Here is a selection of publications where different laminin isoforms are being used to create more authentic cell culture systems
Efficient differentiation of human pluripotent stem cells into cardiomyocytes on cell sorting thermoresponsive surface
Sung T.-C., Su H.C, Ling Q.-D., Kumar S.S., Chang Y., Hsu S.T-., Higuchi A.Biomaterials, 2020
The current differentiation process of human pluripotent stem cells (hPSCs) into cardiomyocytes to enhance the purity of hPSC-derived cardiomyocytes requires some purification processes, which are laborious processes. Here, the authors have developed cell sorting plates, which are prepared from coating thermoresponsive poly(N-isopropylacrylamide) and extracellular matrix proteins vitronectin or Biolaminin 521. The Biolaminin 521-coated surface exhibited higher beating colony numbers than the vitronectin-coated surface. This is explained by the fact that hPSC-derived cardiomyocytes express less integrin αVβ5 but more α6β1, where the main binding sites of rVN and LN-521 are integrin αVβ5 and integrin α6β1, respectively. After hPSCs were induced into cardiomyocytes on the thermoresponsive surface coated with Biolaminin 521 for 15 days, the cells were detached partially from the thermoresponsive surface. The detached cells exhibited a higher cardiomyocyte marker of cTnT than the remaining cells on the thermoresponsive surface as well as the cardiomyocytes after purification using conventional cell selection. The detached cells expressed several cardiomyocyte markers, such as α-actinin, MLC2a, and NKX2.5. This study a promising method for the purification of hPSC-derived cardiomyocytes without conventional laborious processes.
Chemically defined generation of human cardiomyocytes
Burridge P., Elena Matsa E., Shukla P., Lin Z., Churko J., Ebert A., Lan F., Diecke S., Huber B., Mordwinkin N., Plews J., Abilez O., Cui B., Gold J. & Wu J. Nature methods, 2014
Cardiac differentiation strategy using a chemically defined medium consisting of just three components: the basal medium RPMI1640, l-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. this protocol produced contractile sheets of up to 95% TNNT2+ cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell. They first assessed chemically defined pluripotent culture on other defined matrices: rh E-cadherin, rh vitronectin, laminin-521, iMatrix-511, human fibronectin and a fibronectin mimetic. Laminin-based matrices resulted in higher growth rates compared to the vitronectin peptide. Fibronectin-based matrices did not support pluripotent growth. All five suitable matrices supported efficient differentiation in CDM3 but only the laminin-based matrices maintained long-term adhesion (>15 d) during CDM3 cardiac differentiation. The authors state that laminin-521 is an optimal matrix for chemically defined differentiation of human iPSC to cardiomyocytes but they still performed all subsequent characterization on the vitronectin peptide due to cost awareness.
A Chemical Probe that Labels Human Pluripotent Stem Cells
Hirata N., Nakagawa M., Fujibayashi Y., Yamauchi K., Murata A., Minami I., Tomioka M., Kondo T., Kuo T-F., Endo H., Inoue H., Sato S., Ando S., Kawazoe Y., Aiba K., Nagata K., Kawase E., Chang Y-T., Suemori H., Eto K., Nakauchi H., Yamanaka S., Nakatsuji N., Ueda K., Uesugi M. Cell Reports, 2014
The Yamanaka group uses cardiac-specific laminin-211 as the matrix to differentiate iPS cells to cardiomyocytes in a biorelevant environment specific to heart cells. This thus shows that you can use laminin-211 as a cardiac matrix. 326 fluorescent compounds screened to identify a fluorescent probe that is selective for human pluripotent stem cells compared to differentiated cells. hiPSCs were cultured on 3.5 cm culture dishes coated with human laminin 211 and cardiac differentiation was carried out. Cardiac colonies were harvested on day 15 and cultured for 7–10 days in floating culture. A majority of the prepared cells expressed the cardiac markers: cardiac troponin T, a-actinin, and NKX2.5.
In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors
Yap L., Wang J.-W., Moreno-Moral A., de Kleijn D.P.V., Petretto E., Tryggvason K. Cell Reports, 2019
In this article, the authors have developed a high reproducible, chemically defined, xeno-free laminin-based differentiation protocol to generate stem cell-derived cardiovascular progenitors (CVPs). Laminin-221 (LN-221) was identified as the most likely expressed cardiac laminin and the authors show that this protein promotes differentiation of pluripotent human embryonic stem cells (hESCs) toward cardiomyocyte lineage and downregulates pluripotency and teratoma-associated genes. Single-cell RNA sequencing of CVPs derived from hESC lines identified three main progenitor subpopulations. These CVPs were transplanted into myocardial infarction mice, where heart function was improved as measured by echocardiogram and human heart muscle bundle formation was identified histologically. This method may provide clinical-quality cells for use in regenerative cardiology.
The functions of exogenous and endogenous laminin-5 on corneal epithelial cells
Ebihara N, Mizushima H, Miyazaki K, Watanabe Y, Ikawa S, Nakayasu K, Kanai A.Exp. Eye Res., 2000
In this study, the authors investigated the functions of laminin-332 on SV-40 transfected human corneal epithelial cells (HCE cells). We also revealed different functions between exogenous and endogenous laminin-332 on HCE cells. HCE cells themselves secreted laminin-5 endogenously. Exogenously added laminin-5 strongly promoted cell adhesion via a3b1 integrin, cell spreading, assembly of hemidesmosomes, and mildly inhibited cell migration. Using an anti-laminin-5 monoclonal antibody (mAb) or anti-integrin a3b1 mAbs, the blocking of the interaction between endogenously secreted laminin-5 and HCE cells caused strong inhibition of cell migration. Integrin a3b1 and a6b4 were expressed in HCE cells. These results indicated that endogenous (unprocessed) laminin-5 has a crucial role in cell migration on HCE cells via a3b1 integrin. In conclusion, structural differences between exogenous and endogenous laminin-5 regulated their functions on HCE cells. Exogenously added laminin-5 strongly promoted cell adhesion, cell spreading, and assembly of hemidesmosomes. Endogenously secreted laminin-5 had a crucial role in cell migration.
Human laminin β2 deficiency causes congenital nephrosis with mesangial sclerosis and distinct eye abnormalities
Zenker M., Aigner T, Wendler O, Tralau T, Müntefering H, Fenski R, Pitz S, Schumacher V, Royer-Pokora B, Wühl E, Cochat P, Bouvier R, Kraus C, Mark K, Madlon H, Dötsch J, Rascher W, Maruniak-Chudek I, Lennert T, Neumann LM, Reis A.Human Molecular Genetics, 2004
Here, the authors demonstrate that LAMB2 mutations can be consistently found in patients with Pierson syndrome, a newly delineated entity characterized by CNS and distinct ocular anomalies. They point out the importance of laminin β2 for the proper development of structures of the anterior eye segment.
Corneal integrins and their functions
Stepp M.A.Experimental Eye Research, 2005
There is a minimum of 12 different integrin heterodimers reported to be expressed by the major resident cells of the cornea: the corneal and limbal epithelial cells, keratocytes/fibroblasts, and corneal endothelial cells. These different integrin heterodimers play important and varied roles in maintaining the cornea and organizing how its cells interact with their surrounding extracellular matrix to maintain corneal clarity. In this review, an overview of the discovery and functions of integrins is provided along with a description of the current state of our knowledge of this large family of important proteins.
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.
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.
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.