Katsumi Mochitate

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 α5 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 β-cells in vitro. Knockdown of ES cells with integrin β1 (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.

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 β1 (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.

Increases in the mRNA levels of γ-Glutamyltransferase and heme oxygenase-1 in the rat lung after ozone exposure☆

gamma-Glutamyltransferase (GGT) and heme oxygenase-1 (HO-1) are induced by chemical and physical stresses producing an oxidative burden on tissues and cells. Both enzymes are proposed to have an antioxidant role in protecting cells and tissues from oxidative burden. To explore the effects of ozone (O3), the major oxidant in photochemical smog, on the expression of GGT and HO-1 genes in the lung, we exposed rats to 0.4 ppm O3 for up to 7 days. After exposures, mRNA levels of GGT and HO-1 in the lung were measured by RNA blot analysis. Although a 1-day exposure did not change either GGT or HO-1 mRNA levels in the lung, both genes responded to prolonged exposure to O3. GGT mRNA was increased to 149% (P < 0.01) and 158% (P < 0.01) of the control by 3- and 7-day exposures, respectively. HO-1 mRNA was also elevated to 174% (P < 0.01) and 184% (P < 0.001) of the control after 3- and 7-day exposures, respectively. The elevation of GGT and HO-1 mRNA after prolonged exposure to O3 suggests that expression of these genes is not involved in the acute respiratory response, but in the recovery process from lung damage induced by O3.

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.

Transforming growth factor-β1 regulates basement membrane formation by alveolar epithelial cells in vitro

Abstract Immortalized alveolar type II epithelial (SV40-T2) cells formed a continuous, thin lamina densa when they were cultured on collagen fibrils with the supplement of 1.0 ng/ml TGF-β1. Corresponding to lamina densa formation, immuno-histochemical analysis of laminin, type IV collagen, perlecan, and entactin (nidogen) indicated integration of these components in a linear array beneath the SV40-T2 cells. Synthesis of these basement membrane constituents was significantly enhanced by β1 in a dose-dependent manner. On the other hand, β1 did not affect the synthesis of extracellular matrix-regulatory enzymes and their inhibitors, such as type II transglutaminase, matrix metalloproteinase-2, plasminogen activator inhibitor-1, or tissue inhibitor of matrix metalloproteinase-1. These results indicate that basement membrane formation in the presence of 1.0 ng/ml TGF-β1 is attributable to enhanced synthesis of basement membrane constituents. However, formation of a continuous basement membrane was inhibited at a TGF-β1 concentration of 5.1 ng/mi. Synthesis of the basement membrane constituents was further enhanced at this concentration and the extracellular matrix-regulatory enzymes remained unchanged. The deposits of cellular fibronectin and type I collagen beneath SV40-T2 cells were significantly augmented. Thus excessive production of interstitial extracellular matrix components appears to obstruct the integration of basement membrane constituents into a continuous architecture. These results indicate that the basement membrane formation by SV40-T2 cells is achieved at the optimal TGF-β1 concentration.

Effect of aging on nitric oxide production by rat alveolar macrophages.

Nitric oxide (NO) plays an important role in alveolar macrophages (AM)-mediated defense against infection. The elderly become highly susceptible to respiratory tract infection. Inhibition of NO production significantly suppresses defense against infections. Therefore, it is necessary to elucidate the effect of senescence on NO production of AM. The alveolar microenvironment and lymphocytes affect NO production by AM. We examined whether changes in the alveolar microenvironment, lymphocytes, or AM brought about by aging affect NO production by AM. Bronchoalveolar lavage fluid was used as a substitute for the alveolar microenvironment. The results showed that NO production by AM activated by lymph node cells in bronchoalveolar lavage fluid from old rats in response to concanavalin A decreased compared with that of young rats. AM from aged rats produced less NO than AM from young rats. Bronchoalveolar lavage fluid and lymph node cells from aged rats had no effect on the amount of NO produced by AM. Therefore, age-associated decrease in the functional capacity of AM plays a central role in the decrease of NO production.

Long‐term effects of ozone and nitrogen dioxide on the metabolism and population of alveolar macrophages

To investigate how alveolar macrophages adapt themselves to oxidative pollutants in the long term, rats were exposed to a strong oxidant, ozone (O3), or a weak oxidant, nitrogen dioxide (NO2), for a maximum duration of 12 wk. After exposures, alveolar macrophages were collected by pulmonary lavage. Throughout 11 wk of exposure to 0.2 ppm O3, the specific activities of glucose-6-phosphate dehydrogenase (G6PDH) and glutathione peroxidase of the peroxidative metabolic pathway and pyruvate kinase and hexokinase of the glycolytic pathway were 40-70% elevated over the controls in alveolar macrophages. The population of alveolar macrophages was consistently 60% higher than the controls. The small-sized macrophages, immature macrophages, preferentially increased. To the contrary, the thymidine incorporation per cell was always 20-30% lower than in the controls, although the total incorporation remained unchanged. No infiltration of polymorphonuclear leukocytes occurred. By 12 wk of exposures to 1.2 and 4.0 ppm NO2, the population of alveolar macrophages increased 30% over the control. Among the enzymes examined, however, only the G6PDH activity increased 10% for 4.0 ppm NO2. No increase in the enzyme activities occurred for 1.2 ppm NO2. Based on these results, alveolar macrophages adapt themselves to the long-term exposure of O3 or NO2 by recruiting immature macrophages through an apparent influx of monocytes. During the exposure to O3, the peroxidative metabolic and glycolytic pathways are enhanced persistently in alveolar macrophages, whereas both pathways were not enhanced by the exposures to NO2.

Metabolic enhancement and increase of alveolar macrophages induced by ozone

Male Wistar rats were exposed to 0.2 ppm ozone (O3) for 14 days and at intervals alveolar macrophages were collected by bronchoalveolar lavage to examine the effects of O3. The specific activities of glucose-6-phosphate dehydrogenase and glutathione peroxidase of alveolar macrophages increased to 1.6-fold (on the 3rd day) and 1.5-fold (on the 5th day), respectively, those of the control values. Similarly, the specific activities of pyruvate kinase, lactate dehydrogenase, and hexokinase also increased to 1.6-fold, 1.4-fold, and 1.2-fold, respectively, those of the control values on the 3rd day. The activities of all enzymes tested were maintained at significantly higher levels until the 14th day. Furthermore, the incorporation of [14C]thymidine into alveolar macrophages increased twice the control values on the 1st and 3rd days and was almost completely inhibited by the addition of 1.23 x 10(-4) M aphidicolin, a competitive inhibitor of DNA polymerase alpha. The number of alveolar macrophages collected from exposed animals also increased to 1.5-fold that of the control value on the 3rd day and was maintained at significantly higher level until the 14th day. It was noted that alveolar macrophages of small size preferentially increased between the 5th and 14th days. These results show that exposures to 0.2 ppm O3 induced a metabolic enhancement of the peroxidative metabolism, glycolysis, and DNA synthesis in alveolar macrophages and increased the macrophages of small size.

In vivo effects of nitrogen dioxide on membrane constituents in lung and liver of rats

When rats were exposed to 10 ppm NO2 for 7 days, the succinate-cytochrome c reductase activity, the rate-limiting step of mitochondrial succinoxidase, of the liver decreased progressively, reaching 77% (P less than 0.01) of the control level by the fifth day. By contrast, this activity was gradually increased by a 10-day exposure to 4 ppm NO2 and reached 1.14-fold that of the control level at the seventh day. A reduction in the components of electron-transport systems in liver microsomes was found during exposure to NO2 at both concentrations. This reduction was preferential for components of drug-metabolizing systems. The NADPH-cytochrome c reductase activity and the cytochrome P-450 content were decreased to 82% (P less than 0.05) and 76% (P less than 0.05), respectively, at the fifth day of exposure to 10 ppm NO2. Exposure to 4 ppm NO2 also caused a significant decrease in the NADPH-cytochrome c reductase and the cytochrome P-450 content, which were lowered to 84% (P less than 0.05) of the control level at the fourth and seventh days, respectively. Reduction in these components appears to occur even at the concentration of 0.4 ppm at the seventh day. Alterations in the components of mitochondria and microsomes of the lung were considerably different from those of the liver. The protein content of the lung increased to 1.18 (P less than 0.05)- and 1.14 (P less than 0.05)-fold that of the control during exposure to 10 and 4 ppm NO2, respectively. The succinate-cytochrome c reductase activity showed a reduced value, which was 73% (P less than 0.01) of the control level, 1 day after exposure to 10 ppm NO2, Subsequently, the activity increased to 1.14-fold that of the control during exposure to 10 (at the fifth day) and 4 ppm (at the fourth day) NO2. Components of microsomal electron-transport systems also showed a slightly elevated value during exposure to 4 ppm NO2. Seven days after exposure, the cytochrome P-450 content was decreased to 61% (P less than 0.01) of the control level, while other components were retained at control levels. A significant reduction in the cytochrome P-450 content was also observed at the exposure concentration of 1.2 ppm, but not of 0.4 ppm, at the seventh day.

Effects of nitrate and nitrite, chemical intermediates of inhaled nitrogen dioxide, on membrane components of red blood cells of rats

Rat blood was incubated at 37 degrees C for 60 min with either NaNO3 or NaNO2 to examine the relationship between the decrease in the hexose content and Ca2+,Mg2+-ATPase activity of red cell membranes, and NO3- and NO2-. The hexose content decreased depending on the NaNO2 concentration up to 100 microM reaching 76% (p less than 0.05) of the control value. NaNO3 had little effect on the hexose content. On the other hand, the Ca2+,Mg2+-ATPase activity decreased depending on the NaNO3 concentration up to 200 microM, where the activity reached 75% (p less than 0.01) of the control value. The effect of NaNO2 on this activity was smaller than that of NaNO3. The sialic acid content and the Na+,K+-ATPase activity did not show significant alterations by incubation with NaNO2 and NaNO3 at below 100 microM. To examine the in vivo effects of NO2- and NO3-, 50 mM NaNO3 was intravenously injected into rats five times at hourly intervals (dose: 1.0 ml/kg body weight), and blood was collected 1 hr after the last injection. The activities of Ca2+,Mg2+- and Na+,K+-ATPases of red cell membranes were decreased to 68% (p less than 0.05) and 80% of the control value, respectively. Reduction by injection of 50 mM NaNO2 was smaller than that by 50 mM NaNO3. The results show that the hexose content and the Ca2+,Mg2+-ATPase activity of red cell membranes were decreased by NO-x that increased in the blood during short-term exposure of rats to NO2.