Here is a selection of publications where different laminin isoforms were used to create more authentic cell culture systems.
Laminin 511 Precoating Promotes the Functional Recovery of Transplanted Corneal Endothelial Cells
Can Zhao, Qingjun Zhou, Haoyun Duan, Xin Wang, Yanni Jia, Yajie Gong, Wenjing Li, Chunxiao Dong, Zongyi Li, and Weiyun Shi. Tissue Eng Part A, 2020
Abnormal adhesion of grafted corneal endothelial cells (CECs) affects the application of intracameral injection for corneal endothelial dysfunction therapy. The authors explored whether laminin 511 (LN511) improves the therapeutic function of the intracameral CEC injection. Injected LN511 was found to be able to settle and form a coating on the posterior surface of Descemet's membrane (DM). The data suggests that the strategy of LN511 precoating and CECs' intracameral injection could be a potential method for the therapy of corneal endothelial dysfunction.
Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting and functional bioinks
Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting and functional bioinks
Sorkioa A., Kochb L., Koivusaloa L., Deiwickb A., Miettinena S., Chichkovb B., Skottman H.
In this study, the authors produced 3D cornea-mimicking tissues using human stem cells and laser-assisted bioprinting (LaBP). Human embryonic stem cell-derived limbal epithelial stem cells (hESC-LESC) were used as a cell source for printing epithelium-mimicking structures, whereas human adipose tissue-derived stem cells (hASCs) were used for constructing layered stroma-mimicking structures. The authors used two previously established LaBP setups based on laser-induced forward transfer, with different laser wavelengths and appropriate absorption layers. Recombinant human laminin and human-sourced collagen I served as the bases for the functional bioinks. For hESC-LESCs, bioink containing human recombinant laminin-521 was chosen, as laminin is a major component in LESC basement membrane in the native cornea. Three different types of corneal structures was printed: stratified corneal epithelium using hESC-LESCs, lamellar corneal stroma using alternating acellular layers of bioink and layers with hASCs, and finally structures with both a stromal and epithelial part. The printed constructs were evaluated for their microstructure, cell viability and proliferation, and key protein expression. The 3D printed stromal constructs were also implanted into porcine corneal organ cultures. Both cell types maintained good viability after printing. Laser-printed hESC-LESCs showed epithelial cell morphology, expression of Ki67 proliferation marker and co-expression of corneal progenitor markers p63α and p40. Importantly, the printed hESC-LESCs formed a stratified epithelium with apical expression of CK3 and basal expression of the progenitor markers. The structure of the 3D bioprinted stroma demonstrated that the hASCs had organized horizontally as in the native corneal stroma and showed positive labeling for collagen I. After 7 days in porcine organ cultures, the 3D bioprinted stromal structures attached to the host tissue with signs of hASCs migration from the printed structure. This is the first study to demonstrate the feasibility of 3D LaBP for corneal applications using human stem cells and the successful fabrication of layered 3D bioprinted tissues mimicking the structure of the native corneal tissue.
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.
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.
Laminin synthesis and the adhesion characteristics of immortalized human corneal epithelial cells to laminin isoforms
Filenius S., Hormia M., Rissanen J., Burgeson R.E., Yamada Y., Araki-Sasaki K., Nakamura M., Virtanen I., Tervo T. Exp. Eye Res., 2001
The authors studied the synthesis of laminins and determined the specific integrins mediating the adhesion of immortalized human corneal epithelial cells to mouse laminin-111, and human laminin-332 and laminin-511. The cells produced a3, b3 and g2 chains of laminin-332, but not laminin-111 and laminin-511. Integrin a3B1 complex-mediated the adhesion of corneal epithelial cells to human laminin-332 and laminin-511. Integrin complex a3B, as well as laminin a3 chain, was also shown to mediate cell adhesion to newly produced endogenous laminin-332. The results also show that among corneal basement membrane laminins, laminin-332 is synthesized by epithelial cells while laminin-511 may be a product of keratocytes.
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.
Changes in Corneal Basal Epithelial Phenotypes in an Altered Basement Membrane
Wang I-J., Jui-Fang Tsai R., Yeh L-K., Yao-Nien Tsai R., Hu F-R., Kao W.W.Y.PLOS ONE, 2011
Analysis of human healthy donors shows that the BM of the limbal epithelium differs from that of the central cornea. Aside from laminin-111 and laminin-332, the limbal BM also contains laminin α2β2 chains, while the corneal BM does not.
Compositional Differences between Infant and Adult Human Corneal Basement Membranes
Kabosova A., Azar D.T., Bannikov G.A., Campbell K.P, Durbeej M., Ghohestani R.F., Jones J.C.R, Kenney M.C, Koch M., Ninomiya Y., Patton B.L., Paulsson M., Sado Y., Sage E.H., Sasaki T., Sorokin L.M., Steiner-Champliaud M-F, Sun T-T, SundarRaj N., Timpl R., Virtanen I., Ljubimov A.V. Invest Ophthalmol Vis Sci. 2007
The purpose of the study was to identify changes in the human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) during postnatal corneal development. Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from α1-α2 to α3-α4 chains after 3 years of life; in the adult, α1-α2 chains were retained only in the limbal BM. Laminin α2 and β2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, β2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin γ3 chain. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult.