Here is a selection of publications where different laminin isoforms were used to create more authentic cell culture systems.

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

    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.

  • Organotypic arrangement of mouse embryonic lung cells on a basement membrane extract: involvement of laminin

    L Schuger, KS O'Shea, BB Nelson, J Varani. Development, 1990

    In this article, the authors cultured single cell suspensions generated by trypsinization of lungs removed from day 12 embryos. The results strongly supports a role for the basement membrane in the organotypic rearrangement of embryonic lung cells and subsequent in vitro cyst formation and budding of the reestablished epithelium.

  • Synergistic activities of alpha3 and alpha6 integrins are required during apical ectodermal ridge formation and organogenesis in the mouse

    Both integrin α6-null mice α3-null mice die at birth, with kidney and lung defects at late stages of development, and skin blistering. To investigate possible overlapping functions between α3 and α6 integrins, we analyzed the phenotype of compound α3−/− and α6−/− mutant embryos. Double homozygous mutant embryos were growth-retarded and displayed several developmental defects not observed in the single mutant animals. The presence of novel phenotypes in double mutant embryos demonstrates the synergism between α3 and α6 integrins and their essential roles in multiple processes during embryogenesis.

  • Identification of laminin domains involved in branching morphogenesis: Effects of anti-laminin monoclonal antibodies on mouse embryonic lung development

    Schuger L., Skubitz A.P.N, O'Shea K.S., Chang J.F., Varani J.Developmental biology, 1991

    Using a series of monoclonal anti-laminin antibodies, the deposition and functional involvement of different laminin domains in the developing lung were investigated. Immunohistochemistry reactivity was largely localized to the basement membrane but was also present diffusely in the extracellular matrix throughout the mesenchyme. Organ cultures of lung explants from Day 12 embryos were used. Although all antibodies penetrated the tissues in culture, only laminin alpha-1 and a-5 antibodies inhibited branching activity. Laminin alpha-2, -3, and -4 antibodies did not alter lung morphogenesis.

  • Laminin α1 Chain Synthesis in the Mouse Developing Lung: Requirement for Epithelial–Mesenchymal Contact and Possible Role in Bronchial Smooth muscle Development

    Schuger L., Skubitz A.P.N., Zhang J., Sorokin L., He L. The Journal of Cell Biology, 1997

    The present study shows that whereas a1 and a2 laminins are synthesized in the mouse developing lung and in epithelial-mesenchymal co-cultures derived from it, epithelial and mesenchymal monocultures lose their ability to synthesize the laminin α1 chain. Synthesis of laminin α1 chain, however, returns upon re-establishment of epithelial-mesenchymal contact. Cell-cell contact is critical since laminin α1 chain is not detected in monocultures exposed to coculture-conditioned medium or in epithelial-mesenchymal cocultures in which heterotypic cell-cell contact is prevented by an interposing filter. Immunohistochemical studies on cocultures treated with brefeldin A, an inhibitor of protein secretion, indicated both epithelial and mesenchymal cells synthesize laminin α1 chain upon heterotypic cell-cell contact. Lung explants exposed to monoclonal antibodies to laminin α1 chain exhibited alterations in peribronchial cell shape and decreased smooth muscle development, as indicated by low levels of smooth muscle a actin and desmin. Taken together, the data suggest that laminin α1 chain synthesis is regulated by epithelial-mesenchymal interaction and may play a role in airway smooth muscle development.

  • Epithelial laminin α5 is necessary for distal epithelial cell maturation, VEGF production, and alveolization in the developing murine lung

    Nguyen N.M., Kelley D.G, Schlueter J.A., Meyer M.J., Senior R.M., Miner J.H.Developmental Biology, 2005

    Laminin α5 is prominent in the basement membrane of alveolar walls, airways, and pleura in developing and adult lung. To identify roles for laminin α5 in lung development, the authors have generated an inducible lung epithelial cell-specific Lama5 null mouse. Lama5 null embryos exposed to doxycycline from E6.5 died a few hours after birth. Compared to control littermates, Lama5 null mice lungs had dilated, enlarged distal airspaces, but the basement membrane ultrastructure was preserved. Distal epithelial cell differentiation was perturbed, with a marked reduction of alveolar type II cells and a virtual absence of type I cells. Cell proliferation was reduced and apoptosis was increased. Capillary density was diminished, and this was associated with a decrease in total lung VEGF production. Overall, these findings indicate that epithelial laminin α5, independent of its structural function, is necessary for murine lung development, and suggest a role for laminin α5 in signaling pathways that promote alveolar epithelial cell differentiation and VEGF expression.

  • Lung development in laminin γ2 deficiency: abnormal tracheal hemidesmosomes with normal branching morphogenesis and epithelial differentiation

    Nguyen N.M., Pulkkinen L., Schlueter J.A, Meneguzzi G., Uitto J., Senior R.M.Respiratory Research, 2006

    Laminin-332 is prominent in the basement membrane of alveolar walls and airways of developing and adult lung. A function for laminin γ2 in lung development has been hypothesized. Compared to littermate controls, Lamc2-/- lungs were similar in morphology during embryonic life. Epithelial differentiation did not reveal a difference between Lamc2-/- and littermate control lungs. Likewise, vascular development, smooth muscle cell differentiation, and elastic fiber formation looked similar, as did airway basement membrane ultrastructure. Branching morphogenesis by lung bud culture was similar in Lamc2-/- and littermate control lungs. These findings indicate that laminin γ2 and laminin-332, despite their prominence in the lung, have a minimal role in lung development through the saccular stage.

  • Differential Expression of Laminins and Their Integrin Receptors in Developing and Adult Human Lung

    Virtanen I., Laitinen A., Tani T., PiUikko P., Laitinen L.A., Burgeson R.E., LehtoV-P.Am. J. Respir. Cell Mol. BioI. 1996

    Here, the authors studied the expression of different laminin chains and their integrin receptors in fetal and adult lung by immunohistochemistry. Special attention was focused on the changes in the expression of these proteins during the development from the pseudoglandular (PG) and canalicular stages to adult lung and on the possible implications of the changes for the normal lung development. The most significant changes in the expression pattern were found during the development from the PO stage to the canalicular stage. Basement membranes (BM) of both the epithelial buds and the becoming bronchi showed reactivity for laminin α1, a3, and B3 chains at all stages. The a2 chain was expressed only in the epithelial buds at the PO stage, and could not be found in any epithelial structures at the canalicular stage. Similarly, at the PG stage the B2 chain was expressed in BMs of both epithelial buds and bronchi but disappeared from the bronchial BM before the canalicular stage. The B1 chain appeared in the bronchial BM first in the mature lung. There were considerable changes in the expression of integrins (lnt) comcomitantly with alterations in the composition of the BMs. At the PG stage, the epithelial buds showed reactivity for integrin a2, a3, and a6 subunits, but at the canalicular stage the integrin a2 and a6 subunits disappeared, and only the a3 integrin subunit was found in evolving alveolar walls; integrin a6 was found in capillaries.  Our results suggest that there are major changes in the expression of laminin and their integrin receptors during morphogenesis of the lung, which may be important for normal development.

  • Expression Patterns of Laminin α1 and α5 in Human Lung during Development

    Pierce R.A, Griffin G.L., Miner J.H., Senior R.M.
    Am. J. Respir. Cell Mol. Biol., 2000

    To determine the temporal and spatial patterns of laminin α1 and laminin α5 in developing human lung, the authors prepared complementary DNA probes specific for laminin α1 and α5 messenger RNAs (mRNAs). Laminin α1 mRNA was prominent in first trimester fetal lung but was not detectable at 23 wk or at later times. In contrast, laminin α5 mRNA was readily detected in early fetal lung and remained present thereafter. Immunohistochemical staining demonstrated laminin a 1 only in early fetal lung, whereas laminin α5 was persistent from the early fetal period. In situ hybridization localized laminin α1 expression to the distal epithelium in the first-trimester lung, and laminin α5 to all epithelium and developing pulmonary arteries from the first trimester through the perinatal period. These studies indicate that laminin α1 expression is restricted to early human lung morphogenesis, whereas the expression of laminin α5 in the human lung is continuous from early lung development through adult life. It is evident that laminin α1 and laminin α5 have different roles in the development of the human lung.

  • Laminin isoforms and lung development: All isoforms are not equal

    Developmental Biology, 2006

    The embryonic lung has abundant laminin isoforms. Studies of embryonic lung explants and organotypic co-cultures show that laminin α1 and laminin 111 are important for epithelial branching morphogenesis and that laminin α2 and laminin 211 have a role in smooth muscle cell differentiation. In vivo studies of laminin α5-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. Laminin α4 null mice do not have obvious lung abnormalities and laminin γ2 null mice have only minimal changes in lung development. It is clear that multiple laminin isoforms are crucial for lung development and that different laminin isoforms exhibit specific, nonoverlapping functions.