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

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  • Chronic stress induced disturbances in Laminin: a significant contributor to modulating microglial pro-inflammatory tone?

    Pietrogrande G., Mabotuwana N., Zhao Z., Mahmoud A., Johnson S.J., Nilsson M., Walker F.R.Brain, Behavior, and Immunity, 2017.

    In this study, Pietrogrande and colleagues have addressed the potential role of the extracellular matrix protein Laminin as a crucial factor to drive microglia into an inflamed state. Chronic restraint stress of C57BL6 adult mice over six weeks resulted in elevated levels of Laminin-α1 and pro-inflammatory markers such as TNF-α and iNOS, quantified by qPCR and western blot. Immunolabeling of Laminin-α1 identified pyramidal neurons and dentate gyrus to be their primary source within the hippocampus. Furthermore, Iba-1 staining of microglia revealed that chronic stress also strongly reduced the total branch length (15%), number of primary branches (47%) and number of branching points (68%) when compared to microglia of control mice. In vitro, primary microglia and BV2 cells grown on Laminin-111 expressed higher levels of TNF-α, IL-1β, and iNOS. In addition, LPS activation of microglia coated on Laminin-111 led to an increased pro-inflammatory state represented by higher pro-inflammatory cytokines level and phagocytic capability, both before and after stimulation. Interestingly, similar to observations made in vivo, microglia cultured on Laminin- 111 had fewer ramifications compared to control. These results, thus, expose the capability of chronic restraint stress in modulating Laminin within the CNS, an effect that has implications for understanding environmental mediated disturbances of microglial function.

  • Human diseases reveal novel roles for neural lamininsCortical deficiency of laminin γ1 impairs the AKT/GSK-3β signaling pathway and leads to defects in neurite outgrowth and neuronal migration

    Chen Z.L., Haegeli V., Yu H., Strickland S.Developmental Biology, 2009

    In this study, Chen and colleagues demonstrate the importance of laminin γ1 in the cerebral cortex and its absence leading to defects in neuritogenesis and neuronal migration. Mice lacking laminin γ1 gene expression suffered from disrupted cortical layers and impaired axonal pathfinding. Such loss during development has been shown to greatly affect the FAK and paxillin mediated integrin signaling mechanisms. Furthermore, mutant mice also display reduced phosphorylation of GSK-3β and AKT proteins. These data clearly show the participation of both integrin signaling and AKT/GSK-3β pathway in the regulation of neurite growth and neuronal migration by laminins.

  • Laminin/β1 integrin signal triggers axon formation by promoting microtubule assembly and stabilization

    Lei W.L., Xing S.G., Deng C.Y., Ju X.C., Jiang X.Y., Luo Z.G.Cell Research 2012

    In this study, the authors present several lines of evidence implicating the indispensable role of laminin in promoting neural polarization through integrin b1 (Itgb1) mediated microtubule assembly and stabilization. Laminin coated substrates (either in stripes or gradient) could initiate directional axon growth in undifferentiated neurites of both cultured hippocampal neurons and cortical slices in an Itgb1 dependent manner. Impairing endogenous laminin function either by treatment with exogenous laminins or by abolishing Itgb1 signaling using siRNA, resulted in defective axonal formation. Conditional knock out mice with abrogated Itgb1 expression in dorsal telencephalic progenitors displayed defective expression/activity of neuronal polarity related proteins, SAD and LKB1 kinases in addition to abnormal axonal development of cortical pyramidal neurons. These results not only identify laminin/ integrin b1 signaling as a crucial step in axon initiation and development but also link extracellular matrix adhesion to cytoskeleton remodeling that occurs during neuronal polarization.

  • β2 and γ3 laminins are critical cortical basement membrane components ablation of Lamb2 and Lamc3 genes disrupt cortical lamination and produces dysplasia

    Radner S., Banos C., Bachay G., Li Y.N., Hunter D.D., Brunken W.J., Yee K.T.Developmental Neurobiology 2012

    Here, the authors demonstrate the significance of laminin b2 and g3 expression in maintaining a functional cortical pial basement membrane to which Cajal Retzius and radial glial cells attach and in turn guide neural development. Several isoforms of laminins, those containing b2 and g3 in particular, have been isolated from the brain underlining their importance in CNS functions. In the present study, the authors employ a reverse genetic approach where mice with a homozygous deletion of b2 and g3 genes displayed cortical laminar disorganization. In addition, the ablation of both these laminin chains resulted in the incidence of human cobblestone lissencephaly. Interestingly, heterozygous mice also exhibited disruption of cortical neurons with lesser severity. In fact, similar to b2 distribution, g3 was also observed to be localized in the developing cortex. Mutation in the binding site of the laminin g1 gene results in abnormal cortex lamination.

  • Axon guidance of rat cortical neurons by microcontact printed gradients

    Fricke R., Zentis P.D., Rajappa L.T., Hofmann B., Banzet M., Offenhäusser A., Meffert S.H.Biomaterials, 2011

    Substrate-bound gradients play a crucial role in the axon guidance mechanism eventually leading to the development of complex neural circuits. In this study, the authors have grown single embryonic rat cortical neurons on a discontinuous substrate-bound gradient primarily comprising of laminin/poly-L-lysine (PLL) or PLL alone and examined the corresponding effects on neurite growth and axon guidance. Though different patterns of the substrate-bound gradient in terms of slope, width, and length had varying outcomes, they allowed neural adhesion, controlled neurite growth and guided up to 84% of the axons. The presence of laminin clearly had additional effects on both neurite growth and axon directionality when compared to PLL alone. The authors have thus mimicked the in vivo protein gradient conditions involved in creating defined neural networks during CNS development and successfully established an optimal model that could be used to guide axons of single multipolar neurons in vitro.

  • Modulation of Synapsin I Gene Expression in Rat Cortical Neurons by Extracellular Matrix

    Savettieri G., Mazzola G.A., Rodriguez Sanchez M.B., Caruso G., Di Liegro I., Cestelli A.Cellular and Molecular Neurobiology, 1998

    Here the authors have attempted to dissect the complex interaction between the genetic program and environmental cues involved in neuronal differentiation. In this study, neurons isolated from fetal rat brain (embryonic day 16) cortices were cultured on six different extracellular components, such as laminin-111, collagen, and poly-D-lysine in a chemically defined, neuron-specific medium. Among the ECM components tested, laminin allowed both firm attachment and extensive neurite outgrowth from neuronal cell bodies. Furthermore, laminin strongly increased gene expression the synapse-specific protein,  Synapsin I, a well-known central marker for neuronal differentiation. These results highlight the significance of extracellular matrix components, particularly laminins, for the extension of neurite networks and neuronal differentiation of primary cortical neurons.

  • Neurons From Human Pluripotent Stem Cells Under Xeno-Free Conditions Restore Motor Deficits in Parkinsonian Rodents

    Niclis J.C., Gantner C.W., Alsanie W.F., McDougall S.J., Bye C.R., Elefanty A.G., Stanley E.G., Haynes J.M., Pouton C.W., Thompson L.H., Parish C.L.Stem cells translational medicine, 2016

    In this study, the authors describe the first fully defined feeder- and xenogeneic-free protocol for the generation of vmDA neurons from hPSCs. The protocol is translational across multiple embryonic and induced hPSC lines. hPSCs were cultured xeno-free on laminin-521 in TeSR2. For vmDA differentiation, two xeno-free matrix proteins, vitronectin, and human laminin-521 were compared for their ability to replace Matrigel. Both matrices facilitated appropriate patterning, however, only laminin-521 supported the long-term attachment of neural precursors. This “next-generation” protocol consistently increases both the yield and proportion of vmDAneural progenitors (OTX2/FOXA2/LMX1A) and neurons (FOXA2/TH/PITX3) that display classical vmDA metabolic and electrophysiological properties. The mechanism underlying these improvements are identified and demonstrate clinical applicability with the first report of scalability and cryopreservation of bona fide vmDA progenitors at a time amenable to transplantation. Finally, transplantation of xeno-free vmDA progenitors from LMX1A- and PITX3-eGFP reporter lines into Parkinsonian rodents demonstrate improved engraftment outcomes and restoration of motor deficits.

  • Niche-derived laminin-511 promotes midbrain dopaminergic neuron survival and differentiation through YAP

    Zhang D., Yang S., Toledo E.M., Gyllborg D., Saltó C., Villaescusa J.C., Arenas E.Sci Signal. 2017

    The authors investigated the mechanisms controlling the survival of mDA neurons using embryonic and mDA neurons, midbrain tissue from mice, and differentiated human neural stem cells. The work identifies laminin511-YAP as a key pathway by which niche signals control the survival and differentiation of mDA neurons. Laminin alpha-5 is present in the extracellular matrix surrounding mDA neurons and indeed, the authors found laminin-511 promoted the survival and differentiation of mDA neurons via a novel pathway involving YAP, miR-130a, and PTEN. Laminin-511 bound to integrin a3b1 and activated the transcriptional cofactor YAP. Laminin511-YAP signaling enhanced cell survival by inducing the expression of the microRNA miR-130a, which suppressed the synthesis of the cell death–associated protein PTEN. In addition, laminin511-YAP signaling increased the expression of transcription factors critical for mDA identities, such as LMX1A and PITX3, and prevented the loss of mDA neurons in response to oxidative stress, a finding that warrants further investigation to assess the therapeutic potential for PD patients. The authors propose that by enhancing laminin511-YAP signaling, it may be possible to prevent mDA neuron degeneration in PD or enhance the survival of mDA neurons in cell replacement therapies.

  • Predictive Markers Guide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson’s Disease

    Kirkeby A., Nolbrant S., Tiklova K., Heuer A., Kee N., Cardoso T., Rylander Ottosson D., Lelos M.J., Rifes P., Dunnett S.B., Grealish S., Perlmann T., Parmar M. Cell Stem Cell, 2016

    Here, the authors developed a good manufacturing practice (GMP) differentiation protocol for the highly efficient and reproducible production of transplantable dopamine progenitors from hESCs on laminin-111. They identified predictive markers expressed in dopamine neuron progenitors that correlate with graft outcome in an animal model of Parkinson’s disease. Timed FGF8b resulted in a high yield of caudal VM cells and good graft outcomes correlate with markers of caudal VM and MHB. Commonly used markers did not accurately predict in vivo subtype-specific maturation. Instead, we identified a specific set of markers associated with the caudal midbrain that correlate with high dopaminergic yield after transplantation in vivo. Using these markers, GMP-adapted dopamine differentiation protocol was developed.

  • Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation

    Nolbrant S., Heuer A., Parmar M., Kirkeby A. Nature Protocols, 2017

    Generation of precisely patterned neural cells from human pluripotent stem cells (hPSCs) is instrumental in developing disease models and stem cell therapies. Here, the authors provide a detailed 16-d protocol for obtaining high-purity ventral midbrain (VM) dopamine (DA) progenitors for intracerebral transplantation into animal models and for in vitro maturation into neurons. They have successfully transplanted such cells into the rat; however, in principle, the cells can be used for transplantation into any animal model, and the protocol is designed to also be compatible with clinical transplantation into humans. They show how to precisely set the balance of patterning factors to obtain specifically the caudal VM progenitors that give rise to DA-rich grafts. By specifying how to perform quality control (QC), troubleshooting, and adaptation of the procedure, this protocol will facilitate implementation in different laboratories and with a variety of hPSC lines. To facilitate the reproducibility of experiments and enable the shipping of cells between centers, the authors present a method for cryopreservation of the progenitors for subsequent direct transplantation or terminal differentiation into DA neurons. This protocol is free of xeno-derived products and can be performed under good manufacturing practice (GMP) conditions.