Publications

Generation of a CHIP isogenic human iPSC-derived cortical neuron model for functional proteomics

Catarina Dias, Erisa Nita. Jakub Faktor, Lenka Hernychova, Tilo Kunath, Kathryn L. Ball. STAR Protocols, 2022

This protocol describes the production of gene-edited cells using CRISPR-Cas9 and a patient-derived induced pluripotent stem cell (iPSC) line. Biolaminin 521 was used as the matrix to improve single cell survival and pluripotency. The resulting panel of iPSC lines was differentiated into cortical neurons with the Biolaminin 111 culture matrix. The overall aim was to identify protein and pathway targets for the neuroprotective E3-ubiquitin ligase CHIP, which is important in healthy brain aging. The protocol can be adapted to other proteins and pluripotent stem cell lines.

Functional characterization of human pluripotent stem cell-derived cortical networks differentiated on laminin-521 substrate: comparison to rat cortical cultures

Tanja Hyvärinen, Anu Hyysalo, Fikret Emre Kapucu, Laura Aarnos, Andrey Vinogradov, Stephen J Eglen, Laura Ylä-Outinen, Susanna Narkilahti. Sci Rep, 2019

In this article, differentiation of functionally active hPSC-derived cortical networks on defined laminin-521 substrate is reported. They assessed compared the activity development of hPSC-derived networks to that of widely used rat embryonic cortical cultures using microelectrode array (MEA) measurements. The authors conclude that hPSC-derived neural cultures produced with a defined protocol generate cortical type network activity, and they could be applied as a human-specific model for pharmacological studies and modeling network dysfunctions.

β1 Integrin Maintains Integrity of the Embryonic Neocortical Stem Cell Niche

Loulier et al.
PLOS, 2009

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.

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.

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.

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.

Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture

Nasu M., Takata N., Danjo T., Sakaguchi H., Kadoshima T., Futaki S., Sekiguchi K., Eiraku M., Sasai Y. PLoS ONE, 2012

Here the authors report substantial supporting effects of the extracellular matrix (ECM) protein laminin on the continuous formation of properly polarized cortical NE in a floating aggregate culture of mESCs. The laminin protein used here contained laminin 111 that was generated from cultured cells and co-purified with entactin (nidogen-1/2), a laminin-binding protein. The addition of purified laminin and entactin, even at low concentrations, stabilized the formation of continuous cortical NE as well as the maintenance of the basement membrane and prevented rosette formation. The results indicate that laminin-entactin ECM promotes the formation of structurally stable telencephalic tissues in 3D mESC culture, and supports the morphogenetic recapitulation of cortical development.

Laminin chains in the basement membranes of human thymus

Virtanen I., Lohi J., Tani T., Sariola H., Burgeson R.E., Lehto V-P Histochemical Journal, 1996

The laminin a2 chain has been suggested to be the only laminin a chain expressed in mouse and human thymus. Here, the authors use monoclonal antibodies to study the expression of laminin chains in samples of fetal and 6-year-old human thymus. The subepithelial basement membrane of the capsule of fetal 16- to 18-week thymus presented a bright immunoreactivity for laminin a1, a3, B1, B3 and g1 chains but not for a2 chain, suggesting the expression of laminin-111 and -332. Most cortical and medullary epithelial cells, including Hassall's corpuscles, however, lacked laminin immunoreactivity. In thymic specimens from 6-year-old children, immunoreactivity for the laminin a1, a3, B1, B3 and g1 chains were invariably found in the subepithelial basement membrane of the capsule and that for laminin a2 chain was now also distinct but more heterogeneous. The present results show that the thymic subepithelial basement membrane of the capsule presents properties that are commonly seen in stratified and combined epithelia, and are compatible with suggestions of the antigenic similarity of thymic epithelial cells and keratinocytes.

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.