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.

 

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.

 

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 explains 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%.

 

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.

 

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.

 

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.

 

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. 

 

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.

 

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 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.