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Neurons and Glial Cells

Laminin α5 substrates promote survival, network formation and functional development of human pluripotent stem cell-derived neurons in vitro

Anu Hyysalo A., Ristola M., Mäkinen M.E.-L., Häyrynen S., Nykter M., Narkilahti S.
Stem Cell Research, 2017

Laminins are one of the major protein groups in the extracellular matrix (ECM) and specific laminin isoforms are crucial for neuronal functions in the central nervous system in vivo. In the present study, we compared recombinant human laminin isoforms (LN211, LN332, LN411, LN511, and LN521) and laminin isoform fragment (LN511- E8) in in vitro cultures of human pluripotent stem cell (hPSC)-derived neurons. We showed that laminin substrates containing the α5-chain are important for neuronal attachment, viability and network formation, as detected by phase contrast imaging, viability staining, and immunocytochemistry. Gene expression analysis showed that the molecular mechanisms involved in the preference of hPSC-derived neurons for specific laminin isoforms could be related to ECM remodeling and cell adhesion. Importantly, the microelectrode array analysis revealed the widest distribution of electrophysiologically active neurons on laminin α5 substrates, indicating most efficient development of neuronal network functionality. This study shows that specific laminin α5 substrates provide a controlled in vitro culture environment for hPSC-derived neurons. These substrates can be utilized not only to enhance the production of functional hPSC-derived neurons for in vitro applications like disease modeling, toxicological studies, and drug discovery, but also to produce clinical grade hPSC-derived cells for regenerative medicine applications.

    

Laminin-332 coordinates mechanotransduction and growth cone bifurcation in sensory neurons

Chiang L.Y., Poole K., Oliveira B.E., Duarte N., Sierra Y.A., Bruckner-Tuderman L., Koch M., Hu J., Lewin G.R.
Nature Neuroscience 2011

In this study, laminin 332 produced by mouse epidermal keratinocytes was found to be non-permissive to the transduction of mechanical force into electrical signals (mechanotransduction) in cultured dorsal root ganglion (DRG) sensory neurons in vitro. In addition, the inhibitory effect of laminin 332 was more pronounced on the sensory neurons with rapidly adapting mechanosensitive currents compared to the intermediately and slowly adapting counterparts. Despite observing the slowly adapting mechanosensitive currents in sensory neurons grown on primary keratinocytes or keratinocyte derived matrix, the kinetics of such currents were slower than the control. Loss of laminin 332 alone was sufficient to recapitulate the suppressive effect on rapidly adapting mechanosensitive currents. Furthermore, such an effect was independent of both a3B1 and a6B4 interaction with laminin 332. Interestingly, this mechanotransduction inhibition was carried out at local sites by preventing the formation of protein tethers that connect the sensory membrane to the laminin substrate. Experiments involving cultured sensory neurons on laminin- 1 or -332 matrices revealed the suppressive effect of laminin 332 on the sensory neuron bifurcation. Subsequently, loss of this laminin in humans resulted in increased sensory terminal branching and possible de-repression of mechanosensitive currents. These data thus prove the crucial role of laminin 332 in suppressing mechanosensitivity and axonal branching of sensory neurons at the dermo-epidermal border and also serve as an explanation for the extreme pain experienced by JEB patients with laminin- 332 deficiency.

 

Aligned Poly(ε-caprolactone) Nanofibers Guide the Orientation and Migration of Human Pluripotent Stem Cell-Derived Neurons, Astrocytes, and Oligodendrocyte Precursor Cells In Vitro

Hyysalo A., Ristola M., Jpki T., Honkanen M., Vippola M., Narkilahti S.
Macromolecular Bioscience, 2017

Biomaterials can be used as supporting scaffolds, as they mimic the characteristics of the natural cellular environment. In this study, hPSC-derived neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) are cultured on aligned poly(ε-caprolactone) nanofiber platforms, which guide cell orientation to resemble that of spinal cord in vivo. Human neurons and astrocytes require extracellular matrix molecule coating for the nanofibers, but OPCs grow on nanofibers without additional treatment. Clinically relevant human recombinant laminin substrates (laminin-521 and laminin-511) are compatible with PCL nanofibers and can be used for efficient culturing of hPSC-derived neurons and astrocytes on PCL nanofibers. Furthermore, the nanofiber platform is combined with a 3D hydrogel scaffold with controlled thickness, and nanofiber-mediated orientation of hPSC-derived neurons is also demonstrated in a 3D environment.

  

Human mesenchymal cells from adipose tissue deposit laminin and promote regeneration of injured spinal cord in rats

Menezes K., Nascimento M.A., Gonçalves J.P., Cruz A.S., Lopes D.V., Curzio B., Bonamino M., de Menezes J.R., Borojevic R., Rossi M.I., Coelho-Sampaio T.
PLOS ONE, 2014

Here the authors investigated the regenerative properties of human adipose tissue derived stromal cells (hADSCs) in a rat model of spinal cord compression. Cells were delivered directly into the spinal parenchyma immediately after injury. Human ADSCs promoted functional recovery, tissue preservation, and axonal regeneration. Analysis of the cord tissue showed an abundant deposition of laminin of human origin at the lesion site and spinal midline; the appearance of cell clusters composed of neural precursors in the areas of laminin deposition, and the appearance of blood vessels with separated basement membranes along the spinal axis. These effects were also observed after injection of hADSCs into non-injured spinal cord. Considering that laminin is a well-known inducer of axonal growth, as well a component of the extracellular matrix associated to neural progenitors, the authors propose that it can be the paracrine factor mediating the pro-regenerative effects of hADSCs in spinal cord injury.

 

Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization

Glasgow H.L., Whitney M.A., Gross L.A., Friedman B., Adams S.R., Crisp J.L., Hussain T., Frei A.P., Novy K., Wollscheid B., Nguyen Q.T., Tsien R.Y.
PNAS, 2106

Here, the extracellular matrix proteins laminin-421 and -211 were identified as NP41 binding targets, and TRICEPS-based glycoprotein capture supported laminin-421 as the primary binding target. Fluorescently labeled nerve-binding peptide NP41 holds promise to reduce surgical nerve damage and facilitate nerve repair. Clinical translation hinges on identification of binding targets to assess potential toxicity and understand the mechanism. For target identification, the authors developed a receptor capture method, enabling covalent tagging and identification of proteins within close proximity to a bound ligand. The results explain the ability of NP41 to highlight degenerated nerve “ghosts” months after transection that were invisible to the unaided eye but contain laminins. Targeting extracellular matrix is advantageous for clinical imaging agents, likely reducing undesirable neurological effects. 

 

Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells

Manno, Gyllborg, Codeluppi, Nishimura, Salto, Zeisel, Borm, Stott, Toledo, Villaescusa, Lönnerberg, Ryge, Barker, Arenas, Linnarsson.
Cell, 2016

Manno and colleagues used single-cell RNA sequencing to examine ventral midbrain development in human and mouse. They found that cell types and gene expression were generally conserved across species, but with clear differences in cell proliferation, developmental timing, and dopaminergic neuron development. Additionally, they quantitatively assessed the fidelity of dopaminergic neurons derived from human pluripotent stem cells, at a single-cell level. The study provides insight into the molecular programs controlling human midbrain development and provides a foundation for the development of cell replacement therapies.

 

A Novel In Vitro Method for Detecting Undifferentiated Human Pluripotent Stem Cells as Impurities in Cell Therapy Products Using a Highly Efficient Culture System

Tano K., Yasuda S., Kuroda T., Saito H., Umezawa A., Sato Y.
PLOS ONE, 2014

Tano and colleagues show a novel approach based on LN-521 for direct and sensitive detection of trace amounts of residual undifferentiated hPSCs for cell therapy products. The presence of contaminating hPSCs in cell therapy products is a major quality concern associated with tumorigenicity and this first in vitro assay is direct, simple and cost-effective. The highly efficient culture system using LN-521 detected colony forming hPSCs spiked into primary human MSCs or neurons at a ratio as low as 0.001%–0.01%.

  

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 we report substantial supporting effects of the extracellular matrix (ECM) protein laminin on the continuous formation of properly polarized cortical NE in 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 basement membrane and prevented rosette formation. Treatment with the neutralizing ß1-integrin antibody impaired the continuous NE formation. The stabilized cortical NE exhibited typical interkinetic nuclear migration of cortical progenitors, as seen in the embryonic cortex. The laminin-treated cortical NE maintained a continuous structure and contained ventricular, basal-progenitor, cortical-plate and Cajal-Retzius cell layers. The cortical NE in this culture was flanked by cortical hem-like tissue. Furthermore, when Shh was added, ventral telencephalic structures such as lateral ganglionic eminence–like tissue formed in the region adjacent to the cortical NE. Thus, our 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-411 Is a Vascular Ligand for MCAM and Facilitates TH17 Cell Entry into the CNS

Flanagan K., Fitzgerald K., Baker J., Regnstrom K., Gardai S., Bard F., Mocci S., Seto P., You M., Larochelle C., Prat A., Chow S., Li L., Vandevert C., Zago W., Lorenzana C., Nishioka C., Hoffman J., Botelho R., Willits C., Tanaka K., Johnston J., Yednock T.
PLOS ONE, 2012

TH17 cells enter tissues to facilitate pathogenic autoimmune responses. Herein, they characterize MCAM (CD146) as an adhesion molecule that defines human TH17 cells. Parental CHO cells, lacking MCAM expression, or CHO cells stably transfected with mouse MCAM were incubated in the presence of laminin-411 or laminin-511. They identify the MCAM ligand as laminin 411, an isoform of laminin expressed within the vascular endothelial basement membranes. hMCAM binds to a ligand in the ECM with identical staining to laminin a4. Moreover, mMCAM colocalizes with laminin 411 on the choroid plexus, and shows no specific binding to tissues from LAMA4 -/- mice. Their data suggest that MCAM and laminin-411 interact to facilitate TH17 cell entry into tissues and promote inflammation. The specific location of laminin 411 in the endothelial basement membrane may either function to augment adhesion of cells attempting CNS endothelial penetration, or serve as an adhesion based gating system to signal appropriate entry mechanisms. As such, modulation of the interaction between MCAM and laminin 411 represents a novel and selective approach that may help to maintain or restore homeostasis to inflamed tissues in autoimmune diseases.

 

Quantification of molecular interactions between ApoE, amyloid-beta (Aβ) and laminin: Relevance to accumulation of Aβ in Alzheimer's disease

Zekonyte J., Sakai K., Nicoll J.A.R., Weller R.O., Carare R.O.
Biochimica et Biophysica Acta, 2015

Accumulation of amyloid-β (Aβ) in plaques in the brain and in artery walls as cerebral amyloid angiopathy indicates a failure of elimination of Aβ from the brain with age and Alzheimer's disease. A major pathway for elimination of Aβ and other soluble metabolites from the brain is along basement membranes within the walls of cerebral arteries that represent the lymphatic drainage pathways for the brain. Since Aβ40 is the predominant type of Aβ found in cerebral amyloid angiopathy, in the present study we tested the hypothesis that interactions of Aβ40 with protein components of cerebral vascular basement membranes, such as laminin, are stronger in the presence of ApoE3 than in the presence of ApoE4. Proteins (LN-511, ApoE3, or ApoE4) were immobilized on AFM probes using the amine–amine reactive linker aldehyde–PEG–NHS. Force-spectroscopy experiments were performed to study the reciprocal influence of different isoforms of ApoE and Aβ on their binding interactions with laminin-511. The results show that apolipoprotein E co-localizes with Aβ in basement membrane drainage pathways in the walls of arteries. Moreover, all AFM measurements demonstrate that Aβ + ApoE3 complex has a stronger binding to laminin-511 than Aβ + ApoE4. These results suggest that perivascular elimination of ApoE4/Aβ complexes would be less efficient than with other isoforms of apolipoprotein E, thus endowing a higher risk for Alzheimer's disease. Therapeutic correction for ApoE4/Aβ/laminin interactions may increase the efficiency of elimination of Aβ in the prevention of Alzheimer's disease.

 

Laminin and growth factor receptor activation stimulates differential growth responses in subpopulations of adult DRG neurons

Tucker B.A., Rahimtula M., Mearow K.M.
European Journal of Neuroscience, 2006

Here they show laminin-induced neurite outgrowth and its relation to three known DRG neuronal types. They also show PI3K pathway is responsible. They also discuss this in the light of possible therapeutic targets. The study is limited in that that they only use invitrogen laminin (purified laminin-111) and isoform-specific effects cannot be seen, but they come to a number of highly interesting conclusions: 1) The current findings provide strong support for the use of the ECM molecule laminin in conjunction with NGF and GDNF in order to stimulate optimal levels of axon growth from all populations of regenerating sensory neurons. 2) identified intracellular signaling components that provide potential therapeutic targets when attempting to stimulate regeneration of peripheral axons. Pharmacological alterations of the PI 3-K/Akt pathway resulting in activation of either PI 3-K or Akt could be beneficial. 3) Laminin-induced neurite growth occurs in the absence of added trophic factors only in heavy-chain neurofilament-positive and calcitonin gene-related peptide-positive DRG neurons [nerve growth factor (NGF)-responsive population]. In contrast, laminin alone is not sufficient to stimulate significant neurite growth from lectin Griffonia simplicifolia IB4-positive neurons (IB4+ve), although it is still required to elicit a growth response from these cells in the presence of glial-derived neurotrophic factor. 

 

Adult SVZ Stem Cells Lie in a Vascular Niche: A Quantitative Analysis of Niche Cell-Cell Interactions

Shen Q., Wang Y., Kokovay E., Lin G., Chuang S-M. Goderie S.K., Roysam B., Temple S.
Cell Stem Cell, 2008

Here, they examine the relationship of adult SVZ NSC lineage cells to blood vessels using confocal imaging of SVZ whole mounts in which the normal 3D relationships of cells are preserved. Neural stem cells (NSCs) in the adult subventricular zone (SVZ) lie close to blood vessels in a vascular-derived laminin-rich niche. Cells expressing stem cell markers, including GFAP, and proliferation markers are closely apposed to the laminin-containing extracellular matrix (ECM) surrounding vascular endothelial cells. Adult SVZ progenitor cells express the laminin receptor a6B1 integrin, and blocking this inhibits their adhesion to endothelial cells, altering their position and proliferation in vivo.

 

Laminin enhances the growth of human neural stem cells in defined culture media 

Hall et al.
BMC Neuroscience, 2008

 

Transcriptomes of germinal zones of human and mouse fetal neocortex suggest a role of extracellular matrix in progenitor self-renewal

Fietz et al.
PNAS. 2012.

 

The hippocampal laminin matrix is dynamic and critical for neuronal survival

Chen et al.
Mol Biol Cell., 2003

 

Hippocampal neurons: Laminin chain expression suggests that laminin-10 is a major isoform in the mouse hippocampus and is degraded by the tissue plasminogen activator/plasmin protease cascade during excitotoxic injury

Indyk et al.
Neuroscience, 2003

 

Functional Diversity of Laminins 

Domogatskaya et al.
Annu. Rev. Cell Dev. Biol., 2012

 

β1 Integrin Maintains Integrity of the Embryonic Neocortical Stem Cell Niche

Loulier et al.
PLOS, 2009

 

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 reproducibility of experiments and enable 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.

 

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 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 high yield of caudal VM cells and good graft outcome 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, a GMP-adapted dopamine differentiation protocol was developed.

 

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 identity, 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 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.

  

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 demonstrates improved engraftment outcomes and restoration of motor deficits.

 

A defined xeno-free and feeder-free culture system for the derivation, expansion and direct differentiation of transgene-free patient-specific induced pluripotent stem cells

Lu H.F., Chai C., Lim T.C., Leong M.F., Lim J.K., Gao S., Lim K.L., Wan A.C.
Biomaterials, 2014

Reprogramming of iPSCs on LN-521 and direct differentiation to dopaminergic cells on laminin-521.This article demonstrates LN-521 as an optimal defined, xeno- and feeder-free matrix for the reprogramming of human iPS cells. LN-521 achieves high-efficiency reprogramming in different media, fast and easy expansion as well as direct differentiation to dopaminergic neurons on LN-521. The authors conclude that the efficient transgene-free hiPSC derivation and expansion on LN-521 enables clinical applications useful for human patient iPSCs and derivatives for cellular therapy.

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 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 highlights the significance of extracellular matrix components, in particularly laminins, ifor 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 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 upto 84% of the axons. 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 disrupts 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 reverse genetic approach where mice with homozygous deletion of b2 and g3 genes displayed cortical laminar disorganization. In addition, 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 laminin g1 gene results in abnormal cortex lamination.

 

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 a 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.

 

Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks

Shi Y., Kirwan P., Livesey F.J.
Nature Protocols, 2012

Here, the authors provide a detailed protocol for generation of both deep- and upper-layer excitatory neurons from human human pluripotent stem cells (PSCs) that are both electrically active and form functional cortical circuits. The authors prove that their protocol helps to generate both primary neuroepithelial cells and secondary cortical stem/progenitor cells from hPSCs. Laminin coated dishes were used to plate neuroepithelial cells derived from PSCs and maintained in neural induction/maintenance medium for neurogenesis. Furthermore, addition of vitamin A has been shown to be pivotal for the efficient induction of secondary cortical stem/progenitor cells and cortical neurogenesis. The time frame for cortical neurogenesis and functional synapse formation closely mimic human cortical development in utero and part from facilitating a better understanding of disease mechanisms, this culture system could be instrumental for tissue engineering of cortical implants.

 

Sustained synchronized neuronal network activity in a human astrocyte co-culture system

Kuijlaars J., Oyelami T., Diels A., Rohrbacher J., Versweyveld S., Meneghello G., Tuefferd M., Verstraelen P., Detrez J.R., Verschuuren M., De Vos W.H., Meert T., Peeters P.J., Cik M., Nuydens R., Brône B., Verheyen A.
Scientific reports (Nature), 2016

In this report, Kuijlaars J et al have developed a human in vitro co-culture model comprising of human induced pluripotent stem cells (hiPSCs) and astrocytes that could recapitulate sustained activity, bursting frequency and calcium oscillation synchronization of cortical neurons over time. In this co-culture model, the neuroepithelial sheets derived from hiPSCs were transferred to laminin coated plates and grown until their differentiation into cortical neurons expressing key markers such as TBR1, CTIP2 and SATB2. Another important feature is the balanced existence of both GABAergic and glutaminergic neurons which is critical for the maturity and function of a neuronal network.

 

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-111 on the continuous formation of properly polarized cortical NE in floating aggregate culture of mESCs. 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 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 basement membrane and prevented rosette formation. Treatment with the neutralizing ß1-integrin antibody impaired the continuous NE formation. The stabilized cortical NE exhibited typical interkinetic nuclear migration of cortical progenitors, as seen in the embryonic cortex. The laminin-treated cortical NE maintained a continuous structure and contained ventricular, basal-progenitor, cortical-plate and Cajal-Retzius cell layers. When Shh was added, ventral telencephalic structures such as lateral ganglionic eminence–like tissue formed in the region adjacent to the cortical NE.

 

Human diseases reveal novel roles for neural laminins

Colognato H., ffrench-Constant C., Feltri M.L.
Trends in Neurosciences, 2005

Cortical abnormalities have been linked to transgenic mice lacking  alpha 6 beta 1 and alpha 3 beta 1 receptors . Notably, laminin 111, 411 (alpha 6 beta 1) and 332, 511 and 521 (alpha 3 beta 1) are the corresponding ligands.

 

Cortical 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.

 

The hippocampal laminin matrix is dynamic and critical for neuronal survival. Chen et al., Mol Biol Cell. 2003


Laminin chain expression suggests that laminin-10 is a major isoform in the mouse hippocampus and is degraded by the tissue plasminogen activator/plasmin protease cascade during excitotoxic injury. Indyk et al. Neuroscience. 2003


Differential effects of laminin isoforms on axon and dendrite development in hippocampal neurons. Fusaoka-Nishioka et al., Neurosci Res. 2011


Functional Diversityof Laminins. Domogatskaya et al., Annu. Rev. Cell Dev. Biol. 2012

Spinal cord neurons: Impeded interaction between Schwann cells and axons in the absence of laminin alpha4. Wallquist et al., J Neurosci. 2005


Laminins 2 (alpha2beta1gamma1, Lm-211) and 8 (alpha4beta1gamma1, Lm-411) are synthesized and secreted by tooth pulp fibroblasts and differentially promote neurite outgrowth from trigeminal ganglion sensory neurons. Fried et al., Exp Cell Res. 2005


Integrin-laminin interactions controlling neurite outgrowth from adult DRG neurons in vitro. Plantman etal., Mol Cell Neurosci. 2008


Properly formed but improperly localized synaptic specializations in the absence of laminin alpha4. Patton et al., Nat Neurosci. 2001


Induction, assembly, maturation and maintenance of a postsynaptic apparatus. Sanes and Lichtman, Nature Reviews Neuroscience, 2001


Functional Diversityof Laminins. Domogatskaya et al., Annu. Rev. Cell Dev. Biol. 2012


Laminin-332 coordinates mechanotransduction and growth cone bifurcation in sensory neurons. Chiang et al. Nature Neuroscience, 2011


Loss of laminin-α4 results in pre- and postsynaptic modifications at the neuromuscular junction. Chand et al. FASEB Journal, 2017

Human iPS-Derived Astroglia from a Stable Neural Precursor State Show Improved Functionality Compared with Conventional Astrocytic Models

Lundin A., Delsing L., Clausen M., Ricchiuto P., Sanchez J., Sabirsh A., Ding M., Synnergren J., Zetterberg H., Brolén G., Hicks R., Herland A., Falk A.
Stem Cell Reports, 2018

hiPSC-derived astrocytes provide a promising source of astrocytes for screening models, but generally the protocols are extensive and use undefined conditions, introducing variability. Here, the authors report human induced pluripotent stem cell (hiPSC)-derived astroglia (NES-Astro) developed under defined conditions through long-term neuroepithelial-like stem (ltNES) cells. NES-Astro and astrocytic models from primary sources, astrocytoma (CCF-STTG1), and hiPSCs were characterized. The authors show (1) the application of ltNES in directed glia generation, with (2) high reproducibility across hiPSC lines, and (3) proven astrocyte functions including SLC1A3-driven glutamate uptake, immune competence, and calcium responsiveness to neurotransmitters. With the use of human recombinant laminin-521, they also show (4) the use of an animal-free culture system applicable for good manufacturing practice and clinical adaptation, and (5) NES-Astro as a cryopreservable cell model for direct application in pharmacological HTS. The authors also assessed modulation of astrocyte biology using APOE-annotated compounds, confirming hits of the cholesterol biosynthesis pathway in adult and hiPSC-derived astrocytes. Current astrocytic in vitro models are questioned for lack of biological characterization. Principal component analysis (PCA) reflect the differences between the five main model types used in this study. Additionally, the PCA results showed a high homogeneity between ltNES lines generated from various donors, but more importantly a high reproducibility by the tight clustering between the NES-Astro lines of different genotypes.

 

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 proinflammatory 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.

 

Aligned Poly(ε-caprolactone) Nanofibers Guide the Orientation and Migration of Human Pluripotent Stem Cell-Derived Neurons, Astrocytes, and Oligodendrocyte Precursor Cells In Vitro

Hyysalo A., Ristola M., Jpki T., Honkanen M., Vippola M., Narkilahti S.
Macromolecular Bioscience, 2017

Biomaterials can be used as supporting scaffolds, as they mimic the characteristics of the natural cellular environment. In this study, hPSC-derived neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) are cultured on aligned poly(ε-caprolactone) nanofiber platforms, which guide cell orientation to resemble that of spinal cord in vivo. Human neurons and astrocytes require extracellular matrix molecule coating for the nanofibers, but OPCs grow on nanofibers without additional treatment. Clinically relevant human recombinant laminin substrates (laminin-521 and laminin-511) are compatible with PCL nanofibers and can be used for efficient culturing of hPSC-derived neurons and astrocytes on PCL nanofibers. Furthermore, the nanofiber platform is combined with a 3D hydrogel scaffold with controlled thickness, and nanofiber-mediated orientation of hPSC-derived neurons is also demonstrated in a 3D environment.

 

YAP and TAZ control peripheral myelination and the expression of laminin receptors in Schwann cells

Poitelon Y., Lopez-Anido C., Catignas K., Berti C., Palmisano M., Williamson C., Ameroso D., Abiko K., Hwang Y., Gregorieff A., Wrana JL., Asmani M., Zhao R., Sim FJ., Wrabetz L., Svaren J., Feltri ML.
Nature Neuroscience, 2016

A mechanistic article just published by Poitelon and colleagues in Nat Neurosci, adding further evidence for the importance of laminin-211 for radial sorting and proper axon myelination by Schwann cells. The authors show that laminin-211 in combination with mechanical stimuli activate and modulate Yap and Taz, that are downstream effectors in the Hippo pathway, required for radial sorting fo axons and subsequent myelination.

 

Laminin 211 inhibits protein kinase A in Schwann cells to modulate neuregulin 1 type III-driven myelination.

Ghidinelli M., Poitelon Y., Shin YK., Ameroso D., Williamson C., Ferri C., Pellegatta M., Espino K., Mogha A., Monk K., Podini P., Taveggia C., Nave K.A., Wrabetz L., Park H.T., Feltri M.L.
PLoS Biology, 2017

Ghidinelli and colleagues, through their in vivo and in vitro studies have clearly shown that Laminin 211, apart from promotion, can also inhibit myelination by modulating neuregulin 1 type III activity via PKA signaling and thereby prevent inappropriate and excessive myelin wrapping of small nerve fibers.

 

The adhesion GPCR GPR126 has distinct, domain-dependent functions in Schwann cell development mediated by interaction with Laminin-211

Petersen S.C., Luo R., Liebscher I., Giera S., Jeong S-J., Mogha A., Ghidinelli M., Feltri M.L., Schöneberg T., Piao X., Monk K.R.
Neuron 2015

The authors demonstrate that the binding of Laminin-211 to the GPR126 either suppress or promote the activation GPR126-CTF and is laminin-211 concentration dependent. Schwann cell myelination starts with activation of the GPR126 which results in cleavage into two fragments, NTF and CTF. NTF initiates radial, axonal sorting and CTF promotes the axonal wrapping thus driving the terminal differentiation. The study beautifully shows the that laminin-211 is a fundamental regulator of Schwann cell biology and that its actual binding to the GPR126 regulate radial sorting and myelination. Laminin-211 as a novel ligand for GPR126 that differentially modulates receptor signaling to control both early and late Schwann cell development. GPR126 is an adhesion GPCR required for Schwann cell myelination.

 

Ablation of astrocytic laminin impairs vascular smooth muscle cell function and leads to hemorrhagic stroke

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 cells maintenance. In vitro, both astrocytes and astrocytic laminin promoted brain vascular smooth muscle cells differentiation.

 

Astrocytic laminin regulates pericyte differentiation and maintains blood brain barrier integrity

Yao Y., Chen Z-L., Norris E.H., Strickland S.
Nature comm, 2014

Here they show that lack of astrocytic laminin, a brain-specific BM component, induces BBB breakdown. Use conditional knockout mice and an acute adenovirus-mediated knockdown model. Using functional blocking antibody and RNAi, we further demonstrate that astrocytic laminin, by binding to integrin a2 receptor, prevents pericyte differentiation from the BBB-stabilizing resting stage to the BBB-disrupting contractile stage, and thus maintains the integrity of BBB. Loss of astrocytic laminin also decreases aquaporin-4 (AQP4) and tight junction protein expression. These results indicate that astrocytic laminin maintains the integrity of BBB through, at least in part, regulation of pericyte differentiation.

 

Laminins containing the β2 and γ3 chains regulate astrocyte migration and angiogenesis in the retina

Gnanaguru G., Bachay G., Biswas S., Pinzón-Duarte G., Hunter D.D. Brunken W.J.
Development, 2013

Astrocytes regulates retinal vascular development. Generated extrinsically to the retina, astrocytes migrate into the retina through the optic nerve head. In this study, we show that astrocytes migrate within a laminin-containing basement membrane. Genetic deletion of the laminin β2 and γ3 chains affects astrocyte migration and spatial distribution. We show that laminins act as haptotactic factors in vitro in an isoform-specific manner, inducing astrocyte migration and promoting astrocyte differentiation. The addition of exogenous laminins to laminin-null retinal explants rescues astrocyte migration and spatial patterning. Furthermore, we show that the loss of laminins reduces β1 integrin expression in astrocytes which can be restored when astrocytes are culture on LN-521 or Matrigel. Finally, they show that laminins containing β2 and γ3 chains regulate subsequent retinal blood vessel growth and maintain vascular integrity. These in vivo and in vitro studies demonstrate clearly that laminins containing β2 and γ3 chains are indispensable for migration and spatial organization of astrocytes and that they play a crucial role during retinal angiogenesis in vivo.

 

The hippocampal laminin matrix is dynamic and critical for neuronal survival. Chen et al., Mol Biol Cell. 2003

 

Impeded interaction between Schwann cells and axons in the absence of laminin alpha4. Wallquist et al., J Neurosci. 2005

 

Laminins 2 (alpha2beta1gamma1, Lm-211) and 8 (alpha4beta1gamma1, Lm-411) are synthesized and secreted by tooth pulp fibroblasts and differentially promote neurite outgrowth from trigeminal ganglion sensory neurons. Fried et al., Exp Cell Res. 2005

 

Integrin-laminin interactions controlling neurite outgrowth from adult DRG neurons in vitro. Plantman etal., Mol Cell Neurosci. 2008

 

Properly formed but improperly localized synaptic specializations in the absence of laminin alpha4. Patton et al., Nat Neurosci. 2001

 

Astrocytic laminin regulates pericyte differentiation and maintains blood brain barrier integrity. Yao et al., Nature Communications 2014

 

Laminin alters fyn regulatory mechanisms and promotes oligodendrocyte development. Relucio et al., J Neurosci. 2009

 

α6β1 and α7β1 integrins are required in Schwann cells to sort axons. Pellegatta et al., J Neurosci. 2013

 

The adhesion GPCR GPR126 has distinct, domain-dependent functions in Schwann cell development mediated by interaction with Laminin-211. Petersen et al., Neuron 2015

Characterization and functional analysis of laminin isoforms in human bone marrow. Siler et al., Blood. 2000


Cytokines regulate microglial adhesion to laminin and astrocyte extracellular matrix via protein kinase C-dependent activation of the alpha6beta1 integrin. Milner and Campbell, J Neurosci. 2002


Monocytic cells synthesize, adhere to, and migrate on laminin-8 (alpha 4 beta 1 gamma 1). Pedraza et al., J Immunol. 2000


Laminin isoform-specific promotion of adhesion and migration of human bone marrow progenitor cells. Gu et al., Blood. 2003


Functional Diversity of Laminins. Domogatskaya et al., Annu. Rev. Cell Dev. Biol. 2012


Origin and differentiation of microglia. Ginhoux et al., Front  Cell Neurosci. 2013

The extracellular matrix protein laminin-10 promotes blood–brain barrier repair after hypoxia and inflammation in vitro

Kangwantas K., Pinteaux E., Penny J.
Journal of neuroinflammation 2016

Integrity of the BBB is primarily maintained by brain endothelial cells, the tight junctions between them and their attachment to the blood vessel basement membrane (mainly composed fibronectin, collagen IV, and laminin-411 and -511). Here the authors used an in vitro model of the BBB, composed of primary rat brain endothelial cells grown on these different ECM proteins. The in vitro BBB model was exposed to oxygen-glucose deprivation with or without reoxygenation, and in the absence or the presence of IL-1β in order to mimic the ischemic and inflammatory conditions that occur during stroke. They show that LN-511 plays a key role in maintenance of BBB integrity and that it’s a key ECM molecule involved in BBB repair after hypoxic injury and inflammation.

 

Endothelial Cell Laminin Isoforms, Laminins 8 and 10, Play Decisive Roles in T Cell Recruitment Across the Blood–Brain Barrier in Experimental Autoimmune Encephalomyelitis

Sixt M., Engelhardt B., Pausch F., Hallmann R., Wendler O., Sorokin L.M
J Cell Biol., 2001

Blood–brain barrier endothelial cell basement membranes in development of inflammatory lesions in the central nervous system (CNS). Laminin-411 and laminin-511 are described as the major laminin isoforms in vascular basement membranes. Their expression was influenced by pro-inflammatory cytokines or angiostatic agents. Inflammatory cuffs occurred exclusively around endothelial basement membranes containing laminin-411, whereas in the presence of laminin-511 no infiltration was detectable. Integrin α6 and β-dystroglycan were prominent in CNS blood vessels, whereas no staining was observed for integrin α3, α7, and β4 subunits. One of the major laminin receptors, integrin α6β1, was localized predominantly on the endothelial cells, where it is likely to mediate interactions with the endothelial cell laminin-411 and 511, whereas astrocyte endfeet appear to utilize a different receptor for interactions with the parenchymal laminins. β-Dystroglycan occurred predominantly on astrocyte endfeet.

 

In vitro models of the blood-brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use. Helms et al., J Cereb Blood Flow Metab. 2016

 

The extracellular matrix protein laminin α2 regulates the maturation and function of the blood-brain barrier. Menezes et al., J Neurosci. 2014

 

Expression and function of laminins in the embryonic and mature vasculature. Hallmann et al., Physiol Rev. 2005

 

Functionality of endothelial cells and pericytes from human pluripotent stem cells demonstrated in cultured vascular plexus and zebrafish xenografts. Orlova et al., Arterioscler Thromb Vasc Biol. 2014

 

Astrocytic laminin regulates pericyte differentiation and maintains blood brain barrier integrity. Yao et al., Nat Commun. 2014