Human Placenta Laminin-111 as a Multifunctional Protein for Tissue Engineering and Regenerative Medicine.

Laminins are major components of all basement membranes surrounding nerve or vascular tissues. In particular laminin-111, the prototype of the family, facilitates a large spectrum of fundamental cellular responses in all eukaryotic cells. Laminin-111 is a biomaterial frequently used in research, however it is primarily isolated from non-human origin or produced with time-intensive recombinant techniques at low yield.Here, we describe an effective method for isolating laminin-111 from human placenta, a clinical waste material, for various tissue engineering applications. By extraction with Tris-NaCl buffer combined with non-protein-denaturation ammonium sulfate precipitation and rapid tangential flow filtration steps, we could effectively isolate native laminin-111 within only 4 days. The resulting material was biochemically characterized using a combination of dot blot, SDS-PAGE, Western blot and HPLC-based amino acid analysis. Cytocompatibility studies demonstrated that the isolated laminin-111 promotes rapid and efficient adhesion of primary Schwann cells. In addition, the bioactivity of the isolated laminin-111 was demonstrated by (a) using the material as a substrate for outgrowth of NG 108-15 neuronal cell lines and (b) promoting the formation of interconnected vascular networks by GFP-expressing human umbilical vein endothelial cells.In summary, the isolation procedure of laminin-111 as described here from human placenta tissue, fulfills many demands for various tissue engineering and regenerative medicine approaches and therefore may represent a human alternative to various classically used xenogenic standard materials.

Isolation and analysis of laminins.

Laminins are large glycoproteins forming structural and signaling networks with two major physiological roles: one role crucial for the formation and stability of basement membranes and the other role, as crucial as the first, in cell anchorage and signaling. Laminins come in several flavors as 16 different isoforms are known, each with both common and unique functions. Here the most current techniques for purification and identification of laminins in tissues and cultivated cells as well as for testing the cell adhesion-promoting activity of laminins will be described.

Utility of the laminin immunohistochemical stain in distinguishing invasive from noninvasive urothelial carcinoma.

BACKGROUND: To study the utility of the laminin immunostain in distinguishing invasive from noninvasive urothelial carcinoma (UC). The distinction is difficult but clinically significant as it can affect the decision to administer intravesical Bacillus Calmette-Guerin or can even lead to cystectomy. MATERIALS AND METHODS: Representative sections of the transurethral resection of bladder tumor specimens from 25 cases of formalin-fixed paraffin-embedded invasive UCs and 25 cases of noninvasive UCs were selected for immunohistochemical (IHC) staining with laminin (Ventana, Oro Valley, AZ, USA). These cases were selected using a computer-assisted search of our laboratory information system (Cerner CoPath). Tissue from five paraffin-embedded tissue blocks containing unremarkable urothelial-lined bladder parenchyma was chosen as controls. RESULTS: All five control cases demonstrated crisp linear staining of the basement membrane underlying the unremarkable urothelium. Similar findings were also noted in the 25 cases of noninvasive UC. All 25 cases of the invasive UC demonstrated a complete absence of the staining around invasive and malignant urothelial cells. Laminin staining was also noted in both the muscularis mucosae and the detrusor muscle, although the pattern of staining in these areas was granular and was distinguishable from the crisp linear staining of the basement membrane. CONCLUSION: Laminin IHC staining can be useful in differentiating invasive from noninvasive UC.

An efficient electrochemiluminescence amplification strategy via bis-co-reaction accelerator for sensitive detection of laminin to monitor overnutrition associated liver damage.

With the world wildly improvement in dietary and nutrition status, it couldn't be ignored that the chronic liver disease (CLD) resulted from the overnutrition. In order to estimate nutrition status for healthy living, an efficient and sensitive electrochemiluminescence (ECL) sandwich immunosensor of laminin (LN), a marker of CLD, was proposed for early diagnosis of CLD. In this work, the anodic ECL behavior of perylene derivative using H2O2 as co-reactant was demonstrated and the possible ECL mechanism was proposed. Furthermore, a significantly amplified ECL response could be obtained via Ag and Fe-Fe2O3 nanoparticles as bis-co-reaction accelerator. As a result, the proposed ECL immunosensor performed good sensitivity and accuracy with a detection limit down to 0.03pg/mL. Moreover, this immunosensor was successfully employed to monitor patient serum, which exhibited an alternative avenue for the early diagnosis of other diseases via proteins, nucleotide sequence, microRNA and cells.

Static and Dynamic Assays of Cell Adhesion Relevant to the Vasculature.

Methods are described for analyzing adhesion of isolated cells (such as leukocytes, tumor cells, or precursor cells) to purified adhesion receptors or cultured endothelial cells. "Static" assays (where cells are allowed to settle on the adhesive substrates) and flow-based assays (where cells are perfused over the substrates) are compared. Direct observations of the time course of adhesion and migration can be made when purified proteins or endothelial cells are cultured in plates, after cells are allowed to settle onto them for a desired period. In the flow-based assay, cells are perfused through coated glass capillaries, flow-channels incorporating coated plates, or commercially available preformed channels. Again, direct video-microscopic observations are made. In this assay various stages of capture, immobilization, and migration can be followed. In general, the static systems have higher throughput and greatest ease of use, but yield less detailed information, while the flow-based assay is most difficult to set up but is most physiologically relevant if one is interested in the dynamics of adhesion in the vasculature.

Immobilization and therapeutic passive stretching generate thickening and increase the expression of laminin and dystrophin in skeletal muscle

Extracellular matrix and costamere proteins transmit the concentric, isometric, and eccentric forces produced by active muscle contraction. The expression of these proteins after application of passive tension stimuli to muscle remains unknown. This study investigated the expression of laminin and dystrophin in the soleus muscle of rats immobilized with the right ankle in plantar flexion for 10 days and subsequent remobilization, either by isolated free movement in a cage or associated with passive stretching for up to 10 days. The intensity of the macrophage response was also evaluated. One hundred and twenty-eight female Wistar rats were divided into 8 groups: free for 10 days; immobilized for 10 days; immobilized/free for 1, 3, or 10 days; or immobilized/stretched/free for 1, 3, or 10 days. After the experimental procedures, muscle tissue was processed for immunofluorescence (dystrophin/laminin/CD68) and Western blot analysis (dystrophin/laminin). Immobilization increased the expression of dystrophin and laminin but did not alter the number of macrophages in the muscle. In the stretched muscle groups, there was an increase in dystrophin and the number of macrophages after 3 days compared with the other groups; dystrophin showed a discontinuous labeling pattern, and laminin was found in the intracellular space. The amount of laminin was increased in the muscles treated by immobilization followed by free movement for 10 days. In the initial stages of postimmobilization (1 and 3 days), an exacerbated macrophage response and an increase of dystrophin suggested that the therapeutic stretching technique induced additional stress in the muscle fibers and costameres.

Immobilization and therapeutic passive stretching generate thickening and increase the expression of laminin and dystrophin in skeletal muscle.

Extracellular matrix and costamere proteins transmit the concentric, isometric, and eccentric forces produced by active muscle contraction. The expression of these proteins after application of passive tension stimuli to muscle remains unknown. This study investigated the expression of laminin and dystrophin in the soleus muscle of rats immobilized with the right ankle in plantar flexion for 10 days and subsequent remobilization, either by isolated free movement in a cage or associated with passive stretching for up to 10 days. The intensity of the macrophage response was also evaluated. One hundred and twenty-eight female Wistar rats were divided into 8 groups: free for 10 days; immobilized for 10 days; immobilized/free for 1, 3, or 10 days; or immobilized/stretched/free for 1, 3, or 10 days. After the experimental procedures, muscle tissue was processed for immunofluorescence (dystrophin/laminin/CD68) and Western blot analysis (dystrophin/laminin). Immobilization increased the expression of dystrophin and laminin but did not alter the number of macrophages in the muscle. In the stretched muscle groups, there was an increase in dystrophin and the number of macrophages after 3 days compared with the other groups; dystrophin showed a discontinuous labeling pattern, and laminin was found in the intracellular space. The amount of laminin was increased in the muscles treated by immobilization followed by free movement for 10 days. In the initial stages of postimmobilization (1 and 3 days), an exacerbated macrophage response and an increase of dystrophin suggested that the therapeutic stretching technique induced additional stress in the muscle fibers and costameres.

Differentiation of human pluripotent stem cells to retinal pigmented epithelium in defined conditions using purified extracellular matrix proteins.

A potential application of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is the generation of retinal pigmented epithelium (RPE) to treat age-related macular degeneration (AMD), a common but incurable retinal disease. RPE cells derived from hESCs (hESC-RPEs) and iPSCs (iPSC-RPEs) express essential RPE markers and can rescue visual function in animal models. However, standard differentiation protocols yield RPE cells at low frequency, especially from iPSC lines, and the common use of Matrigel and xenogeneic feeder cells is not compatible with clinical applications. The extracellular matrix (ECM) can affect differentiation, and therefore changes in ECM composition may improve the frequency of stem cell-RPE differentiation. We selected several purified ECM proteins and substrates, based on the in vivo RPE ECM environment, and tested their ability to support iPSC-RPE differentiation and maintenance. iPSCs differentiated on nearly all tested substrates developed pigmented regions, with Matrigel and mouse laminin-111 supporting the highest pigmentation frequencies. Although iPSC-RPEs cultured on the majority of the tested substrates expressed key RPE genes, only six substrates supported development of confluent monolayers with normal RPE morphology, including Matrigel and mouse laminin-111. iPSCs differentiated on mouse laminin-111 produced iPSC-RPEs expressing RPE proteins, and hESCs differentiated on mouse laminin-111 resulted in high yields of functional hESC-RPEs. This identification of key ECM proteins may assist with future scaffold designs and provide peptide sequences for use in synthetic, xeno-free, GMP-compliant generation of RPE from human pluripotent stem cells relevant to clinical translation.

Rat mammary extracellular matrix composition and response to ibuprofen treatment during postpartum involution by differential GeLC-MS/MS analysis.

Breast cancer patients diagnosed within five years following pregnancy have increased metastasis and decreased survival. A hallmark of postpartum biology that may contribute to this poor prognosis is mammary gland involution, involving massive epithelial cell death and dramatic stromal remodeling. Previous studies show pro-tumorigenic properties of extracellular matrix (ECM) isolated from rodent mammary glands undergoing postpartum involution. More recent work demonstrates systemic ibuprofen treatment during involution decreases its tumor-promotional nature. Utilizing a proteomics approach, we identified relative differences in the composition of mammary ECM isolated from nulliparous rats and those undergoing postpartum involution, with and without ibuprofen treatment. GeLC-MS/MS experiments resulted in 20327 peptide identifications that mapped to 884 proteins with a <0.02% false discovery rate. Label-free quantification yielded several ECM differences between nulliparous and involuting glands related to collagen-fiber organization, cell motility and attachment, and cytokine regulation. Increases in known pro-tumorigenic ECM proteins osteopontin, tenascin-C, and laminin-α1 and pro-inflammatory proteins STAT3 and CD68 further identify candidate mediators of breast cancer progression specific to the involution window. With postpartum ibuprofen treatment, decreases in tenascin-C and three laminin chains were revealed. Our data suggest novel ECM mediators of breast cancer progression and demonstrate a protective influence of ibuprofen on mammary ECM composition.

The potential of laminin-2-biomimetic short peptide to promote cell adhesion, spreading and migration by inducing membrane recruitment and phosphorylation of PKCδ.

Laminin α2 chain plays an important role in basement membrane assembly and peripheral myelinogenesis; however, the integrin binding motif within human laminin α2 chain and the signaling pathways downstream of this ligand-receptor interaction are poorly understood. We identified a motif, RNIPPFEGCIWN (Ln2-LG3-P2), within LG3 domain of human laminin α2 chain as a major site for both α3ß1 integrin and cellular activities such as cell adhesion, spreading, and migration. Binding of α3ß1 integrin with Ln2-LG3-P2 induced the membrane recruitment of protein kinase Cδ (PKCδ) and stimulated its tyrosine phosphorylation. The cellular activities induced by Ln2-LG3-P2 and the phosphorylation of focal adhesion kinase (FAK) were inhibited by rottlerin, a PKCδ inhibitor, but not by Gö6976, a PKCα/ß inhibitor. These results indicate that RNIPPFEGCIWN motif within human laminin α2 chain is a major ligand for α3ß1 integrin, and that binding of α3ß1 integrin mediates cellular activities through membrane recruitment and tyrosine phosphorylation of PKCδ and FAK phosphorylation.

Cell surface proteoglycans syndecan-1 and -4 bind overlapping but distinct sites in laminin α3 LG45 protein domain.

Keratinocyte migration during epidermal repair depends on interactions between cellular heparan sulfate proteoglycan receptors, syndecan-1 and -4, and the C-terminal globular domains (LG45) of the extracellular matrix protein laminin 332. This study investigates the molecular basis of the binding specificity of the syndecan-1 and -4 receptors expressed by human keratinocytes. We used site-directed mutagenesis to alter a recombinant LG45 protein by substituting the most critical basic residues with glutamine. All proteins were expressed in mammalian cells, purified, and characterized biochemically. We used in vitro binding assays, including surface plasmon resonance, to examine interactions between mutated LG45 and heparan sulfates, syndecan-1 and -4. We identify a major heparin binding domain on the outer edge of a ß-strand of LG45 surrounded by a track of converging low affinity residues. This domain harbors distinctive syndecan-1 and -4 binding-specific sequences. This is the first study to demonstrate a binding specificity of two proteoglycans produced by a single cell type. In addition, we found that although syndecan-1 interacts exclusively through its glycosaminoglycan chains, syndecan-4 binding relies on both its core protein and its heparan sulfate chains. These results suggest that LG45 may trigger different signals toward keratinocytes depending on its interaction with syndecan-1 or -4.

Adsorption of HSA, IgG and laminin-1 on model hydroxyapatite surfaces--effects of surface characteristics.

Ellipsometry and mechanically assisted sodium dodecyl sulphate elution was utilized to study the adsorption of human serum albumin (HSA), human immunoglobulin G (IgG), and laminin-1, as well as competitive adsorption from a mixture of these proteins on spin-coated and sintered hydroxyapatite (HA) surfaces, respectively. The HA surfaces were characterized with respect to wettability and roughness by means of water contact angles and atomic force microscopy, respectively. Both surface types were hydrophilic, and the average roughness (Sa) and surface enlargement (Sdr) were lower for the sintered compared to the spin-coated HA surfaces. The adsorbed amounts on the sintered HA increased as follows: HSA < laminin-1 < IgG < the protein mixture. For the competitive adsorption experiments, the adsorbed fractions increased accordingly: HSA < laminin-1 < IgG on both types of HA substratum. However, a higher relative amount of HSA and laminin-1 and a lower relative amount of IgG was found on the spin-coated surfaces compared to the sintered surfaces. The effects observed could be ascribed to differences in surface roughness and chemical composition between the two types of HA substratum, and could have an influence on selection of future implant surface coatings.

Substrates for human pluripotent stem cell cultures in conditioned medium of mesenchymal stem cells.

We aimed to establish a culture system of human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), free from xenogeneic proteins, Matrigel(™) and conditioned medium of mouse embryonic fibroblasts. The conditioned culture medium consisted of mesenchymal stem cells derived from human bone marrow. We examined surface properties suitable for hPSC culture by using self-assembled monolayers (SAMs) of alkanethiols with four different functional groups: CH(3), OH, COOH and NH(2). hPSCs neither adhered nor proliferated on surfaces with a water contact angle higher than 40°. Based on this finding, the contact angle of a polystyrene (PSt) culture dish was reduced to less than 40°, and COOH and OH groups were introduced to its surface by oxygen plasma treatment, making the PSt dish suitable for hPSC culture. This combination of a PSt dish treated with oxygen plasma treatment and conditioned medium of mesenchymal stem cells achieved a long-term maintenance of hPSCs without differentiation.

The use of human hepatocytes to investigate bile acid synthesis.

De novo synthesis of bile acids is a liver-specific function that is difficult to maintain in cultured cells. There are significant species differences in both types of bile acids formed and more importantly in the regulation of bile acid homeostasis. This highlights the need for a good human in vitro model. Isolated primary human hepatocytes have the capacity to synthesize normal conjugated bile acids at a rate similar to that in vivo. In this chapter we describe the importance of different culture conditions such as choice of substrate, media and supplements on the total bile acid production as wells as the bile acid composition.

Laminin-121--recombinant expression and interactions with integrins.

Laminin-121, previously referred as to laminin-3, was expressed recombinantly in human embryonic kidney (HEK) 293 cells by triple transfection of full-length cDNAs encoding mouse laminin α1, ß2 and γ1 chains. The recombinant laminin-121 was purified using Heparin-Sepharose followed by molecular sieve chromatography and shown to be correctly folded by electron microscopy and circular dichroism (CD). The CD spectra of recombinant laminin-121 were very similar to those of laminin-111 isolated from Engelbreth-Holm-Swarm tumor (EHS-laminin) but its T(m) value was smaller than EHS-laminin and recombinant lamnin-111 suggesting that the replacement of the ß chain reduced the stability of the coiled-coil structure of laminin-121. Its binding to integrins was compared with EHS-laminin, laminin-3A32 purified from murine epidermal cell line and recombinantly expressed laminins-111, -211 and -221. Laminin-121 showed the highest affinity to α6ß1 and α7ß1 integrins and furthermore, laminin-121 most effectively supported neurite outgrowth. Together, this suggests that the ß2 laminins have higher affinity for integrins than the ß1 laminins.

Simplified purification procedure of laminin-332 and laminin-511 from human cell lines.

Laminins are glycoproteins expressed in the basement membrane of multiple epithelial tissues. Previously described purification procedures for the human laminin variants laminin-5 (LN-332) and laminin-10 (LN-511) use tissue as starting material and have multiple steps. We demonstrate a two-step laminin immunoaffinity purification method to produce consistent quantities of intact and biologically active LN-332 and LN-511 from human keratinocyte (HaCaT) and human lung carcinoma (A549) cell lines, respectively. The purification of LN-332 and LN-551 was demonstrated by PAGE analysis, silver staining and Western blot analysis. The purification procedure includes instruction on removing a cell adhesion contaminant known as galectin-3 binding protein from purified LN-511. The biological activity of purified laminin was tested in a standard cell adhesion assay and compared to commercially available LN-111. This rapid and reproducible purification method will contribute to understanding the role of LN-332 and LN-511 in cell behavior, signaling, and gene expression.

Crystal structure and cell surface anchorage sites of laminin alpha1LG4-5.

The laminin G-like (LG) domains of laminin-111, a glycoprotein widely expressed during embryogenesis, provide cell anchoring and receptor binding sites that are involved in basement membrane assembly and cell signaling. We now report the crystal structure of the laminin alpha1LG4-5 domains and provide a mutational analysis of heparin, alpha-dystroglycan, and galactosylsulfatide binding. The two domains of alpha1LG4-5 are arranged in a V-shaped fashion similar to that observed with laminin alpha2 LG4-5 but with a substantially different interdomain angle. Recombinant alpha1LG4-5 binding to heparin, alpha-dystroglycan, and sulfatides was dependent upon both shared and unique contributions from basic residues distributed in several clusters on the surface of LG4. For heparin, the greatest contribution was detected from two clusters, 2719RKR and 2791KRK. Binding to alpha-dystroglycan was particularly dependent on basic residues within 2719RKR, 2831RAR, and 2858KDR. Binding to galactosylsulfatide was most affected by mutations in 2831RAR and 2766KGRTK but not in 2719RKR. The combined analysis of structure and activities reveal differences in LG domain interactions that should enable dissection of biological roles of different laminin ligands.

Characterization of commercial laminin preparations from human placenta in comparison to recombinant laminins 2 (alpha2beta1gamma1), 8 (alpha4beta1gamma1), 10 (alpha5beta1gamma1).

Laminins, a family of large heterotrimeric (alphabetagamma) proteins, are major components of basement membranes implicated in a variety of cellular functions. Different commercial laminin preparations isolated from human placenta have been widely used in functional studies but their molecular properties are poorly known. In the present study, we characterized several of these preparations by ELISA, silver staining and Western blotting, in comparison to mouse laminin 1 (alpha1beta1gamma1), and recombinant human laminins 2 (alpha2beta1gamma1), 8 (alpha4beta1gamma1) and 10 (alpha5beta1gamma1). The cell migration-promoting activity of different batches was also tested. The placenta laminin preparations differed from one another and consisted of highly fragmented proteins, a mixture of laminin isoforms, and/or contaminating fibronectin. Major functional differences between batches were also observed, reflecting molecular heterogeneity. Previous data obtained in functional studies using these preparations need to be interpreted with caution and may require revision, and future functional studies demand prior molecular characterization of the laminins, particularly their alpha-chain.