Detection of cellular damage by hydrogen peroxide using SV40-T2 cells on shear horizontal surface acoustic wave (SH-SAW) sensor.

The rat lung epithelial cell line SV40-T2 was used to develop a cellular biosensing system to assay for environmental toxicants. The novel approach on which this system is based involves direct attachment of cultured rat or human cells onto a cell-adhesive matrix on the device through which shear horizontal surface acoustic waves (SH-SAW) are transmitted using 50 MHz SAW resonator. This novel design enables sensitive monitoring of changes of the electrophysical characteristics of cells, such as their conductivity and relative permittivity. A time-dependent change of phase of SAW and change of insertion loss (change of amplitude) were observed when the cells were treated with 0.5 or 1.0 mM H2O2. The change of insertion loss was biphasic, with an early phase (1-3 h) and a late phase (3-6 h). The late phase coincided with the destruction of cell-cell tight junctions detected by measurement of the transepithelial electrical resistance and paracellular permeability; in contrast, the early phase coincided with the destruction of intracellular actin filaments by H2O2. The early-phase effect of H2O2 on phase shift may be attributable to the change of intracellular permittivity by a change of cellular polarity. Immunofluorescence microscopy showed the disappearance of zonula occludens protein 1 from the region of cell-cell contact. These results suggest the correlation between the change of insertion loss as an SAW parameter and the destruction of tight junctions of the cells on the SH-SAW device in the late phase.

Efficient differentiation of embryonic stem cells into hepatic cells in vitro using a feeder-free basement membrane substratum.

The endoderm-inducing effect of the mesoderm-derived supportive cell line M15 on embryonic stem (ES) cells is partly mediated through the extracellular matrix, of which laminin α5 is a crucial component. Mouse ES or induced pluripotent stem cells cultured on a synthesized basement membrane (sBM) substratum, using an HEK293 cell line (rLN10-293 cell) stably expressing laminin-511, could differentiate into definitive endoderm and subsequently into pancreatic lineages. In this study, we investigated the differentiation on sBM of mouse and human ES cells into hepatic lineages. The results indicated that the BM components played an important role in supporting the regional-specific differentiation of ES cells into hepatic endoderm. We show here that knockdown of integrin ß1 (Itgb1) in ES cells reduced their differentiation into hepatic lineages and that this is mediated through Akt signaling activation. Moreover, under optimal conditions, human ES cells differentiated to express mature hepatocyte markers and secreted high levels of albumin. This novel procedure for inducing hepatic differentiation will be useful for elucidating the molecular mechanisms controlling lineage-specific fates during gut regionalization. It could also represent an attractive approach to providing a surrogate cell source, not only for regenerative medicine, but also for pharmaceutical and toxicologic studies.

Synthesized basement membranes direct the differentiation of mouse embryonic stem cells into pancreatic lineages.

We previously reported that embryonic stem (ES) cells cultured on M15 cells, a mesoderm-derived supportive cell line, were efficiently differentiated towards an endodermal fate, finally adopting the specific lineages of various digestive organs such as the pancreas and liver. We show here that the endoderm-inducing activity of M15 cells is in part mediated through the extracellular matrices, and that laminin alpha5 is one of the crucial components. In an attempt to establish a feeder-free ES-cell procedure for pancreatic differentiation, we used a synthesized basement membrane (sBM) substratum using an HEK293 cell line stably expressing laminin-511. On the sBM, mouse ES or induced pluripotent stem (iPS) cells sequentially differentiated into the definitive endoderm, pancreatic progenitor cells, and then insulin-expressing pancreatic beta-cells in vitro. Knockdown of ES cells with integrin beta1 (Itgb1) reduces differentiation towards pancreatic cells. Heparan sulfate proteoglycan 2 (HSPG2) knockdown and heparitinase treatment synergistically decreased the number of Pdx1-expressing cells. These findings indicate that components of the basement membrane have an important role in the differentiation of definitive endoderm lineages. This novel procedure will be useful for the study of pancreatic differentiation of ES or iPS cells and the generation of potential sources of surrogate cells for regenerative medicine.

Interleukin-1beta and tumor necrosis factor-alpha have opposite effects on fibroblasts and epithelial cells during basement membrane formation.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are typical proinflammatory cytokines that influence various cellular functions, including metabolism of the extracellular matrix. We examined the roles of IL-1beta and TNF-alpha in basement membrane formation in an in vitro model of alveolar epithelial tissue composed of alveolar epithelial cells and pulmonary fibroblasts. Formation of the basement membrane by immortalized rat alveolar type II epithelial (SV40-T2) cells, which ordinarily do not form a continuous basement membrane, was dose-dependently upregulated in the presence of 2 ng/ml IL-1beta or 5 ng/ml TNF-alpha. IL-1beta or TNF-alpha alone induced increased secretion of type IV collagen, laminin-1, and nidogen-1/entactin, all of which contributed to this upregulation. In contrast, while SV40-T2 cells cultured with a fibroblasts-embedded type I collagen gel were able to form a continuous basement membrane, they failed to form a continuous basement membrane in the presence of IL-1beta or TNF-alpha. Fibroblasts treated with IL-1beta or TNF-alpha secreted matrix metalloproteinase (MMP)-9 and MMP-2, and these MMPs inhibited basement membrane formation and degraded the basement membrane architecture. Neither IL-1beta- nor TNF-alpha-treated SV40-T2 cells increased the secretion of MMP-9 and MMP-2. These results suggest that IL-1beta participates in basement membrane formation in two ways. One is the induction of MMP-2 and MMP-9 secretion by fibroblasts, which inhibits basement membrane formation, and the other is induction of basement membrane component secretion from alveolar epithelial cells to enhance basement membrane formation.

Seamless signal transduction from live cells to an NO sensor via a cell-adhesive sensing matrix.

A smart live-cell assay was developed as a cellular biosensing system. This system is based on novel tactics: the direct assembly of human cultured cells onto a cell-adhesive sensing matrix. This novel design provides considerable advantages, among them the possibility of capturing molecular signals immediately after they are secreted from living cells. The design also helps preserve all cellular characteristics intact. In this study, a cell-adhesive NO sensing matrix, acting as both an NO-permeable membrane and a cell-adhesive scaffold, was designed using functional polymers and a short peptide sequence derived from extracellular matrix (ECM) proteins. Using the cell-adhesive NO sensing matrix, we constructed a cellular biosensing system based on in situ monitoring of NO released from a human umbilical vein endothelial cell (HUVEC) layer. HUVECs were employed as an organ-functional model of a blood vessel in view of screening vasodilatory substances for clinical purposes. In our novel system, the electrochemical NO sensor is adjacent to the NO-producing cells, which allows the sensing device to achieve superior sensitivity and precise response to a very low number of NO molecules. Our design enables the fixing of the exact distance between the organ-functional model and the chemical sensor without cumbersome manipulations. Consequently, this cellular biosensing system may be readily applicable to high-throughput analysis in the field of drug screening.

Role of basement membrane in EMMPRIN/CD147 induction in rat tracheal epithelial cells.

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a glycosylated transmembrane protein known to induce matrix metalloproteinases (MMPs). Although the expression of EMMPRIN is physiologically limited to fetal lung epithelium, the transcriptional regulation of this protein remains to be elucidated. We hypothesized that the interaction of epithelial cells with the basement membrane regulates EMMPRIN expression. The basement membrane has highly integrated architecture composed of specific extracellular matrix, such as laminins and type IV collagen, and exhibits multiple functions. We previously developed a structured basement membrane mimic, a synthesized basement membrane (sBM) substratum, in which laminin-111, a unique component of embryonic lungs, is incorporated. In the present study we quantified expression of EMMPRIN mRNA of rat tracheal epithelial cells cultured on sBM, laminin-111, type IV collagen, or laminin-332. EMMPRIN was upregulated on sBM and laminin-111, although this was not accompanied by MMP-9 induction. In contrast, type IV collagen and laminin-332 did not induce EMMPRIN. These findings suggest potential roles for basement membrane in the transcriptional regulation of tracheal epithelial EMMPRIN.

Differentiation of tracheal basal cells to ciliated cells and tissue reconstruction on the synthesized basement membrane substratum in vitro.

Although lung epithelial cells directly attach to the basement membrane underneath in vivo, harvested epithelial cells are typically cultured on type I collagen gel (Col I-gel) in vitro. Recently we developed new culture substratum, designated as "synthesized Basement Membrane" (sBM), that has bared lamina densa on fibrillar collagen. To validate the usefulness of sBM substratum in airway tissue reconstitution in vitro, we cultured rat tracheal epithelial cells on sBM substratum and Col I-gel. When starting the air-liquid interface culture, most of the epithelial cells were squamous and positive for the basal cell marker cytokeratin 14 (CK14). After 14 days on sBM substratum, CK14-positive cells differentiated not only to Clara and mucous cells, but also to ciliated cells. Those differentiated cells formed pseudostratified-like epithelium and the remaining CK14-positive cells were polarized to the basal side. However, on Col I-gel, the CK14-positive cells were still squamous and not polarized, and ciliated cells did not appear. In conclusion, we established a new culture model on sBM substratum in which basal cells could differentiate to ciliated cells. The application of sBM substratum is useful in the study of the airway epithelial cell differentiation in vitro.

Homeobox B3, FoxA1 and FoxA2 interactions in epithelial lung cell differentiation of the multipotent M3E3/C3 cell line.

HOM/C homeobox (Hox) and forkhead box (Fox) factors are reported to be expressed in the foregut endoderm and are subsequently detected in a spatio-temporal pattern during lung development. Some of these factors were reported to influence the expression of lung marker proteins or to modulate lung development. To clarify the molecular mechanisms for generating functional lung cells from progenitor cell populations, we introduced the forkhead box factors, FoxA1 and FoxA2, and the homeobox factor, HoxB3, into the differentiation process in a multipotent hamster lung epithelial M3E3/C3 cell line. Ectopic expression of FoxA2 promoted differentiation to Clara-like cells with up-regulation of the expression of the lung marker proteins, Clara cell-specific 10-kDa protein and surfactant protein-B. In contrast, FoxA1 repressed the differentiation. HoxB3 transfection induced FoxA2 expression transiently at the pre-differentiation stage. The endogenous HoxB3 expression level decreased at later stages of Clara-like cell differentiation, and the attenuation was enhanced by FoxA2 transfection. HoxB3 is a putative upstream regulator that enhances FoxA2 expression at the pre-differentiation stage. In addition, we found that the expression of HoxA4, HoxA5, and HoxC9 increased differentially during Clara-like cell differentiation. These results suggest that HoxB3 may be a putative positive regulator of FoxA2 expression at the pre-differentiation stage, and those interactions of Fox factors and Hox factors could participate in Clara cell differentiation.

Hepatocyte growth factor inhibits the formation of the basement membrane of alveolar epithelial cells in vitro.

Hepatocyte growth factor (HGF) is a pulmotrophic factor for the regeneration of injured pulmonary tissue. We investigated the role of HGF in basement membrane formation during wound healing by immortalized alveolar type II epithelial cells that could form a continuous basement membrane when they were cultured on collagen fibrils in the presence of entactin-contaminated laminin-1. Cells cultured with 5.0 ng/ml HGF neither formed a continuous basement membrane on collagen fibrils nor maintained a continuous basement membrane architecture on a basement membrane substratum. The cells showed increased secretion of matrix metalloproteinase-9 and urokinase-type plasminogen activator, and the HGF-induced inhibition of basement membrane formation was attenuated by addition of 200 ng/ml tissue inhibitor of matrix metalloproteinase-1. Cells sequentially exposed to HGF and 1.0 ng/ml transforming growth factor-beta1 had enhanced basement membrane formation compared with those receiving these reagents in the reverse order or concurrently. HGF simultaneously stimulated proliferation and migration of the cells so that it advanced wound closure on the basement membrane substratum. The present results indicate that the role of HGF in wound healing is the stimulation of reepithelization, but this factor may also contribute to the degradation of the basement membrane.

Laminin alpha-chain expression and basement membrane formation by MLE-15 respiratory epithelial cells.

Basement membranes have a critical role in alveolar structure and function. Alveolar type II cells make basement membrane constituents, including laminin, but relatively little is known about the production of basement membrane proteins by murine alveolar type II cells and a convenient system is not available to study basement membrane production by murine alveolar type II cells. To facilitate study of basement membrane production, with particular focus on laminin chains, we examined transformed murine distal respiratory epithelial cells (MLE-15), which have many structural and biochemical features of alveolar type II cells. We found that MLE-15 cells produce laminin-alpha5, a trace amount of laminin-alpha3, laminins-beta1 and -gamma1, type IV collagen, and perlecan. Transforming growth factor-beta1 significantly induces expression of laminin-alpha1. When grown on a fibroblast-embedded collagen gel, MLE-15 cells assemble a basement membrane-like layer containing laminin-alpha5. These findings indicate that MLE-15 cells will be useful in modeling basement membrane production and assembly by alveolar type II cells.