Publications
Here is a selection of publications where different laminin isoforms were used to create more authentic cell culture systems.
Imaging-Based Screen Identifies Laminin 411 as a Physiologically Relevant Niche Factor with Importance for i-Hep Applications
Ong J., Paola Serra M., Segal J., Cujba A.-M., Seng Ng S., Butler R., Millar V., Hatch S., Zimri S., Koike H., Chan K., Bonham A., Walk M., Voss T., Heaton N., Mitry R., Dhawan A., Ebner D., Danovi D., Nakauchi H., Rashid S.T.Stem Cell Reports, 2018
Here, the authors show that extracellular niche factors likely play a critical role in bridging this gap in functional differences between hepatocytes derived from induced pluripotent stem cells (i-Heps) and primary cells. They defined a profile of healthy, freshly isolated primary hepatocytes (Hepatocyte Likeness Index; HLI) that cells of interest can be compared against for high-throughput screening. They applied this platform to screen hepatocyte niche factors for their ability to drive i-Heps closer to that target and validated their findings in a pharma-like screening environment. The HLI was applied in a targeted screen of extracellular niche factors to identify substrates driving i-Heps closer to the standard. Results from the screen performed highlighted the important role played by laminins where laminin 411 was identified as a key niche protein. Laminin 411 is a component of the hepatic niche. Laminin 411 advanced i-Heps toward functional significance and prolonged survival of hepatic progenitor cells, contributing to better i-Hep-based drug-screening applications. This paper underscores the importance of combining substrates, soluble factors, and HCA when developing iPSC applications.
Impaired integrin α5β1-mediated hepatocyte growth factor release by stellate cells of the aged liver
Rohn F., Kordes C., Buschmann T., Reichert D., Wammers M., Poschmann G., Stühler K., Benk A.S., Geiger F., Spatz J.P., Häussinger D.Aging Cell, 2020
Laminin proteins are critically involved in HSC function which is further illustrated in this article. Here, the authors illustrate the mechanistic link between fluid mechanical forces, loss of important extracellular matrix proteins, such as laminins, and hepatic aging, and how it all leads to an impaired liver regeneration potential. The authors provide evidence that integrin α5β1 is an important mechanosensor in hepatic stellate cells (HSC) involved in shear stress-induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α5 and β1 decreases in liver and HSC from aged rats. CRISPR/Cas9-mediated integrin α5 and β1 knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α5 and laminin gene expression whereas integrin β1 remains unaffected. In the aged liver, laminin β2 and γ1 protein chains as components of laminin-521 are lowered. The integrin α5 knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin-521 enhances HGF expression in HSC, demonstrating that these laminin proteins are critically involved in HSC function. During aging, HSC acquires a senescence-associated secretory phenotype and lower their growth factor expression essential for tissue repair. These findings suggest that impaired mechanosensing via integrin α5β1 in HSC contributes to the age-related reduction of ECM and HGF release that could affect liver regeneration.
Laminin-521 promotes quiescence in isolated stellate cells from rat liver
Rohn F., Kordes C., Castoldi M., Götze S., Poschmann G., Stühler K., Herebian D., Benk A.S., Geiger F., Zhang T. Spatz J.P., Häussinger D.Biomaterials, 2018
Here, the authors show that Biolaminin 521 supports the quiescent state of HSC and that laminin a5 can be regarded as an important element of their niche in the space of Dissé. Hepatic stellate cells (HSC) are liver-resident mesenchymal stem cells, which reside in a quiescent state on a basement membrane-like structure in the space of Dissé. In the present study, a laminin a5 chain was detected in the space of Dissé of normal rat liver. Since HSC is critical for liver regeneration and can contribute to fibrosis in chronic liver diseases, the effect of laminins on HSC maintenance was investigated. Isolated rat HSC were seeded on uncoated polystyrene (PS) plates or PS plates coated with either laminin-521 (PS/LN-521) or laminin-211 (PS/LN-211). PS/LN-521 improved HSC adhesion and better preserved their retinoid stores as well as quiescence- and stem cell-associated phenotype, whereas HSC on PS/LN-211 or PS developed into myofibroblasts-like cells. To improve the homogeneity, as well as the presentation of laminin molecules on the culture surface to HSC, laminin-functionalized, gold-nanostructured glass surfaces, were generated. This approach further enhanced the expression of quiescence-associated genes in HSC.
Laminin-521 Promotes Rat Bone Marrow Mesenchymal Stem Cell Sheet Formation on Light-induced Cell Sheet Technology
Jiang Z., Xi Y., Lai K., Wang Y., Wang H., Yang G.BioMed Research International, 2016
The cell sheet technology is an area of research that is of great interest for tissue engineering and regenerative medicine. Here, the authors investigated the effects of an ECM coating on rat bone marrow mesenchymal stem cells (rBMSC) cultured on light-induced TiO2 nanodot films. Cell sheets can be detached on a TiO2 nanodot-coated quartz substrate by using UV365 illumination. The effects of rat fibronectin, human recombinant laminin-521, -511, -421, and -111 on the formation of cell sheets were investigated and also compared to uncoated films. The result showed the highest success for laminin-521. rBMSCs rapidly attached and spread on films coated with laminin-521 (1.2 ug/ml) and formed intact cell sheets after 5 days of culture. Laminin-521 promotes the formation of rBMSC sheets with good viability under hyperconfluent conditions (4 to 8 layers of cells). The cells also maintained multilineage potential, including osteogenic, adipogenic, and chondrogenic differentiation. The cell sheets formed had rich ECM (including collagen I) and cells were connected with each other in dense network-like tissue. rBMSC cultured on uncoated surface and cells partially detached and failed to form cell sheets. In summary, laminin-521 and UV365 illumination systems provided a simple, rapid, and effective cell sheets strategy.
Bone Marrow Mesenchymal Stem Cells Adhesion Assay
Yang Z. and Xiao R. Bio-protocol, 2016
Here, the authors present a protocol for the culture of bone marrow MSC (BM-MSCs) on laminin-521 or laminin-511. The protocol is based on the method by Siler et al., 2000, and can easily be translated to MSCs from other origin or alternative ECMs coating. Both laminin isoforms show a significantly better efficient attachment compared to uncoated wells and also support seeding of a lower cell number compared to uncoated plats. The BM-MSCs adhere to the laminin-coated wells within 10 min, while for non-coated wells, it may take a longer time. In this protocol, a final coating concentration of 2 μg/cm2 is used but can effectively be lowered 4-10 times without loss of function.
CD49f Acts as an Inflammation Sensor to Regulate Differentiation, Adhesion, and Migration of Human Mesenchymal Stem Cells
Yang Z., Dong P., Fu X., Li Q., Ma S., Wu D., Kang N., Liu X., Yan L., Xiao R.Stem Cells, 2015
Here, we studied the role of CD49f (also known as integrin α6) in bone marrow MSCs. CD49f is preferentially expressed in fetal cells rather than adult cells, CD49f‐positive BM-MSCs possess higher CFU‐F formation ability and differentiation potential than CD49f negative cells, and the CD49f expression of BM-MSCs gradually decreases during in vitro passaging. An adhesion assay showed strong adhesion of BM-MSCs to both laminin 511 and 521 that were significantly higher than the control group coated with BSA, and the adhesion occurred evenly throughout the well. Pre‐blocking of CD49f on BM-MSCs inhibited the adhesion of fetal BM-MSCs to laminin 511 and 521. Also, CD49f knockdown dramatically decreased the differentiation of BMSCs. Inflammation (TNF-a) down-regulated CD49f in BMSCs with impaired differentiation, decreased adhesion to laminins and increased migration. This study provides evidence for CD49f as a stemness marker of BMSCs which is correlated with cell adhesion on laminin-521 and -511.
Release of Matrix Metalloproteinase-8 During Physiological Trafficking and Induced Mobilization of Human Hematopoietic Stem Cells
Steinl C., Essl M., Schreiber T.D., Geiger K., Prokop L., Stevanovic´ S., Pötz O., Abele H., Wessels J.T, Aicher W.K., Klein G.Stem Cells Dev., 2013
In the present study, we provide evidence that the collagenase MMP-8 is involved in stem cell mobilization. Cell–matrix adhesion assays were performed with LM-511 where MACS-isolated CD34+ cells were allowed to attach to immobilized LM-511. A rapid release of MMP-8 from isolated neutrophil granulocytes can be observed during an in vitro culture. Activated MMP-8 degrades LM-511 but without influencing the HSC progenitor–matrix adhesion. This since MMP-8 strongly digests the LM-a5 chain but leaves the LG1–3 domains intact.
Laminin expression during bone marrow mononuclear cell transplantation in hepatectomized rats
Carvalho S., Cortez E., Stumbo A.C., Thole A., Caetano C., Marques R., Pelajo-Machado M., Cristóvao Porto L., Carvalho L.Cell Biology International, 2008
The aim of this study was to investigate laminin expression during liver regeneration induced by partial hepatectomy followed by bone marrow mononuclear cell (BMMNC) transplantation (injected into recently hepatectomyzed rats via the portal vein). Results showed that 15 min after partial hepatectomy, a transplanted CD34+ HSC was found in contact with laminin, which was localized in the portal and centrilobular veins of rat livers. Furthermore, 1 and 3 days after hepatectomy, transplanted BMMNCs were found in the hepatic sinusoids (endothelia) expressing laminin. These results strongly suggest that laminin might be an important extracellular matrix component for bone marrow cell attachment and migration in the injured liver.
Laminin isoform-specific promotion of adhesion and migration of human bone marrow progenitor cells
Gu Y-C., Kortesmaa J., Tryggvason K., Persson J., Ekblom P., Jacobsen S-E., Ekblom M.HEMATOPOIESIS, 2003
Here they studied human bone marrow cell adhesion to laminin-511/521, laminin-411, laminin-111, and fibronectin. About 35% to 40% of CD34+ and CD34+CD38- stem and progenitor cells adhered to laminin-511/521, and 45% to 50% adhered to fibronectin, whereas they adhered less to laminin-411 and laminin-111. Adhesion of CD34+CD38- cells to laminin-511/521 was maximal without integrin activation, whereas adhesion to other proteins was dependent on protein kinase C activation. Integrin a6 chain expressed on most CD34+ and CD34+CD38-cells. Laminin-511/521 was highly adhesive to lineage-committed myelomonocytic and erythroid progenitor cells and most lymphoid and myeloid cell lines studied, whereas fibronectin and laminin-411 were less adhesive. Laminin-511/521was a ubiquitous adhesive protein for differentiated precursors of both B-lymphocytic, erythroid, megakaryocytic, and myelomonocytic cell lineages, whereas adhesion to laminin-411 and laminin-111 was restricted to a few cell lines. CD34+ cell migration was greatly enhanced through membranes coated with Laminin-511/521 and laminin-411. Our findings raise the possibility that a4 and a5 laminins are involved in mobilization and homing of hematopoietic progenitor cells.
Contribution of α6 integrins to hematopoietic stem and progenitor cell homing to bone marrow and collaboration with α4 integrins
Qian H., Tryggvason K., Jacobsen S.E., Ekblom M.Blood, 2006
The integrin a6 chain is ubiquitously expressed in human and mouse hematopoietic stem and progenitor cells. The integrin a6 chain is ubiquitously expressed in human HPCs. Laminin-411 and -511, are present in subendothelial basement membranes of sinusoids in bone marrow, at sites of hematopoietic cell development and trafficking and might, therefore, regulate HSC functions. In this paper, they show that mouse HSC and progenitors express a6B1 integrin which mediates high cell adhesion to laminin-511 and 521 and to laminin-411 to a lower extent. Blocking of a6 significantly reduced progenitor cell homing to bone marrow in mice. Integrin a4 receptors are also important for homing of HSCs to bone marrow (but not to the spleen). The first data showing that a6 integrins (LN521/511 binding) function in vivo as hematopoietic stem and progenitor cell homing receptors. Also, show the role of integrin a4 receptor for homing of long-term multilineage reconstituting HSCs and collaboration of these 2 integrins in homing of short-term HSCs.