineering as a synthetic basement membrane.

The importance of laminin-containing basement membranes (BM) for adult muscle function is well established, in particular due to the severe phenotype of congenital muscular dystrophies in patients with mutations disrupting the BM-muscle cell interaction. Developing muscles in the embryo are also dependent on an intact BM. However, the processes controlled by BM-muscle cell interactions in the embryo are only beginning to be elucidated. In this review, we focus on the myotomal BM to illustrate the critical role of laminin-111 in BM assembly and function at the surface of embryonic muscle cells. The myotomal BM provides also an interesting paradigm to study the complex interplay between laminins-containing BM and growth factor-mediated signaling and activity.

Breast cancer progression involves genetic changes and changes in the extracellular matrix (ECM). To test the importance of the ECM in tumor cell dissemination, we cultured epithelium from primary human breast carcinomas in different ECM gels. We used basement membrane gels to model the normal microenvironment and collagen I to model the stromal ECM. In basement membrane gels, malignant epithelium either was indolent or grew collectively, without protrusions. In collagen I, epithelium from the same tumor invaded with protrusions and disseminated cells. Importantly, collagen I induced a similar initial response of protrusions and dissemination in both normal and malignant mammary epithelium. However, dissemination of normal cells into collagen I was transient and ceased as laminin 111 localized to the basal surface, whereas dissemination of carcinoma cells was sustained throughout culture, and laminin 111 was not detected. Despite the large impact of ECM on migration strategy, transcriptome analysis of our 3D cultures revealed few ECM-dependent changes in RNA expression. However, we observed many differences between normal and malignant epithelium, including reduced expression of cell-adhesion genes in tumors. Therefore, we tested whether deletion of an adhesion gene could induce sustained dissemination of nontransformed cells into collagen I. We found that deletion of P-cadherin was sufficient for sustained dissemination, but exclusively into collagen I. Our data reveal that metastatic tumors preferentially disseminate in specific ECM microenvironments. Furthermore, these data suggest that breaks in the basement membrane could induce invasion and dissemination via the resulting direct contact between cancer cells and collagen I.

Laminin-111, a multifunctional matrix protein, has diverse biological functions. Previously, we have identified various biologically active sequences in laminin-111 by a systematic peptide screening. We also demonstrated that peptide-conjugated chitosan matrices enhance the biological functions of the active sequences and are useful as a scaffold. Here, we conjugated sixty biologically active laminin-111 peptides onto chitosan matrices. The twenty-nine peptide-chitosan matrices promoted various biological activities, including cell attachment, spreading, and neurite outgrowth. The biological activities of peptide-chitosan matrices depend on the peptide. These peptide-chitosan matrices are categorized into six groups depending on their biological activities. Next, we conjugated five active peptides, which showed strong cell attachment activity in the each group, onto a single chitosan matrix to mimic the multiple activities of laminin-111. The mixed peptides-chitosan matrix significantly promoted cell attachment and cell spreading over that observed with the individual peptides. We also demonstrated that a mixed peptides-chitosan matrix, using four neurite outgrowth-promoting peptides each from a different group, enhanced the activity. These data suggest that the mixed peptides synergistically induce laminin-like biological activities on a chitosan matrix. The active peptides-chitosan matrices described here have potential for use as biomaterial for tissue engineering and regeneration.

Extracellular matrix (ECM) components play an important role in the regulation and maintenance of neural stem cells (NSCs). Laminin, an ECM component, is a key factor in promoting axonal regeneration and differentiation of NSCs. Since NSCs cannot be easily harvested with low morbidity, adipose-derived stem cells have been suggested for therapeutic applications of neural tissue damage. Therefore, the potential of laminin-111 to enhance the production of neurotrophin-3 (NT3) and its related signal pathways from canine adipose tissue-derived stem cells (cADSCs) was investigated. Laminin-111 enhanced NT3 production in neural induction medium (NIM). Treatment of NIM or laminin-111 on cADSCs distinctively changed integrin β1 mRNA and protein expression levels. In addition, laminin-111-induced Akt phosphorylation was inhibited by integrin β1 small interfering RNA (siRNA) and PI3K inhibitors (LY294002 and wortmannin). Furthermore, increased phosphorylations of mTOR and p70S6K by laminin-111 were blocked by inhibitors or specific siRNA, respectively. Moreover, laminin-111-induced NT3 production was blocked by these inhibitors. In experiments to induce the differentiation of cADSCs, laminin-111 increased the expression of neuronal markers β 3 tubulin, MAP2, and NeuN, and decreased the expression of the NSC markers nestin and vimentin. In conclusion, laminin-111 increases NT3 production through Akt, mTOR, and p70S6K pathways via integrin β1 in cADSCs cultured in NIM. J. Cell. Physiol. 226: 3251–3260, 2011. © 2011 Wiley Periodicals, Inc.

We had developed a conditional Laminin α 1 knockout-mouse model (Lama1(cko)) bypassing embryonic lethality of Lama1 deficient mice to study the role of this crucial laminin chain during late developmental phases and organogenesis. Here, we report a strong defect in the organization of the adult cerebellum of Lama1(cko) mice. Our study of the postnatal cerebellum of Lama1(cko) animals revealed a disrupted basement membrane correlated to an unexpected excessive proliferation of granule cell precursors in the external granular layer (EGL). This was counteracted by a massive cell death occurring between the postnatal day 7 (P7) and day 20 (P20) resulting in a net balance of less cells and a smaller cerebellum. Our data show that the absence of Lama1 has an impact on the Bergmann glia scaffold that aberrantly develops. This phenotype is presumably responsible for the observed misplacing of granule cells that may explain the overall perturbation of the layering of the cerebellum and an aberrant folia formation.

Organization into polarized three-dimensional structures defines whether epithelial cells are normal or malignant. In a model of morphogenesis, we show that inhibiting key signaling pathways in human breast cancer cells leads to "phenotypic reversion" of the malignant cells. Using architecture as an endpoint, we report that, in all cases, signaling through Raf/MEK/ERK disrupted tissue polarity via matrix metalloproteinase9 (MMP9) activity. Induction of Raf or activation of an engineered, functionally inducible MMP9 in nonmalignant cells led to loss of tissue polarity, and reinitiated proliferation. Conversely, inhibition of Raf or MMP9 with small molecule inhibitors or shRNAs restored the ability of cancer cells to form polarized quiescent structures. Silencing MMP9 expression also reduced tumor growth dramatically in a murine xenograft model. LC-MS/MS analysis comparing conditioned medium from nonmalignant cells with or without active MMP9 revealed laminin 111 (LM1) as an important target of MMP9. LM1 has been implicated in acinar morphogenesis; thus, its degradation by MMP9 provides a mechanism for loss of tissue polarity and reinitiation of growth associated with MMP9 activity. These findings underscore the importance of the dynamic reciprocity between the extracellular matrix integrity, tissue polarity, and Raf/MEK/ERK and MMP9 activities, providing an axis for either tissue homeostasis or malignant progression. © 2010 by Cold Spring Harbor Laboratory Press.

Duchenne muscular dystrophy (DMD) still needs effective treatments, and myoblast transplantation (MT) is considered as an approach to repair damaged skeletal muscles. DMD is due to the complete loss of dystrophin from muscles. The lack of link between the contracting apparatus and the extracellular matrix leads to frequent damage to the sarcolemma triggering muscle fiber necrosis. Laminins are major proteins in the extracellular matrix. Laminin-111 is normally present in skeletal and cardiac muscles in mice and humans but only during embryonic development. In this study, we showed that intramuscular injection of laminin-111 increased muscle strength and resistance in mdx mice. We also used laminin-111 as a coadjuvant in MT, and we showed this protein decreased considerably the repetitive cycles of degeneration, inflammatory reaction, and regeneration. Moreover, MT is significantly improved. To explain the improvement, we confirmed with the same myoblast cell batch that laminin-111 improves proliferation and drastically increases migration in vitro. These results are extremely important because DMD could be treated only by the injection of a recombinant protein, a simple and safe therapy to prevent loss of muscle function. Moreover, the improvement in MT would be significant to treat the muscles of DMD patients who are already weak.

Laminins are cell adhesion molecules that comprise a family of glycoproteins found predominantly in basement membranes, which are the thin sheets of extracellular matrix that underlie epithelial and endothelial cells and surround muscle cells, Schwann cells, and fat cells. Many laminins self-assemble to form networks that remain in close association with cells through interactions with cell surface receptors. Laminins are vital for many physiological functions. They are essential for early embryonic development and organogenesis and have crucial functions in several tissues including muscle, nerve, skin, kidney, lung, and the vasculature. A great wealth of data on laminins is available, and an in-depth description is not attempted here. In this review, I will instead provide a snapshot of laminin structure, tissue distribution, and interactions with other matrix molecules and receptors and briefly describe laminin mutations in mice and humans. Several illuminating and timely reviews are cited that can be consulted for references to original articles and more detailed information concerning laminins.

Laminin-111 promotes the malignant phenotype, and a 12-mer synthetic peptide (AG73, RKRLQVQLSIRT) from the carboxyl terminus of the alpha1 chain increases B16F10 melanoma metastasis to the lung and liver. Using an antibody array, fibronectin was identified as an up-regulated protein in B16F10 cells after incubation with this peptide. The increased fibronectin is cell-associated with no increase in soluble fibronectin. The AG73 peptide increased the number and size of bone metastases with both B16F10 melanoma and MDA-231 breast carcinoma cells in an intracardiac injection model. Using siRNA transfection, we found that a reduction in fibronectin expression did not reduce bone metastasis in the presence of the metastasis-promoting peptide AG73. We conclude that the laminin peptide AG73 increases metastasis independently of fibronectin expression.

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a lethal muscle-wasting disease that is caused by mutations in the LAMA2 gene, resulting in the loss of laminin-α2 protein. MDC1A patients exhibit severe muscle weakness from birth, are confined to a wheelchair, require ventilator assistance, and have reduced life expectancy. There are currently no effective treatments or cures for MDC1A. Laminin-α2 is required for the formation of heterotrimeric laminin-211 (ie, α2, β1, and γ1) and laminin-221 (ie, α2, β2, and γ1), which are major constituents of skeletal muscle basal lamina. Laminin-111 (ie, α1, β1, and γ1) is the predominant laminin isoform in embryonic skeletal muscle and supports normal skeletal muscle development in laminin-α2-deficient muscle but is absent from adult skeletal muscle. In this study, we determined whether treatment with Engelbreth-Holm-Swarm-derived mouse laminin-111 protein could rescue MDC1A in the dy(W-/-) mouse model. We demonstrate that laminin-111 protein systemically delivered to the muscles of laminin-α2-deficient mice prevents muscle pathology, improves muscle strength, and dramatically increases life expectancy. Laminin-111 also prevented apoptosis in laminin-α2-deficient mouse muscle and primary human MDC1A myogenic cells, which indicates a conserved mechanism of action and cross-reactivity between species. Our results demonstrate that laminin-111 can serve as an effective protein substitution therapy for the treatment of muscular dystrophy in the dy(W-/-) mouse model and establish the potential for its use in the treatment of MDC1A.

Summary The establishment of self-renewing hepatoblast-like cells (HBCs) from human pluripotent stem cells (PSCs) would realize a stable supply of hepatocyte-like cells for medical applications. However, the functional characterization of human PSC-derived HBCs was not enough. To purify and expand human PSC-derived HBCs, human PSC-derived HBCs were cultured on dishes coated with various types of human recombinant laminins (LN). Human PSC-derived HBCs attached to human laminin-111 (LN111)-coated dish via integrin alpha 6 and beta 1 and were purified and expanded by culturing on the LN111-coated dish, but not by culturing on dishes coated with other laminin isoforms. By culturing on the LN111-coated dish, human PSC-derived HBCs were maintained for more than 3 months and had the ability to differentiate into both hepatocyte-like cells and cholangiocyte-like cells. These expandable human PSC-derived HBCs would be manageable tools for drug screening, experimental platforms to elucidate mechanisms of hepatoblasts, and cell sources for hepatic regenerative therapy., Graphical Abstract, Highlights • Hepatoblast-like cells were generated from human ES/iPS cells • Hepatoblast-like cells are proliferated and maintained on human Laminin 111 • Hepatoblast-like cells are able to differentiate into hepatic and biliary lineages • Hepatoblast-like cells could integrate into mouse liver parenchyma, The establishment of self-renewing hepatoblast-like cells (HBCs) from human embryonic stem cells/human induced pluripotent stem cells would realize a stable supply of hepatocyte-like cells for medical applications. The HBCs could be purified and expanded by culturing on the laminin-111-coated dish. The HBCs were maintained for more than 3 months and had the ability to differentiate into both hepatocyte- and cholangiocyte-like cells.

Background 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. Methods 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. Results 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. Conclusions 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. Electronic supplementary material The online version of this article (doi:10.1186/s13395-016-0116-4) contains supplementary material, which is available to authorized users.