Here is a selection of publications where different laminin isoforms are being used to create more authentic cell culture systems
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.
Characterization and functional analysis of laminin isoforms in human bone marrow
Siler U., Seiffert M., Puch S., Richards A., Torok-Storb B., Müller C.A, Sorokin L., Klein G.Blood, 2000
Based on gene expression, laminin-411/421 and laminin-511/521 are the most abundant laminin isoforms synthesized by human bone marrow stromal cells. Laminin-511/521 preparations showed strong adhesive interactions with human CD34+ cell lines. Antibodies against the B1 integrin subunit inhibited these interactions. In addition to its adhesion-mediating properties, laminin-511/521 preparations also showed a mitogenic activity for human hematopoietic progenitor cells. Other laminin isoforms tested, especially laminin-111 and laminin-211 and 221, showed only weak or no adhesive interactions with the hematopoietic cell lines tested and are suggested to play a minor role in the hematopoietic microenvironment. In the bone marrow, LN‐511 and 521 are the major laminins and show strongly adhesive and mitogenic activities toward early developing HSCs.
Integrin α6β1 Is the Main Receptor for Vascular Laminins and Plays a Role in Platelet Adhesion, Activation, and Arterial Thrombosis
Schaff et al.Circulation. 2013
Here, the authors show that laminin-411, laminin-511, and laminin-521, but not laminin-211, allow efficient platelet adhesion and activation across a wide range of arterial wall shear rates. Adhesion was critically dependent on integrin α6β1. Platelets from integrin α6 knockout mice failed to adhere to laminin-411, laminin-511, and laminin-521 but responded normally to a series of agonists. α6β1-Deficient mice presented a marked decrease in arterial thrombosis in 3 models of injury of the carotid, aorta, and mesenteric arterioles. The authors thus reveal an unsuspected important contribution of laminins to thrombus formation in vivo.
Generation of HLA-Universal iPSC-Derived Megakaryocytes and Platelets for Survival Under Refractoriness Conditions
Börger A-K., Eicke D., Wolf C., Gras C., Aufderbeck S., Schulze K., Engels L., Eiz-Vesper B., Schambach A., Guzman C.A., Lachmann N., Moritz T., Martin U., Blasczyk R., Figueiredo C. Molecular Medicine, 2016
Here, the authors have established a protocol for the generation of megakaryocytes (MK) and platelets (PLTs) from an HLA-universal and virtually unlimited cell source (iPSCs) under xeno-free and defined conditions to facilitate their future translation into clinical application. HLA-universal iPSC was generated and cultured on laminin-521. The expression of HLA class I was silenced (shRNA) by up to 82% and remained stable during iPSC cultivation. The generation of MK and PLTs from an HLA-universal iPSC were conducted on laminin-521. On d19, differentiation rates of MKs and PLTs with means of 58% and 76% were observed, respectively. HLA-universal iPSC-derived MKs showed polyploidy with DNA contents higher than 4n and formed proPLTs. Importantly, differentiated MKs remained silenced for HLA class I expression. HLA-universal MKs produced functional PLTs. Notably, iPSC-derived HLA-universal MKs were capable to escape antibody-mediated complement- and cellular-dependent cytotoxicity. Furthermore, HLA-universal MKs were able to produce PLTs after in vivo transfusion in a mouse model indicating that they might be used as an alternative to PLT transfusion.