Publications
Here is a selection of publications where different laminin isoforms were used to create more authentic cell culture systems.
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 is 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.
In vitro models of the blood-brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use
Hans C Helms, N Joan Abbott, Malgorzata Burek, Romeo Cecchelli, Pierre-Olivier Couraud, Maria A Deli, Carola Förster, Hans J Galla, Ignacio A Romero, Eric V Shusta, Matthew J Stebbins, Elodie Vandenhaute, Babette Weksler, Birger Brodin. J Cereb Blood Flow Metab, 2016
This review gives an overview of established in vitro blood-brain barrier models with a focus on their validation regarding a set of well-established blood-brain barrier characteristics. The authors also provide advantages and drawbacks of the different models described.
Functionality of endothelial cells and pericytes from human pluripotent stem cells demonstrated in cultured vascular plexus and zebrafish xenografts
Valeria V Orlova, Yvette Drabsch, Christian Freund, Sandra Petrus-Reurer, Francijna E van den Hil, Suchitra Muenthaisong, Peter Ten Dijke, Christine L Mummery. Arterioscler Thromb Vasc Biol, 2014
This article describes simultaneous derivation of endothelial cells and pericytes from hiPSCs of different tissue origin.
Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signaling 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 signaling in vivo 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 signaling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis.
Laminin-511 and -521 Enable Efficient In Vitro Expansion of Human Corneal Endothelial Cells
Okumura N., Kakutani K., Numata R., Nakahara M., Schlötzer-Schrehardt U., Kruse F., Kinoshita S., Koizumi N. IVOS Cornea, 2015
The purpose of this study was to investigate the usefulness of laminin isoforms as substrates for culturing human corneal endothelial cells (HCECs) for clinical applications. 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 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 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 the 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 were 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.
Adhesion, Migration, and Proliferation of Cultured Human Corneal Endothelial Cells by Laminin-5
Yamaguchi M., Ebihara N., Shima N., Kimoto M., Funaki T., Yokoo S., Murakami A., Yamagami S. Investigative Ophthalmology and Visual Science, 2011
Here, the authors investigate the expression of laminin-332 and its receptors by human corneal endothelial cells (HCECs) and the effect on adhesion, proliferation, and migration of cultured HCECs. Adult HCECs expressed the laminin-332 receptor a3B1 integrin, but not laminin-332 itself. Laminin-332 is expressed in the basement membrane of the corneal epithelium, but not in the corneal endothelium. Significantly more HCEC cells became adherent to recombinant laminin-332-coated dishes than to uncoated dishes in the cell adhesion assay. The proliferation of cultured HCECs was moderately promoted by laminin-332. A significantly higher percentage of wound closure was obtained with medium containing soluble laminin-332 than with the control medium in the wound-healing assay. To conclude, recombinant laminin-332 promotes adhesion, migration, and moderate proliferation of cultured HCECs. The results suggest that immature or undifferentiated HCECs express laminin-332, whereas it is suppressed during development or differentiation.
Enhanced xeno-free differentiation of hiPSC-derived astroglia applied in a blood–brain barrier model
Louise Delsing, Therése Kallur, Henrik Zetterberg, Ryan Hicks, Jane Synnergren. Fluids Barriers CNS, 2019
This study shows that astroglia cells differentiated on Biolaminin 521 display an improved phenotype compared to a mouse EHS-extracted laminin L2020 product. Especially, cells differentiated on Biolaminin 521 had a higher secretion of factors important for BBB formation, such as GFAP, S100B, and Angiopoietin-1, than cells differentiated on the laminin extract. In addition, glutamate uptake and the ability to induce the expression of junction proteins in endothelial cells were affected by the culture matrix choice. The study showed differentiation of functional astroglia from three different human induced pluripotent stem cell lines which were used in a blood-brain barrier model.
Protocol for automated production of human stem cell derived liver spheres
Jose Meseguer-Ripolles, Alvile Kasarinaite, Baltasar Lucendo-Villarin, David C Hay. STAR Protoc, 2021
In this article, the authors describe how they produce human liver spheres from pluripotent stem cell-derived hepatic progenitors, endothelial cells, and hepatic stellate cells, using LN521 in the differentiation protocol. Their process is automated using liquid handling and pipetting systems, permitting cost-effective scale-up and reducing sphere variability.
