Publications

Here is a selection of publications where different laminin isoforms are being used to create more authentic cell culture systems

  • Area of interest

  • Highly sensitive droplet digital PCR method for detection of residual undifferentiated cells in cardiomyocytes derived from human pluripotent stem cells

    Kuroda T., Yasuda S., Matsuyama S., Tano K., Kusakawa S., Sawa Y., Kawamata S., Sato Y. Regenerative Therapy, 2015

    Human iPSCs were maintained on laminin-521 in Essential 8 medium. Cardiac differentiation on laminin-521 and Matrigel. Adult human cardiomyocytes (Promocell, Heidelberg) were cultured on laminin-211 in the Promocell myocyte growth medium. The authors have established a sensitive assay for the detection of the residual undifferentiated hiPSCs in cardiomyocytes, using droplet digital PCR (ddPCR). LIN28 was the most sensitive marker of residual undifferentiated cells in hiPSC-derived cardiomyocytes but also in other tissues such as liver, heart, pancreas, kidney, spinal cord, corneal epithelium, and lung. Hence, the LIN28/ddPCR method is applicable to the quality control of hiPSC-derived cell therapy products.

  • A Small Molecule that Promotes Cardiac Differentiation of Human Pluripotent Stem Cells under Defined, Cytokine- and Xeno-free Conditions

    Minami I., Yamada K., Otsuji T.G., Yamamoto T., Shen Y., Otsuka S., Kadota S., Morone N., Barve M., Asai Y., Tenkova-Heuser T., Heuser J.E., Uesugi M., Aiba K., and Nakatsuji N. Cell Reports, 2012

    In this study, the report of a small molecule that promotes cardiac differentiation of hPSCs. By using this chemical, a xeno-free and cytokine-free cardiac differentiation protocol were achieved. In a part of the study cardiac differentiation on the surface coating with gelatin or human laminin-211. Cell attachment on gelatin or laminin-211 is essential for efficient differentiation, suggesting that mechanotransduction or integrin signaling from interaction with these substrates might be important. iPSCs were differentiated into cardiac colonies on human laminin 211-coated dishes in cytokine-free and xeno-free defined medium containing HAS.

  • 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 report 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. LN221 promoted the differentiation of pluripotent human embryonic stem cells (hESCs) toward cardiomyocyte lineage and downregulated 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.