In order to apply human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to regenerative medicine, the cells should be produced under restricted conditions conforming to GMP guidelines. Since the conventional culture system has some issues that need to be addressed to achieve this goal, we developed a novel culture system. We found that recombinant laminin-511 E8 fragments are useful matrices for maintaining hESCs and hiPSCs when used in combination with a completely xeno-free (Xf) medium, StemFit™. Using this system, hESCs and hiPSCs can be easily and stably passaged by dissociating the cells into single cells for long periods, without any karyotype abnormalities. Human iPSCs could be generated under feeder-free (Ff) and Xf culture systems from human primary fibroblasts and blood cells, and they possessed differentiation abilities. These results indicate that hiPSCs can be generated and maintained under this novel Ff and Xf culture system.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have the potential to provide an infinite source of tissues for regenerative medicine. Although defined xeno-free media have been developed, culture conditions for reliable propagation of hESCs still require considerable improvement. Here we show that recombinant E8 fragments of laminin isoforms (LM-E8s), which are the minimum fragments conferring integrin-binding activity, promote greater adhesion of hESCs and hiPSCs than do Matrigel and intact laminin isoforms. Furthermore, LM-E8s sustain long-term self-renewal of hESCs and hiPSCs in defined xeno-free media with dissociated cell passaging. We successfully maintained three hESC and two hiPSC lines on LM-E8s in three defined media for 10 passages. hESCs maintained high level expression of pluripotency markers, had a normal karyotype after 30 passages and could differentiate into all three germ layers. This culture system allows robust proliferation of hESCs and hiPSCs for therapeutic applications.

Anti-p200 pemphigoid has been characterized by autoantibodies to an unidentified 200-kDa protein (p200) of the dermal؊epidermal junction. The objective of this study was to identify p200. We performed 2D gel electrophoresis of dermal extracts and immunoblotting with patients' sera, followed by MS analysis of a unique protein band. The protein band corresponded to laminin ␥1. Anti-laminin ␥1 mAb reacted with the anti-p200 immunoprecipitates by immunoblotting. Sera from 32 patients with anti-p200 pemphigoid showed 90% reactivity to the recombinant products of laminin ␥1. None of the healthy control sera reacted with laminin ␥1. By immunoblotting, reactivity of a patient's serum with p200 was competitively inhibited by adding anti-laminin ␥1 C-terminus mAb. Purified anti-p200 IgG also inhibited the reactivity of this mAb to dermal laminin ␥1. Most laminin ␥1-positive sera showed reactivity with recombinant laminin ␥1 C-terminal E8 fragment. Reactivity of patients' sera and purified IgG to dermal laminin ␥1 was higher than reactivity to blood vessel laminin ␥1 under reducing conditions. These results suggest that laminin ␥1 is the autoantigen for patients with anti-p200 pemphigoid. The autoantibodies may specifically recognize dermal laminin ␥1 with unique posttranslational modifications. The epitope is localized to the 246 C-terminal amino acids within the coiled-coil domain. The 9 C-terminal residues are known to be critically involved in laminin recognition by integrins.autoimmune disease ͉ basement membrane ͉ bullous pemphigoid ͉ proteomics

Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (␣, ␤, and ␥), in which three laminin globular modules in the ␣ chain and the Glu residue in the C-terminal tail of the ␥ chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the ␤ chain is involved in laminin-integrin interactions. We compared the binding affinities of pairs of laminin isoforms containing the ␤1 or ␤2 chain toward a panel of laminin-binding integrins, and we found that ␤2 chain-containing laminins (␤2-laminins) bound more avidly to ␣3␤1 and ␣7X2␤1 integrins than ␤1 chain-containing laminins (␤1-laminins), whereas ␣6␤1, ␣6␤4, and ␣7X1␤1 integrins did not show any preference toward ␤2-laminins. Because ␣3␤1 contains the "X2-type" variable region in the ␣3 subunit and ␣6␤1 and ␣6␤4 contain the "X1-type" region in the ␣6 subunit, we hypothesized that only integrins containing the X2-type region were capable of discriminating between ␤1-laminins and ␤2-laminins. In support of this possibility, a putative X2-type variant of ␣6␤1 was produced and found to bind preferentially to ␤2-laminins. Production of a series of swap mutants between the ␤1 and ␤2 chains revealed that the C-terminal 20 amino acids in the coiled-coil domain were responsible for the enhanced integrin binding by ␤2-laminins. Taken together, the results provide evidence that the C-terminal region of ␤ chains is involved in laminin recognition by integrins and modulates the binding affinities of laminins toward X2-type integrins.Laminins are large glycoproteins exclusively localized in basement membranes, which represent thin sheets of extracellular matrix bound by a variety of cell types, including epithelial, endothelial, muscle, and glial cells. Laminins are composed of three polypeptide chains (␣, ␤, and ␥), which assemble into a disulfide-bonded heterotrimer with a cross-shaped structure.There are five ␣ chains (␣1-␣5), three ␤ chains (␤1-␤3), and three ␥ chains (␥1-␥3) in mammals (1, 2), combinations of which give rise to at least 12 distinct isoforms expressed in tissue-specific and developmentally regulated manners (1, 3).Laminins play pivotal roles in embryonic development. Mice deficient in expression of the ␥1 chain, which is present in most laminin isoforms except for laminin-332 2 and some ␥3 chaincontaining isoforms, fail to deposit basement membranes and die at the peri-implantation stage of embryonic development (4). Gene knockouts of other laminin chains also result in severe phenotypes. Mice deficient in the ␣5 chain die around embryonic day 17 because of multiple developmental abnormalities, including failure of neural tube closure and digit separation and abnormal placental, kidney, and lung morphogenesis (5-7). Mice lacking the ␣2 chain show adult lethality because of severe and progressive skeletal muscle degeneration (8, 9). These phenotypes can be accounted for by defects in the physical strength of basement membra...

Integrin-growth factor receptor cross-talk plays a role in growth factor signaling, but the specifics are unclear. In a current model, integrins and growth factor receptors independently bind to their ligands (extracellular matrix and growth factors, respectively). We discovered that neuregulin-1 (NRG1), either as an isolated EGF-like domain or as a native multi-domain form, binds to integrins ␣v␤3 (with a K D of 1.36 ؋ 10 ؊7 M) and ␣6␤4. Docking simulation predicted that three Lys residues at positions 180, 184, and 186 of the EGF-like domain are involved in integrin binding. Mutating these residues to Glu individually or in combination markedly suppressed integrin binding and ErbB3 phosphorylation. Mutating all three Lys residues to Glu (the 3KE mutation) did not affect the ability of NRG1 to bind to ErbB3 but markedly reduced the ability of NRG1 to induce ErbB3 phosphorylation and AKT and Erk1/2 activation in MCF-7 and T47D human breast cancer cells. This suggests that direct integrin binding to NRG1 is critical for NRG1/ErbB signaling. Notably, stimulation of cells with WT NRG1 induced co-precipitation of ErbB3 with ␣6␤4 and with ␣v␤3 to a much lower extent. This suggests that WT NRG1 induces integrin-NRG1-ErbB3 ternary complex formation. In contrast, the 3KE mutant was much less effective in inducing ternary complex formation than WT NRG1, suggesting that this process depends on the ability of NRG1 to bind to integrins. These results suggest that direct NRG1-integrin interaction mediates integrin-ErbB cross-talk and that ␣6␤4 plays a major role in NRG-ErbB signaling in these cancer cells.The neuregulins (NRGs) 2 are a family of four structurally related proteins that are part of the EGF family of proteins (NRG1-4) (1-4). Transmembrane NRGs typically function as precursor molecules that are cleaved by metalloproteases. This results in the release of the extracellular domain that may subsequently bind to nearby receptors (autocrine/paracrine action). NRGs contain an epidermal growth factor (EGF)-like motif that binds and activates receptor-tyrosine kinases in the EGF receptor (ErbBs) family. Neuregulin-1 (NRG1) binds to ErbB3 and ErbB4. NRG1 has 11 isoforms (5). NRG1 plays essential roles in the nervous system, heart, and breast. NRG1 signaling is involved in the development and functions of several other organ systems and human diseases, including schizophrenia (6), coronary heart diseases (7), and cancer (8). Targeted deletion of ErbB2, ErbB3, ErbB4, or NRG1 in mice leads to developmental abnormalities that are severe in the nervous system and lethal in the cardiovascular system (9 -11). In cancer the interaction between ErbB receptors and ligands such as NRGs plays an important role in tumor growth. The EGF-like motif of NRGs is essential and sufficient for receptor binding and activation as well as promoting tumorigenesis (12). The presence of the autocrine loop is one of the causes that induces aberrant ErbB receptor activation and has been correlated with cancer development and progression. Disrupting...

Laminins are the major cell adhesive proteins in basement membranes, and consist of three subunits termed ␣, ␤, and ␥. Recently, we found that the Glu residue at the third position from the C termini of the ␥1 and ␥2 chains is critically involved in integrin binding by laminins. However, the ␥3 chain lacks this Glu residue, suggesting that laminin isoforms containing the ␥3 chain may be unable to bind to integrins. To address this possibility, we expressed the E8 fragment of laminin-213 and found that it was incapable of binding to integrins. Similarly, the E8 fragment of laminin-113 was expressed and also found to be inactive in binding to integrins, confirming the distinction between the integrin binding activities of ␥3 chain-containing isoforms and those containing the ␥1 or ␥2 chain. To further address the importance of the Glu residue, we swapped the C-terminal four amino acids of the ␥3 chain with the C-terminal nine amino acids of the ␥1 chain, which contain the Glu residue. The resulting chimeric E8 fragment of laminin-213 became fully active in integrin binding, whereas replacement with the nine amino acids of the ␥1 chain after substitution of Gln for the conserved Glu residue failed to restore the integrin binding activity. These results provide both loss-of-function and gainof-function evidence that laminin isoforms containing the ␥3 chain are unable to bind to integrins due to the absence of the conserved Glu residue, which should play a critical role in integrin binding by laminins.Laminins are heterotrimeric glycoproteins found in basement membranes and consist of three covalently linked chains termed ␣, ␤, and ␥. There are five ␣ chains (␣1-␣5), three ␤ chains (␤1-␤3), and three ␥ chains (␥1-␥3) that can give rise to at least 15 different functional laminin isoforms (1-3). These isoforms have been implicated in a wide variety of biological processes involving cell-basement membrane interactions through binding to cell surface receptors including integrins, syndecans, and dystroglycan (1, 4 -9).Integrins are ␣␤ transmembrane receptors that play critical roles in cell matrix adhesion in multicellular organisms. Several members of the integrin family proteins, including ␣3␤1, ␣6␤1, ␣6␤4, and ␣7␤1, serve as laminin receptors on a variety of cell types (10). The putative binding sites for these integrins have been mapped to the globular (G) 3 domain of the laminin ␣ chains (11-16), although trimerization with ␤ and ␥ chains is necessary for the G domain to exert its integrin binding activity (17)(18)(19). Recently, we found that the C-terminal regions of the ␥ chains are critically involved in integrin binding by laminins (20). Briefly, deletion of the C-terminal three but not two amino acids of the ␥1 chain completely abrogated the integrin binding activity of laminin-511 (␣5␤1␥1), while substitution of Gln for Glu-1607, the amino acid residue at the third position from the C terminus of the ␥1 chain, also abolished the integrin binding activity; thereby underscoring a critical role of Glu-1607 in i...

Hepatocyte‐like cells (HLCs) differentiated from human induced pluripotent stem (iPS) cells are expected to be applied for regenerative medicine. In this study, we attempted to generate safe and therapeutically effective human iPS‐HLCs for hepatocyte transplantation. First, human iPS‐HLCs were generated from a human leukocyte antigen‐homozygous donor on the assumption that the allogenic transplantation might be carried out. Highly efficient hepatocyte differentiation was performed under a feeder‐free condition using human recombinant laminin 111, laminin 511, and type IV collagen. The percentage of asialoglycoprotein receptor 1‐positive cells was greater than 80%, while the percentage of residual undifferentiated cells was approximately 0.003%. In addition, no teratoma formation was observed even at 16 weeks after human iPS‐HLC transplantation. Furthermore, harmful genetic somatic single‐nucleotide substitutions were not observed during the hepatocyte differentiation process. We also developed a cryopreservation protocol for hepatoblast‐like cells without negatively affecting their hepatocyte differentiation potential by programming the freezing temperature. To evaluate the therapeutic potential of human iPS‐HLCs, these cells (1 × 106 cells/mouse) were intrasplenically transplanted into acute liver injury mice treated with 3 mL/kg CCl4 only once and chronic liver injury mice treated with 0.6 mL/kg CCl4 twice weekly for 8 weeks. By human iPS‐HLC transplantation, the survival rate of the acute liver injury mice was significantly increased and the liver fibrosis level of chronic liver injury mice was significantly decreased. Conclusion: We were able to generate safe and therapeutically effective human iPS‐HLCs for hepatocyte transplantation. (Hepatology Communications 2017;1:1058–1069)

The Glu residue in the laminin γ-tail forms a bipartite integrin binding site with three globular domains of the α chain.