Biomedical subjects

Hiroo Iwata

Publications and source records attributed to Hiroo Iwata.

At least 19 recordsLinked to original sources

PubMed2007

Antibody arrays for quantitative immunophenotyping.

Detection of multiple surface antigens expressed on living cell is an important step for cell processing and clinical diagnosis. Here we describe the preparation of antibody arrays that allow parallel detection of multiple surface antigens through affinity binding of living cells. An antibody array was fabricated by photo-assisted patterning of an alkanethiol monolayer formed on a gold-coated glass plate and subsequent immobilization of antibodies specific for cell surface antigens in an array format. We demonstrate here that rapid phenotyping can be performed on the array for both adhesion-dependent and non-dependent cells by direct cell binding assays. The density of bound cells on each antibody spot was in accordance with their contents in an original suspension. This result suggests the feasibility of the array-based method for quantitative assessment of multiple antigen expression. These findings will serve to extend the range of fundamental and clinical applications of antibody arrays.

Antigen-Antibody Complex↗

PubMed2006

Combinatorial protein display for the cell-based screening of biomaterials that direct neural stem cell differentiation.

Neural stem cell (NSC) has emerged as a potential source for cell replacement therapy following traumatic injuries and degenerative diseases of the central nervous system. However, clinical applications of NSC further require technological advances especially for controlling differentiation of NSC. This study aimed at developing biomaterials that serve to expand undifferentiated NSC or to induce cells with specific phenotypes. Our approach is to construct composite biomaterials that consist of extracellular matrix components and growth factors. In order to optimize matrix-growth factor combinations, we conducted the parallel and rapid screening of composite biomaterials through assays using cell-based arrays. The photo-assisted patterning of an alkanethiol self-assembled monolayer was employed to achieve site-addressable combinatorial immobilization of natural and synthetic matrices incorporated with growth factors including epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), and neurotrophin-3 (NT-3). NSC obtained from the rat embryonic striatum was cultured directly on the array to screen for cell adhesion, proliferation, and promotion of neuronal and glial specification. The results showed that the significant number of cells adhered to laminin-1, fibronectin, ProNectin, and poly(ethyleneimine). It was found that cells proliferated most extensively on a spot with immobilized EGF among the spots with different matrix-growth factor combinations. The results also showed that neuronal differentiation was promoted on the spots with immobilized NGF or NT-3, and astroglial differentiation with CNTF. Importantly, observed effects of growth factors were frequently altered depending on the type of co-immobilized matrices, suggesting synergic effects of adhesion and growth factor signals.

Adsorption↗

PubMed2006

Encapsulation of islets with ultra-thin polyion complex membrane through poly(ethylene glycol)-phospholipids anchored to cell membrane.

The microencapsulation of islets of Langerhans (islets) has been studied as a safe and simple technique for islet transplantation without the need for immuno-suppressive therapy. However, thinner membranes are desired, because the increased total volume of the implant led to limited transplantation sites. Here, we propose a novel method for microencapsulation by polyion complex membrane formation on islets. Amino group-terminated poly(ethylene glycol)-conjugated phospholipids (PEG-lipids, M(w): 5000) spontaneously formed a thin layer on cells existing in the outer layer of islets when they were added to islet suspension. This layer-by-layer membrane could be further formed on the PEG-lipid layer through polyion complex formation between amino groups at the end of PEG chains, sodium alginate and poly(l-lysine). Islets could be microencapsulated by this method without volume increase. Encapsulation of the islet surface with PEG-lipids and polyion complex membranes did not impair the insulin release function in response to glucose stimulation. Our method is promising to encapsulate islets without affecting cell viability or increasing volume.

Capsules↗

PubMed2006

Surface plasmon resonance-based highly sensitive immunosensing for brain natriuretic peptide using nanobeads for signal amplification.

In this study, we developed a surface plasmon resonance (SPR)-based sensing method with high sensitivity for the detection of brain natriuretic peptide (BNP), the level of which is a useful indicator of heart failure. In our system, BNP was detected by a sandwich-type immunoassay using two kinds of monoclonal antibodies: primary and secondary. The SPR signal was amplified by using streptavidin-conjugated nanobeads approximately 50 nm in diameter that specifically accumulated on the secondary antibody via biotin-avidin bonding. By this method, the SPR signals were highly intensified, and thus picogram levels (pg/ml) of BNP could be detected. The streptavidin nanobead amplification method realizes detection of very low levels of BNP, as is necessary for the clinical diagnosis. It is also expected that our streptavidin nanobead amplification method can be applicable to the detection of other hormones and tumor markers that are present in only low concentrations within the human body.

Antibodies, Monoclonal↗

PubMed2006

Efficient generation of dopaminergic neurons from mouse embryonic stem cells enclosed in hollow fibers.

Transplantation of dopamine neurons is a promising approach to treat Parkinson's disease. Embryonic stem (ES) cells are expected to be a cell source of the dopaminergic neurons. Various difficulties, however, need to be overcome to realize cell therapy of Parkinson's disease using dopaminergic neurons from ES cells. For example, they are highly sensitive to enzymatic treatment and physical dissociation, and the patient's immune system may attack the transplanted cells. In this study, we attempted to induce dopaminergic neurons from mouse ES cells enclosed in hollow fibers using conditioning medium from PA6 cells, the stromal cells derived from skull bone marrow. beta-tubulin type III positive cells and tyrosine hydroxylase positive cells were efficiently derived in hollow fibers after 16 days in culture, and dopamine release was observed when the hollow fibers containing cells were exposed to 56mm KCl for 15min to induce dopamine release through depolarization of the neurons. By our procedure, enclosure of dopaminergic neurons in hollow fibers was easily performed without loss of cells, and the hollow fiber membrane is expected to efficiently protect dopaminergic neurons from mechanical disturbances and attacks by the host immune system. Although there are many issues, especially related to immuno-isolation, that still remain to be addressed, we believe that differentiation of ES cells within hollow fibers is one of the crucial procedures so that cell therapy of Parkinson's disease can be realized.

Animals↗

PubMed2006

Layer-by-layer assembly of cationic lipid and plasmid DNA onto gold surface for stent-assisted gene transfer.

Intravascular stent-assisted gene transfer is an advanced approach for the therapy of vascular diseases such as atherosclerosis and stenosis. This approach requires a stent that allows local and efficient administration of therapeutic genes to the target cells at the vascular wall. To create such a stent, a method was developed for loading plasmid DNA onto the metal surface. The method involves the formation of self-assembled monolayer on the noble metal surface followed by electrostatic layer-by-layer (LBL) assembly of a cationic lipid/plasmid DNA complex and free plasmid DNA. In this in vitro feasibility study, the thin plainer film and the wire of gold were used as a substrate. The LBL assembly process was characterized by surface plasmon resonance spectroscopy and static contact angle measurement. Plasmid DNA loaded in the multilayer exhibited improved resistance against nuclease digestion. When cultured directly on the DNA-loaded surface, cells were transfected to express exogenous gene in the DNA loading-dependent manner. Plasmid DNA could also be transferred to endothelial cells from its apical side by placing the DNA-loaded gold wire onto the cell layer.

Cations↗

PubMed2006

Induction dopamine releasing cells from mouse embryonic stem cells and their long-term culture.

Cell transplantation therapy using dopaminergic neurons derived from embryonic stem (ES) cells for the treatment of Parkinson's disease has been proposed as one of the major applications for stem cell-based therapy. However, the low collection efficiency of neurons from a culture dish and the rejection of cells after transplantation are expected to limit their future clinical applications. To overcome these problems, we examined the induction of neurogenesis of ES cells under free-floating conditions and microencapsulation of the obtained cell aggregates into an agarose hydrogel. Cell aggregates from ES cells were cultured in various media under the free-floating condition. Immunohistochemical staining for tyrosine hydroxylase (TH) and RT-PCR analyses for TH and Nurr1 showed that dopaminergic neurons were induced in ES cell aggregates cultured in a 1:2 mixture of conditioned medium of PA6 stromal cells and Glasgow minimum essential medium (GMEM) after 16 days in culture. The cell aggregates could be collected and were encased within agarose microcapsules without loss of dopaminergic neurons. The cell aggregates with/without microencapsulation were maintained in CM/GMEM for an additional period. KCl stimulation assays were done at day 23, 30, 37, 44, 51, and 58 to examine dopamine release. Dopamine release abilities were well maintained during 58 days of observation. Amounts of dopamine release from encapsulated cell aggregates were slightly higher than those of unencapsulated cell aggregates from day 16 to 58. Although efficacy for immunoisolation of the agarose microcapsules still remains for future in vivo studies, microencapsulation did not adversely affect viability and functions of the dopamine releasing ES cell progeny.

Animals↗

PubMed2006

Survival of microencapsulated islets at 400 days posttransplantation in the omental pouch of NOD mice.

The long-term durability of agarose microencapsulated islets against autoimmunity was evaluated in NOD mice. Islets were isolated from 6-8-week-old prediabetic male NOD mice and microencapsulated in 5% agarose hydrogel. Microencapsulated or nonencapsulated islets were transplanted into the omental pouch of spontaneously diabetic NOD mice. Although the diabetic NOD mice that received nonencapsulated islets experienced a temporary reversal of their hyperglycemic condition, all 10 of these mice returned to hyperglycemia within 3 weeks. In contrast, 9 of 10 mice transplanted with microencapsulated islets maintained normoglycemia for more than 100 days. Islet grafts were removed at 100, 150, 200, 300, and 400 days posttransplantation. A prompt return to hyperglycemia was observed in the mice after graft removal, indicating that the encapsulated islet grafts were responsible for maintaining euglycemia. Histological examination revealed viable islets in the capsules at all time points of graft removal. In addition, beta-cells within the capsules remained well granulated as revealed by the immunohistochemical detection of insulin. No immune cells were detected inside the microcapsules and no morphological irregularities of the microcapsules were observed at any time point, suggesting that the microcapsules successfully protected the islets from cellular immunity. Sufficient vascularization was evident close to the microcapsules. Considerable numbers of islets showed central necrosis at 400 days posttransplantation, although the necrotic islets made up only a small percentage of the islet grafts. Islets with central necrosis also showed abundant insulin production throughout the entire islets, except for the necrotic part. These results demonstrate the long-term durability of agarose microcapsules against autoimmunity in a syngeneic islet transplantation model in NOD mice.

Alginates↗

PubMed2006

One-step induction of neurons from mouse embryonic stem cells in serum-free media containing vitamin B12 and heparin.

We present a simple method for neural cell fate specification directly from mouse embryonic stem cells (ES cells) in serum-free conditions in the absence of embryoid body formation. Dissociated ES cells were cultured in serum-free media supplemented with vitamin B12 and heparin, but without any expensive cytokines. After 14 days in culture, beta-tubulin type III (TuJ1) and tyrosine hydroxylase (TH)-positive colonies were detected by immunocytochemical examinations. In addition, specific gene analyses by RT-PCR demonstrated expression of an early central nerve system, mature neuron, and midbrain dopaminergic neuron-specific molecules (i.e., nestin, middle molecular mass neurofilament protein, Nurr1, and TH, respectively). Dopamine was also detected in the culture media by reverse-phase HPLC analysis. These facts indicate that addition of vitamin B12/heparin to serum-free culture media induced neurons from ES cells, which included cells that released dopamine. Other supplements, such as putrescine, biotin, and Fe2+, could not induce neurons from ES cells by themselves, but produced synergistic effects with vitamin B12/heparin. The rate of TuJ1+/TH+ colony formation was increased threefold and the amounts of dopamine released increased 1.5-fold by the addition of a mixture of putrescine, biotin, and Fe2+ to vitamin B12/heparin culture media. Our method is a simple tool to differentiate ES cells to dopaminergic neurons for the preparation of dopamine-releasing cells for the cell transplantation therapy of Parkinson's disease. In addition, this method can facilitate the discovery of soluble factors and genes that can aid in the induction of the ES cell to its neural fate.

Animals↗

PubMed2005

Layer-by-layer assembly of poly(ethyleneimine) and plasmid DNA onto transparent indium-tin oxide electrodes for temporally and spatially specific gene transfer.

The layer-by-layer assembly technique was used to adsorb alternately poly(ethyleneimine) and plasmid DNA onto the surface of a transparent electrode made of indium-tin oxide. The surface with adsorbed poly(ethyleneimine) and DNA was characterized by X-ray photoelectron spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy, and contact angle measurements. These analyses revealed that the alternate adsorption process generated a multilayered assembly of cationic poly(ethyleneimine) and anionic DNA. For the spatially and temporally specific gene transfer, cells were cultured on the plasmid-loaded electrode and then a short electric pulse was applied to the cell-electrode system. It was shown that, upon electric pulsing, the plasmid was released from the electrode and transferred into the cells, resulting in efficient gene expression even in primary cultured cells. Transfection could be effected for hippocampal neurons after 3-day culture on the plasmid-loaded electrode, which indicated the feasibility of selecting the time of transfection. Our results also showed that electroporation could be performed in a spatially specific manner by using a plasmid-arrayed electrode, demonstrating the feasibility of the method for the fabrication of transfected cell microarrays.

Animals↗

PubMed2005

Immobilization of histidine-tagged recombinant proteins onto micropatterned surfaces for cell-based functional assays.

This letter describes a method for preparing protein microarrays that allow the functional analysis of proteins at a cellular level. This method involves the utilization of recombinant proteins genetically engineered to carry a fusion tag that has an affinity for metal ions. A micropatterned alkanethiol monolayer was used to prepare a microarray having multiple spots with immobilized metal ions. The fusion protein was chelated to the spots under physiological conditions. The feasibility of the method was demonstrated by culturing neural stem cells on the microarray that displayed oligohistidine-tagged epidermal growth factor.

Animals↗

PubMed2005

Collection of neural inducing factors from PA6 cells using heparin solution and their immobilization on plastic culture dishes for the induction of neurons from embryonic stem cells.

Embryonic stem (ES) cells have the ability to replicate themselves and differentiate into various mature cells. Recently, dopaminergic neurons were efficiently induced from ES cells using mouse stromal cells (PA6 cells) as a feeder cell layer. This simple procedure seems to be very efficient to obtain dopamine-releasing cells for future clinical cell transplantation treatment of Parkinson's disease. In this study, we prepared stock solutions containing neural inducing factors (NIFs) by washing PA6 cells with phosphate-buffered saline containing heparin. ES cells grew successfully in culture media supplemented with 33 v/v% NIFs stock solution, and the rate of neural differentiation of ES cell progeny increased with increasing heparin concentration in the culture media. In addition, NIFs-immobilized surfaces were prepared by exposing polyethyleneimine-modified surfaces to NIFs stock solutions. The NIFs-immobilized culture dish effectively supported cell growth as the culture medium supplemented with NIFs stock did, but its induction effect to dopaminergic neurons from ES cells was much smaller than free NIFs. NIFs stock solutions have two different activities. One can stimulate cell growth and the other induces differentiation of ES cells to the neural fate when heparin existed. The former factors were effectively immobilized on the culture dish, but those that induce differentiation may not be. Further optimization is required.

Animals↗

PubMed2005

Parallel analysis of multiple surface markers expressed on rat neural stem cells using antibody microarrays.

Neural stem cells are the attractive cell source for functional regeneration of damaged central nervous tissues by means of cell transplantation or in situ induction of differentiated neural cells. Such stem cell therapies require the prospective identification and isolation of neural stem cells. However they are difficult due to limited information on surface markers. This study aimed at developing an antibody microarray that permits parallel analysis of multiple surface antigens expressed on neural stem cells present in a neurosphere-forming cell population. A microarray was prepared by micro-spotting antibodies directed to surface antigens and ligands for membrane-associated receptors onto the patterned monolayer of alkanethiols self-assembled on a gold-evaporated glass plate. Neurosphere-forming cells were subjected to a cell-binding assay on the microarray followed by immunofluorescent staining of nestin, an intracellular marker of neural stem cells. It was demonstrated that such a cell based assay facilitated to examine the specificity of surface antigens for nestin-positive neural stem cells. Furthermore, the microarray could also be used to assess proliferation capability of cells bound to individual spots. These results suggest that the microarray-based strategy will provide a useful tool for the parallel analysis of surface markers expressed on a specific cell type in a heterogeneous population.

Animals↗

PubMed2005

Preparation and properties of ProNectin F-coated biodegradable hollow fibers.

ProNectin F-coated biodegradable hollow fibers were newly prepared and their cytocompatibility was evaluated in vitro. Although the coating efficiency onto poly(L-lactic acid) (PLLA) and poly(lactide-co-caprolactone) [p(LA/CL)] matrices was similar, the cell adhesion properties were greatly affected by the nature of the polymer substrate. ProNectin F-coated PLLA showed about seven times higher cytocompatibility than ProNectin F-coated p(LA/CL). The single-extruded melt spinning method and the core-sheath bicomponent melt spinning method were employed to prepare PLLA hollow fibers. The effect of the spinning conditions, such as the melt draw ratio, spinneret temperature, and take-up speed, on the diameter and wall thickness of the spun fibers was studied in detail. For single-extruded melt spinning, a segmented type of spinneret was used, and the effect of the flow rate of nitrogen, which was confined in the hollow part of fibers, was studied. X-ray photographs of the drawn hollow fibers, clarified the significant molecular orientation, which was much higher than that in drawn solid PLLA fiber under identical drawing conditions. The morphology and mechanical properties of hollow fibers demonstrated an increase in the tensile strength and a decrease in the thickness of the PLLA wall with increased nitrogen flow rates and melt draw ratios for single-extruded melt spinning. These results indicate the unique characteristics of ProNectin F-coated PLLA hollow fibers, which can be successfully utilized as a biodegradable substrate.

Absorbable Implants↗

PubMed2005

Antibody microarray for correlating cell phenotype with surface marker.

To correlate cell surface markers with the cell phenotype, an antibody microarray prepared by covalently immobilizing antibodies onto a cellulose membrane and subsequent immunocytochemical staining were employed. The direct binding assay of a lymphoblastic leukemia cell line on the microarray showed that the immobilized antibody served to capture cells expressing the specific antigen. The density of bound cells increased linearly with an increasing content of antigen-expressing cells in suspension. The method was further applied to the analysis of surface antigens expressed on neural stem cells. A binding assay was performed with neural cells obtained from the neurosphere culture of the rat fetal striatum on a microarray spotted with eight kinds of antibodies and four different proteins, followed by immunocytochemical staining of cells bound to the microarray using antibodies to the intracellular markers of immature (nestin and vimentin) and mature (beta-tubulin III and glial fibrillary acidic protein) neural cells. As a result, the phenotype of bound cells could be correlated to surface antigen expression, which illustrated the potential of the solid-phase cytometry developed here for the identification of surface markers.

Animals↗

PubMed2005

Interaction of poly(styrene sulfonic acid) with the alternative pathway of the serum complement system.

Bioartificial pancreas, in which the islets of Langerhans are enclosed in artificial membrane to be protected from the host immune system, is expected to be a promising medical device to treat patients who suffer from insulin-dependent diabetes. Our strategy for preparation of a bioartificial pancreas involves utilizing a membrane including polymeric materials that can inhibit the complement reaction. In this study, we examined the effects of poly(styrene sulfonic acid) (PSSa) on the alternative pathway of the serum complement system to identify the mechanism(s) involved. PSSa was dissolved in pooled normal human serum (NHS), and the mixtures were incubated at 37 degrees C for 30 min. Complement activities in sera were determined by hemolytic assays. Amounts of complement activation products released were determined by ELISA. Interactions of PSSa with complement components and fragments were examined with electrophoresis and immunoblotting. From these examinations, it appeared that the manner of PSSa effects on the alternative pathway (AP) highly depends on its concentration. PSSa seemingly acted as an activator when its concentration was 0.005 g/dl to 0.05 g/dl, while it acted as an inhibitor when its concentration was more than 0.1 g/dl. In terms of activation or inhibition of the AP, forming complex of PSSa with factor H induced activation, and that with factor D induced inhibition.

Biocompatible Materials↗

PubMed2005

Interaction of poly(styrene sulfonic acid) with the classical pathway of the serum complement system.

Bioartificial pancreas, in which the islets of Langerhans (islets) are enclosed in artificial membrane to be protected from the host immune system, is expected to be a promising medical device to treat patients who suffer from insulin-dependent diabetes. Our strategy for the preparation of a bioartificial pancreas involves utilizing a membrane including polymeric materials that can inhibit the complement. When we examined a membrane containing poly(styrene sulfonic acid), long survival of islets enclosed in the membrane was observed in recipients carrying antibodies against islet cells. This fact stimulated us to start examinations of effects of PSSa on the complement system. In this study, we examined effects of PSSa on the classical pathway (CP) of the serum complement system to identify the mechanism(s) involved. The electric static interaction between cationic C1q (pI 9.3) and anionic PSSa induces PSSa-C1q complex formation. The dissociation of C1q(r2s2) complex by PSSa results inactivation of the CP activity. Those results indicate that PSSa was not an activator of the CP, but an inhibitor of CP activation. This study clarifies the mechanism by which PSSa protects islets in a microcapsule from the humoral immunity of the recipient carrying anti-islet antibodies. A microcapsule containing PSSa seems to effectively protect the islet from attacks of the host immune system after transplantation carrying antibodies against islet cells.

Animals↗

PubMed2005

A thin carboxymethyl cellulose culture substrate for the cellulase-induced harvesting of an endothelial cell sheet.

Engineered tissues constructed with two-dimensionally organized cells provide promising parts for reconstructing damaged tissues. Here we propose a new method for fabricating a 2D sheet made of an endothelial cell monolayer. First a culture substrate was prepared by treating the glass surface with an amine-terminated organosilicon derivative, followed by the covalent attachment of a thin carboxymethyl cellulose (CMC) layer. Fibronectin was immobilized onto the CMC-coated surface to promote cell adhesion. These surfaces were characterized step by step by means of contact angle measurement and X-ray photoelectron spectroscopy. Porcine aortic endothelial cells adhered to the culture substrate and consequently formed a confluent monolayer. When the substrate-cell composite was immersed in a cellulase solution, a cell sheet was spontaneously detached from the substrate due to enzymatic digestion of the CMC layer. The cell-cell connections were well preserved in the cell sheet, even after detachment from the substrate, most likely due to the fact that cellulase is harmless to mammalian cells. The cell sheet could be transferred to other culture dish with the aid of a hydrophilic membrane support, retaining the proliferation activity of the cells. The results obtained in this study demonstrate that cellulase treatment of the CMC layer is a rational and efficient method for obtaining a 2D cell sheet.

Animals↗