Biorelevant culture of muscle cells on Biolaminin substrates

Laminin 211, 221 and 521 form an important part of the adult skeletal muscle microenvironment

Laminin proteins are key components of the basal lamina surrounding muscle stem cells and adult cells (Sanes, 2003 and Yin, 2013). Laminins are not only structural proteins of the basal lamina but are also signaling molecules that are important for the adhesion, localization, and function of muscle cells in their niche. Laminin-211, -221, -511 and -521 are the main laminin isoforms expressed in the basal lamina of striated muscles. Laminin 421 and laminin 111 are also expressed in small amounts, preferably at junctional regions (Rogers & Nishimune, 2016). Laminin 211 and laminin 221 are the most abundant laminin isoform in the adult skeletal muscle basement membrane with integrin α7β1 as the main receptor (Holmberg & Durbeej, 2013) and an expression deficiency in the alpha-2 laminins results in muscular dystrophy, accompanied by a dilated cardiomyopathy (Oliviéro, 2000). Laminin 211 is located within the basement membrane that surrounds the sarcolemma, whereas laminin 221 predominates in the neuromuscular and myotendinous junctions.

Laminin is crucial for normal muscle function, evident from naturally occurring mutations in laminin genes. Mutations in the gene encoding laminin a2 chain are the most common cause of congenital muscular dystrophy type 1A (Gawlik & Durbeej, 2011). In a publication by Fontes-Oliveira et al. (2017), the authors show that the 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. By culturing MDC1A myotubes on recombinant Biolaminin 211, the mitochondrial function is restored.  Basal respiration, maximum respiration, and ATP production, as well as basal mitochondrial respiration and maximal mitochondrial respiration capacity, are normalized to control levels in the presence of laminin 211.

Due to their vital role for muscle tissue structure, maintenance, and function, laminin serve as an important tool in protocols aiming to improve cell therapy for muscular deceases. Moreover, the intramuscular injection of laminin has shown to improve muscle regeneration and the efficiency of myoblast transplantation (Riederer, 2015). 

Laminin β-2 immunohistochemical staining of human skeletal muscle shows strong membranous immunoreactivity in myocytes. Anti-LAMB2 antibody
AMAb91096 (Atlas Antibodies).

Biolaminin 521 and 221 are superior substrates for both short-term and long-term myogenic cell expansion and differentiation

During myogenesis, laminin 521 and laminin 511 are the main isoforms surrounding myogenic progenitors (Gawlik & Durbeej, 2011). Indeed, recent data presented by researchers at Icagen Inc. demonstrate that the Biolaminin 521 cell culture matrix maintains the differentiation potential of mouse and human satellite cell-derived myoblasts, even during long-term culture expansion (Penton, 2016). Biolaminin 521 supports increased proliferation during expansion and superior differentiation with myotube hypertrophy, larger myotubes and higher amounts of nuclei per myotube. Moreover, Penton et al. show that Biolaminin 521 supports more consistent and reliable differentiation over long-term culture and is the only substrate facilitating high-level fusion following long-term culture. Biolaminin 521 supports increased differentiation potential without altering the traditional Pax7/MyoD paradigm and the results are translational across several mouse backgrounds, human cells, and disease states. 

Researchers at Stanford that developed artificial muscle fibers (AMFs) that mimic the native myofiber of the MuSC niche show that Biolaminin 211 maintains muscle stem cells (MuSCs) in a potent, quiescent state in vitro. AMFs coated with recombinant Biolaminin 211 showed prolonged quiescence in vitro and enhanced potency in vivo (Quarta, 2016).

An improved method for culturing myotubes on laminins for the robust clustering of postsynaptic machinery

Motor neurons from specialized synapses with skeletal muscle fibers called neuromuscular junctions (NMJs). The function of this type of synapse is to transmit signals from the central nervous system to muscles and thus stimulate their contraction. Muscle cells form a thick ECM around the fiber that contains various laminins, collagens, fibronectin, and other glycoproteins. The basal lamina (BL) at the synaptic cleft has a specific molecular composition that contains laminin α4, α5, and β2 isoforms that are mostly absent in extrasynaptic regions of muscle fibers. These ECM components are crucial for the proper development of NMJs and it has been shown that the laminin-dystroglycan interaction is crucial for regulating NMJ developmental remodeling. 

Little is known about the mechanisms of postsynaptic machinery remodeling in vivo. Mouse laminin-111 has been routinely used to induce AChR clustering and is the only in vitro system where the AChR clusters undergo developmental remodeling that resembles mature NMJs, providing the model to study the underlying mechanisms. In a publication in Scientific Reports (Pęziński, 2020) the authors present 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 the 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 advanced method for culturing myotubes that provide an important basis for high-throughput genetic screening and potential drug development for screening for potential therapeutic targets for neuromuscular disorders.

Protocol:

Long-term expansion and differentiation of myogenic progenitors on laminin

What our customers say

“Laminin-521 shifts away from the paradigm that muscle cells lose their ability to differentiate after culturing. Muscle differentiation on laminin-521 is nothing short of spectacular, yielding increased numbers of nuclei per myotube and improved myotube organization.”

Dr. Christopher Penton

Icagen, Inc., Tucson, Arizona, USA


Biolaminin Key Advantages

Laminin 211 has been shown to contribute to maintaining muscle stem cells (MuSCs) in a potent, quiescent state in vitro and enhanced potency in vivo. Culture on Biolaminin 521 gives larger myotubes and higher amounts of nuclei per myotube. Human primary myotubes that were derived from myoblasts obtained from patient biopsies cultured on Biolaminin-121, -211, -221,-511, and -521 were also able to form AChR clusters with a complex topology that contained synaptic podosomes.

Specific laminin isoforms are present in different tissue microenvironments and they are essential for cell survival, proliferation and differentiation. Biolaminin products allow you to imitate the natural cell-matrix interactions in vitro.

All our matrices are chemically defined and animal origin-free, which makes them ideal substrates for each level of the scientific process – from basic research to clinical applications.

Our products have consistent composition and quality. This enables minimized variability between experiments and uniform pluripotency gene expression profiles between different cell lines.

Numerous scientists have found our products and finally succeeded in their specific stem cell application. The power of full-length laminins incorporated into various cell systems is well documented in scientific articles and clinical trials.



  • Biolaminin 211 LN (LN211)

    Human recombinant laminin 211

    Biolaminin 211 supports the growth, survival, and differentiation of a wide range of tissue-specific cell types, including motor neurons, cardiac cells, and skeletal muscle cells.
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  • Biolaminin 221 LN (LN221)

    Human recombinant laminin 221

    Biolaminin 221 supports the growth, survival, and differentiation of a wide range of tissue-specific cell types, including cardiac cells and skeletal muscle cells.
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  • Biolaminin 521 LN (LN521)

    Human recombinant laminin 521

    Biolaminin 521 LN is the natural laminin for pluripotent stem cells and therefore reliably facilitates self-renewal of human ES and iPS cells in a chemically defined, feeder-free and animal origin-free stem cell culture system. LN521 is animal origin-free to the primary level.
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