Pęziński M., DaszczukP., Shankar Pradhan B., Lochmüller H. Prószyński T.J.
Scientific Reports, 2020
This study demonstrates an improved protocol for culturing C2C12 muscle cells that reproducibly promote the formation of complex AChR clusters. The authors tested several laminin isoforms and found that laminin-121, laminin-211, laminin-221, laminin-511, and laminin-521 induced significantly more AChR clusters in C2C12 myotubes than the commonly used laminin-111. Moreover, they found that clusters of postsynaptic machinery that were formed in C2C12 myotubes cultured on laminin-121 and laminin-221 were the most developed. Laminin-421 and laminin-511 were the isoforms that promoted formation of the most podosome-containing AChR clusters in human primary myotubes. Myotubes that were derived from human primary myoblasts obtained from human biopsies also formed AChR clusters in vitro that underwent the remodeling process, thus demonstrating the potential utility of this methodology for further studies that seek to improve diagnoses of neuromuscular disorders and elucidate their underlying mechanisms. Thus, this novel method may facilitate the identification of novel synaptic regulators and the high reproducibility of culturing and robust formation of AChR clusters are important prerequisites for establishing high-throughput screening. The protocol is also useful for obtaining and freezing a large number of cell stocks and utilizing cells for experimentation with a constant and low passage number, which significantly increases experimental reproducibility. The method can be implemented in different formats, such as permanox slides, glass surfaces as well as multi-well culturing dishes. Collectively, these results demonstrate an advancement of culturing myotubes.
Quarta M., Brett J.O., DiMarco R., De Morree A., Boutet S.C. Chacon R. Gibbons M.C., Garcia V.A., Su J., Shrager J.B., Heilshorn S., Rando T.A.
Nat Biotechnol., 2016
Here, the authors describe a system for maintaining muscle stem cells (MuSCs) in vitro in a potent, quiescent state. They screen for factors that could maintain mouse MuSC quiescence and defined a quiescence medium. The authors also designed artificial muscle fibers (AMFs) that mimic the native myofiber of the MuSC niche. Mouse MuSCs maintained in quiescence medium on AMFs showed enhanced potential for engraftment, tissue regeneration, and self-renewal after transplantation in mice. That also evaluated if the muscle fiber specific membrane protein laminin-211 could maintain MuSCs quiescence. The AMFs were coated with recombinant Integrin α4β1 followed by recombinant laminin-211. Indeed, the results showed similarly prolonged quiescence in vitro and enhanced potency in vivo. When seeded onto the laminin-coated AMFs and cultured for three days, mouse MuSCs showed reduced activation as assessed by EdU incorporation, increased viability as assessed by ATP levels, and higher Pax7 and lower MyoD protein expression when compared to AMFs alone or functionalized with integrin α4β1 only.
Penton C.M., Badarinarayana V., Prisco J., Powers E., Pincus M., Allen R.E., August P.R.
Skeletal Muscle, 2016
Here, the authors 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 propagate and preserve myogenic differentiation potential. The 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. Laminin-521 also provides more consistent and reliable differentiation over long-term culture. 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 the low proliferation and poor differentiation in mouse and human cultures. Matrigel performed well in short-term mouse studies but showed high amounts of variability following long-term expansion.
Fontes-Oliveira C.C., Steinz M., Schneiderat P., Mulder H., Durbeej M.
Scientific Reports, 2017
Congenital muscular dystrophy type 1A (MDC1A) is a severe muscle disorder caused by mutations in the laminin α2 gene. Here, the authors have investigated the bioenergetic profile in myogenic cells from MDC1A and LS patients. To confirm that the metabolic alterations were due to deficiency of laminin-211 in MDC1A cells, MDC1A myotubes were cultured in plates coated with recombinant laminin-211. Indeed, basal respiration, maximum respiration, and ATP production, as well as basal mitochondrial respiration and maximal mitochondrial respiration capacity, were normalized to control levels in the presence of laminin-211. The results indicate that absence of laminin α2 chain leads to downregulated PGC1α expression, which impairs mitochondrial biogenesis, causing a reduction of mitochondrial content that finally leads to a bioenergetic inefficiency in myoblasts and myotubes from MDC1A patients. The authors found dysregulated expression of genes related to energy production, apoptosis, and proteasome in myoblasts and myotubes. The data, for the first time, demonstrated an impairment of the bioenergetic status in human MDC1A and LS muscle cells, which could contribute to cell cycle disturbance and increased apoptosis.
Holmberg J. & Durbeej M.
Cell Adh Migr., 2013
This review focus on the importance of laminin-211 for normal skeletal muscle function.
Riederer I., Bonomo A.C., Mouly V., Savino W.
FEBS Lett., 2015
Accumulating data show that the local microenvironment plays a major role during muscle regeneration. In the satellite cell niche, a major extracellular matrix protein is laminin. Human myoblasts transplanted into immunodeficient mice are preferentially located in laminin-enriched areas. Additionally, laminin-111 enhances myoblast proliferation in vitro and increases the expression of the α7β1 integrin-type laminin receptor. Intramuscular injection of laminin-111 ameliorates muscular pathology in mdx mice, protecting muscle fibers from damage. Moreover, transplantation of human myoblasts with laminin-111 into Rag/mdx immunodeficient recipients improved the efficacy of myoblast transplantation, increasing the number of human dystrophin-positive myofibres. Taken together, these data strongly indicate that exogenous laminin can ameliorate the regeneration process in different models of muscular dystrophies and can be instrumental for improving cell therapy aiming at repairing the degeneration/regeneration process in skeletal muscle.
Rogers R.S. & Nishimune H.
Matrix Biology, 2016
The synaptic cleft between a motor neuron and a muscle fiber is filled with the basal lamina. Laminin α4, α5, and β2 chains specifically localize to neuromuscular junctions (NMJs), and these laminin isoforms play a critical role in the maintenance of NMJs and organization of synaptic vesicle release sites known as active zones. These individual laminin chains exert their role in organizing NMJs by binding to their receptors including integrins, dystroglycan, and voltage-gated calcium channels (VGCCs). Disruption of these laminins or the laminin-receptor interaction occurs in neuromuscular diseases including Pierson syndrome and Lambert–Eaton myasthenic syndrome (LEMS).
Seeger T., Hart M., Patarroyo M., Rolauffs B., Aicher W.K., Klein G.
PLOS ONE, 2015
In the sphincter tissue (smooth muscle) a strong expression of the isoforms laminin-211/221, laminin-411/421 and laminin-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms laminin-411 and laminin-511, but not laminin-211. Even after myogenic differentiation, laminin-211 can hardly be detected. All laminin isoforms tested (-211, -411, -511 and -521) showed significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of laminin-521, they had no influence on the proliferation of MSCs cultivated in a myogenic medium. The strongest cellular adhesion of MSCs was to laminin-511 and laminin-521, whereas laminin-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with laminin-211, but it did not affect the interaction with laminin-511 and laminin-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells.
Bolcato-Bellemin A-L., Lefebvre O., Arnold C., Sorokin L., Miner J. H., Kedinger M., Simon-Assmann P.
Developmental Biology, 2003
Here, the function of the laminin a5 chain in the developing intestine was defined by analyzing laminin a5 -/- mutants and by grafting experiments. The authors show that laminin a5 plays a major role in smooth muscle organization and differentiation, as excessive folding of intestinal loops and delay in the expression of specific markers are observed in laminin a5 -/- mice. Loss of a5 expression was paralleled by ectopic or accelerated deposition of laminin a2 and a4 chains; this may explain why no obvious defects were observed in the villous form and enterocytic differentiation. Lack of the laminin a5 chain was accompanied by a decrease in epithelial a3B1 integrin receptor expression adjacent to the epithelial basement membrane and of Lutheran blood group glycoprotein in the smooth muscle cells, indicating that these receptors are likely mediating the a5 interactions. Taken together, the laminin a5 chain is essential for the normal development of the intestinal smooth muscle.
Gawlik K. and Durbeej M.
Skelet Muscle. 2011
In this review, the authors introduce laminin-211 and describe its structure, expression pattern in developing and adult muscle and its receptor interactions. They also discuss the molecular pathogenesis of MDC1A and advances toward the development of treatment.
Ritié L., Spenlé C., Lacroute J.I., Bolcato-Bellemin A-L., Lefebvre O., Bole-Feysot C., Jost B., Klein A., Arnold C., Kedinger M., Bagnard D., Orend G., Simon-Assmann P.
PLOS ONE, 2012
Laminin-511 is highly expressed in the intestine. To understand the mechanistic role of laminin-511 in tissue homeostasis, the researchers used RNA profiling of embryonic intestinal tissue of lama5 knockout mice and identified a lama5 specific gene expression signature. They show that laminin a5 plays a crucial role in both epithelial and mesenchymal (smooth muscle) cell behavior by inhibiting Wnt and activating PI3K signaling. We conclude that conflicting signals are elicited in the absence of lama5, which alter cell adhesion, migration as well as epithelial and muscle differentiation. The LMa5 deficient intestine also displays a smooth muscle defect and myogenic differentiation markers are affected. Laminin-511 supports adhesion of epithelial cells and Akt phosphorylation. Laminin-511 stimulates the spreading of epithelial and muscle cells (compared to laminin-111). Inhibition of Akt with wortmannin abolished spreading of epithelial cells on laminin-511 as evidenced by cell laminin-511 specifically activates Akt through the PI3K pathway in intestinal epithelial but not in mesenchymal cells. Cell migration was also higher on Laminin-511. Laminin-511 also protects cells against H2O2-induced apoptosis.
Chen Z-L., Yao Y., Norris E.H., Kruyer A., Jno-Charles O., Akhmerov A., Strickland S.
J Cell Biol. 2013
Astrocytes express laminin-111 and 211 and assemble basement membranes (BMs) at their endfeet. Here the authors show that ablation of astrocytic laminin disrupted endfeet BMs and led to hemorrhage in deep brain regions of adult mice. The lack of astrocytic laminin led to impaired function of vascular smooth muscle cells, fragmentation and vascular wall disassembly where astrocytes have a closer association with VSMCs in small arterioles. Acute disruption of astrocytic laminin in the striatum of adult mice also impaired vascular smooth muscle cells function, indicating that laminin is necessary for vascular smooth muscle cell maintenance. In vitro, both astrocytes and astrocytic laminin promoted brain vascular smooth muscle cell differentiation.