Differentiation of Human Embryonic Stem Cells to Endothelial Progenitor Cells on Laminins in Defined and Xeno-free Systems

Nguyen M.T.X., Okina E., Chai X., Tan K.H., Hovatta O., Ghosh S., Tryggvason K.
Stem Cell Reports, 2016

Here, the authors developed a chemically defined, xeno-free protocol for differentiation of hESCs to endothelial progenitor cells (EPCs) using LN521 as the main culture substrate. The EPCs derived are functional and expressed both progenitor and mature endothelial markers. The authors were able to generate about 95% functional EPCs defined as VEGFR2+CD34+CD31+VE-Cadherin+. RNA-sequencing analyses of hESCs, EPCs, and primary human umbilical vein endothelial cells show differentiation-related EC expression signatures, regarding basement membrane composition, cell-matrix interactions, and changes in endothelial lineage markers. Six-week continuous culturing allow the hESC derived EPSs to mature further, relative to HUVECs. These results may facilitate production of stable ECs for the treatment of vascular diseases and in vitro cell modeling.

 

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo

Stenzel D., Franco C.A., Estrach S., Mettouchi A., Sauvaget D., Rosewell I., Schertel A., Armer H., Domogatskaya A., Rodin S., Tryggvason K., Collinson L., Sorokin L., Gerhardt H.
EMBO reports, 2011

Here the authors show that laminin α4 regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. α4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4‐mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. The authors conclude that appropriate laminin/integrin‐induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.

 

Laminin isoforms in endothelial and perivascular basement membranes

Yuosif L., Russo J.D., Sorokin L.
Cell Adh Migr., 2013

α4 and α5 chain laminins are the predominant isoforms in the basal lamina of vascular endothelial cells. While the α4 chain is expressed ubiquitously throughout different developmental stages, the prominent expression of α5 chain appears postnatally and its distribution varies with vessel type.

 

Vascular laminins in physiology and pathology

Di Russo J., Hannocks M-J., Luik A-L., Song J., Zhang X., Yousif L., Aspite G., Hallmann R., Sorokin L.
Matrix biology, 2016

Review on vascular laminins. In the healthy vessel, basement membranes (BMs) are the main ECM structures found in the vessel wall, underlying endothelium and surrounding vascular smooth muscle cells. Laminin α4 and α5 chains are the major α chains occurring in endothelial BMs and in vascular smooth muscle BMs. In most cases, laminins α4 and α5 combine with laminin β1 and γ1 chains to form laminins-411 and -511 in endothelial BMs; however, there is also evidence for the expression of laminin β2 in several tissues suggesting the additional existence of laminins-421 and -521 in endothelial BMs. While laminin α2 does not occur in endothelial BMs it can occur in association with perivascular cells including smooth muscle BMs of large arteries, such as the aorta, but not of smaller arterioles. There is no laminin α1 in endothelial or smooth muscle BMs regardless of tissue or species.

 

The Vascular Basement Membrane: A Niche for Insulin Gene Expression and B Cell Proliferation

Nikolova G., Jabs N., Konstantinova I., Domogatskaya A., Tryggvason K., Sorokin L., Fässler R., Gu G., Gerber H-P., Ferrara N., Melton D.A., Lammert E.
Developmental Cell, 2006

Pancreatic islets intimately interact with endothelial cells and differentiation and delamination of insulin-producing B cells from pancreatic epithelium strictly require endothelial cells. Doug Melton and colleagues show that BMs within islets is formed and found exclusively around capillaries but not islet cells. Islet endothelial cells express laminin a4 and a5. Laminins promote insulin gene expression and proliferation in B-cells and B1-intergrin is required for this laminin response.

 

Integrin α6β1 Is the Main Receptor for Vascular Laminins and Plays a Role in Platelet Adhesion, Activation, and Arterial Thrombosis

Schaff M., Tang CJ., Maurer E., Bourdon C., Receveur N., Eckly A., Hechler B., Arnold C., de Arcangelis A., Nieswandt B., Denis C.v., LefebvreO., Georges-Labouesse E., Gachet C., Lanza F., Mangin P.H
Circulation, 2013

Show that laminin-411, laminin-511 and laminin-521, but not laminin-211, allow efficient platelet adhesion and activation across a wide range of arterial wall shear rates. Adhesion was critically dependent on integrin α6β1 and the glycoprotein Ib-IX complex, which binds to plasmatic von Willebrand factor adsorbed on laminins. Glycoprotein VI did not participate in the adhesive process but mediated platelet activation induced by α5-containing laminins. Platelet-specific knockout of integrin α6 failed to adhere to laminin-411, laminin-511, and laminin-521 but responded normally to a series of agonists. α6β1-Deficient mice presented a marked decrease in arterial thrombosis in 3 models of injury of the carotid, aorta, and mesenteric arterioles. The tail bleeding time and blood loss remained unaltered, indicating normal hemostasis. This study reveals an unsuspected important contribution of laminins to thrombus formation in vivo and suggests that targeting their main receptor, integrin α6β1, could represent an alternative antithrombotic strategy with a potentially low bleeding risk.

The formation of quiescent glomerular endothelial cell monolayer in vitro is strongly dependent on the choice of extracellular matrix coating

Pajęcka K., Nygaard Nielsen M., Krarup Hansen T., Williams J.M.
Experimental Cell Research, 2017

Primary glomerular endothelial cells (GEnCs) is an important tool for studying glomerulosclerotic mechanisms and in drug development. Primary GEnCs are commonly cultured either on gelatin or plasma-derived fibronectin coated surfaces, yet neither of the substrates are a major component of the healthy mature GBM. Here, the authors set out to establish a simple, reproducible model of quiescent hGEnC monolayer in vitro by determining the best commercially available ECM substrate- recombinant human laminin (rhLN)- 521, -511, -111, fibronectin (FN), collagen type IV and collagen I - based Attachment Factor. All ECM matrices except recombinant human laminin 111 (rhLN111) supported comparable cell proliferation. Laminin-521, laminin-511 and FN were best at supporting hGEnC attachment and spreading. Culturing hGEnCs on rhLN521, rhLN511 or fibronectin resulted in a physiologically relevant barrier to 70 kDa dextrans which was 82 % tighter than that formed on collagen type IV. Furthermore, only hGEnCs cultured on rhLN521 or rhLN511 showed plasma-membrane localized zonula occludens-1 and vascular endothelial cadherin indicative of proper tight and adherens junctions. The authors recommendations based on the results are that hGEnCs is best cultured on the mature glomerular basement membrane laminin - rhLN521 – which, as the only commercially available ECM, promotes all of the characteristics of the quiescent hGEnC monolayer: cobblestone morphology, well-defined adherens junctions and physiological perm-selectivity.

Laminin-511 and -521 Enable Efficient In Vitro Expansion of Human Corneal Endothelial Cells (HCEC)

Okumura N., Kakutani K., Numata R., Nakahara M., Schlötzer-Schrehardt U., Kruse F., Kinoshita S., Koizumi N.
IVOS Cornea, 2015

Laminin-511 and -521 were expressed in Descemet’s membrane and corneal endothelium. These laminin isoforms significantly enhanced the in vitro adhesion and proliferation, and differentiation of HCECs compared to uncoated control, fibronectin and collagen I. iMatrix also supported HCEC cultivation with a similar efficacy to that obtained with full-length laminin. Functional blocking of a3b1 and a6b1 integrins suppressed the adhesion of HCECs even in the presence of laminin-511.

 

Identification and Potential Application of Human Corneal Endothelial Progenitor Cells

Hara S., Hayashi R., Soma T., Kageyama T., Duncan T., Tsujikawa M., Nishida K.
Stem Cells Dev. 2014

This article demonstrates for the first time that Laminin-511 is an optimal, human matrix for the isolation and expansion of corneal endothelial progenitors. The authors show that the proliferative capacity of these endothelial progenitors is very high on Laminin-511 compared to conventional methods. Laminin-511 can be used to rapidly isolate and expand a homogenous population of a endothelial progenitor cells that can be differentiated to endothelial cells in a biorelevant environment. The authors demonstrate that the proliferative capacity of these endothelial progenitors is very high on Laminin-511 compared to conventional methods. Laminin-511 can thus be used to rapidly isolate and expand a homogenous population of endothelial progenitors that can be differentiated to endothelial cells in a biorelevant environment. Main points of the article are: 1) High proliferative capacity in serum-free media compared to standard methods, 2) Large numbers of cells generated, 3) Facilitates rapid isolation of a homogenous population of endothelial progenitors, 4) Enables differentiation to endothelial cells in a biorelevant environment, 5) Cells can be subcultured for at least 5 passages.

 

The Different Binding Properties of Cultured Human Corneal Endothelial Cell Subpopulations to Descemet’s Membrane Components

Toda M., Ueno M., Yamada J., Hiraga A., Tanaka H., Schlötzer-Schrehardt U., Sotozono C., Kinoshita S., Hamuro J.
Invest Ophthalmol Vis Sci. 2016

In culture, human corneal endothelial cell (cHCEC) tend to enter into cell-state transition (CST), such as epithelial-to-mesenchymal transition (EMT) or fibrosis, thus resulting in the production of different subpopulations. In this study, the authors examined the binding ability of cHCECs subpopulations to major Descemet’s membrane components that distribute to the endothelial face; that is, laminin-511, -411, Type-IV collagen, and proteoglycans. Each subpopulation was prepared by controlling the culture conditions or by using magnetic cell separation, and then confirmed by staining with several cell-surface markers. Binding abilities of HCEC subpopulations were examined by adding the cells to culture plates immobilized with collagens, laminins, or proteoglycans, and then centrifuging the plates. The cHCECs showed best attachment to laminin laminin-521 and -511. The cells showed a weaker binding to laminin-411, laminin-332, Type-IV collagen. The minimum concentrations necessary for the observed cell binding in this study were as follows: laminin-521 and -511, 3 ng/mL; laminin-411, 2.85 ug/mL; Type-IV collagen, 250 ng/mL. Cells suspended in Opti-MEM-I or Opeguard-MA were bound to laminin, yet no binding was observed in cells suspended in BSS-Plus. Both the fully differentiated, mature cHCEC subpopulations and the epithelial-to-mesenchymal– transitioned (EMT)-phenotype subpopulation were found to attach to laminin- or collagen-coated plates. Interestingly, the binding properties to laminins differed among those subpopulations. Although the level of cells adhered to the laminin-411–coated plate was the same among the cHCEC subpopulations, the fully differentiated, mature cHCEC subpopulations was significantly more tightly bound to laminin-511 than was the EMT-phenotype subpopulations. These findings suggest that the binding ability of cHCECs to major Descemet’s membrane components is distinct among cHCEC subpopulations, and that Opti-MEM-I and Opeguard-MA are useful cell-suspension vehicles for cell-injection therapy. This research group focused on developing a novel medical approach, termed cell-injection therapy, for the treatment of patients with endothelial dysfunction.

 

3D map of the human corneal endothelial cell

He Z., Forest F., Gain P., Rageade D., Bernard A., Acquart S., Peoch M., Defoe D.M., Thuret G.
Scientific reports, 2016

Human corneal endothelial cells (CECs) are highly polarized flat cells that separate the cornea from the aqueous humor. Their apical surface, in contact with aqueous humor is hexagonal, whereas their basal surface is irregular. Here, the authors characterized the structure of human CECs in 3D using confocal microscopy of immunostained whole corneas in which cells and their interrelationships remain intact. Hexagonality of the apical surface was maintained by the interaction between tight junctions and a submembraneous network of actomyosin. Lateral membranes presented complex expansions resembling interdigitated foot processes at the basal surface. Integrin α3β1 was the only protein found exclusively at the basal surface, forming an almost homogenous layer that follows the slightly bumpy surface of Descemet’s membrane. Ligands of integrin α3β1, such as laminin-332, laminin-511, and laminin-521 constitute efficient coating substances that improve the yield of in vitro CEC cultures. This first 3D map aids our understanding of the morphologic and functional specificity of CECs and could be used as a reference for characterizing future cell therapy products destined to treat endothelial dysfunctions.

 

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo

Stenzel D., Franco C.A., Estrach S., Mettouchi A., Sauvaget D., Rosewell I., Schertel A., Armer H., Domogatskaya A., Rodin S., Tryggvason K., Collinson L., Sorokin L., Gerhardt H.
EMBO reports, 2011

Here the authors show that laminin α4 regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. α4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4‐mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. The authors conclude that appropriate laminin/integrin‐induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting 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 laminin-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. The brain endothelial cells did not adhere well to laminin-411.

 

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

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.

 

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.