Modification of exosomes with carbonate apatite and a glycan polymer improves transduction efficiency and target cell selectivity.

Exosomes are secreted from a variety of cells and transmit parental cell-derived biomolecules, such as nucleic acids and proteins, to recipient cells in distant organs. In addition to their important roles in both physiological and pathological conditions, exosomes are expected to serve as natural drug carriers without any cytotoxicity, immunogenicity, or tumorigenicity. However, the use of exosomes as drug delivery tools is limited due to the low uptake efficiency of the target cells, insufficient release of the contents from the endosome to the cytosol, and possible adverse effects caused by the delivery to non-target cells. In the present study, we examined the effects of the modification of exosomes with carbonate apatite or a lactose-carrying polymer. Using newly generated monitoring exosomes that contain either firefly luciferase or fused mCherry/enhanced green fluorescent protein, we demonstrated that the modification of exosomes with carbonate apatite improved their release from the endosome into the cytosol in recipient cells. Meanwhile, the modification of exosomes with a lactose-carrying polymer enhanced the selective delivery to parenchymal hepatocytes. These modified exosomes may provide an efficient strategy for macromolecule therapy for incurable diseases that cannot be treated with conventional small-molecule compounds.

An anti-oxidative cell culture dish inhibits intracellular reactive oxygen species accumulation and modulates pluripotency-associated gene expression in mesenchymal stem cells.

Maintenance of the pluripotent state of mesenchymal stem cells (MSCs) during in vitro expansion is an important factor for the successful proliferation of MSCs possessing high differentiation capacity. However, the differentiation potential of MSCs can easily be lost during in vitro expansion, particularly at late passages. Reactive oxygen species (ROS) are signaling molecules that help to maintain MSC function; however, excessive ROS generation can induce senescence and impair both the differentiation capacity and proliferation of MSCs. In this study, we have designed an amphiphilic block copolymer (redox copolymer), which possesses ROS scavenging capacity in the hydrophobic site. When this redox copolymer was coated on cell culture dishes coupled with human E-cadherin chimeric antibody (hE-cad-Fc), it had an antioxidative effect on cultured MSCs. We also confirmed that the redox polymer construct poly(ethylene glycol) tethered chain on the surface prevented nonspecific cell binding, whereas the co-immobilized surface allowed high adhesion of E-cadherin-positive MSCs. Interestingly, the intracellular ROS level was significantly decreased by the prepared cell culture dish, despite ROS being scavenged only on the surface of the dish, on the cell exterior. Consequently, the cultured MSCs retained high expression levels of pluripotency-associated genes, including SOX2.

Detachment of Chain-Forming Neuroblasts by Fyn-Mediated Control of cell-cell Adhesion in the Postnatal Brain.

In the rodent olfactory system, neuroblasts produced in the ventricular-subventricular zone of the postnatal brain migrate tangentially in chain-like cell aggregates toward the olfactory bulb (OB) through the rostral migratory stream (RMS). After reaching the OB, the chains are dissociated and the neuroblasts migrate individually and radially toward their final destination. The cellular and molecular mechanisms controlling cell-cell adhesion during this detachment remain unclear. Here we report that Fyn, a nonreceptor tyrosine kinase, regulates the detachment of neuroblasts from chains in the male and female mouse OB. By performing chemical screening and in vivo loss-of-function and gain-of-function experiments, we found that Fyn promotes somal disengagement from the chains and is involved in neuronal migration from the RMS into the granule cell layer of the OB. Fyn knockdown or Dab1 (disabled-1) deficiency caused p120-catenin to accumulate and adherens junction-like structures to be sustained at the contact sites between neuroblasts. Moreover, a Fyn and N-cadherin double-knockdown experiment indicated that Fyn regulates the N-cadherin-mediated cell adhesion between neuroblasts. These results suggest that the Fyn-mediated control of cell-cell adhesion is critical for the detachment of chain-forming neuroblasts in the postnatal OB.SIGNIFICANCE STATEMENT In the postnatal brain, newly born neurons (neuroblasts) migrate in chain-like cell aggregates toward their destination, where they are dissociated into individual cells and mature. The cellular and molecular mechanisms controlling the detachment of neuroblasts from chains are not understood. Here we show that Fyn, a nonreceptor tyrosine kinase, promotes the somal detachment of neuroblasts from chains, and that this regulation is critical for the efficient migration of neuroblasts to their destination. We further show that Fyn and Dab1 (disabled-1) decrease the cell-cell adhesion between chain-forming neuroblasts, which involves adherens junction-like structures. Our results suggest that Fyn-mediated regulation of the cell-cell adhesion of neuroblasts is critical for their detachment from chains in the postnatal brain.

Enhanced Biological Functions of Human Mesenchymal Stem-Cell Aggregates Incorporating E-Cadherin-Modified PLGA Microparticles.

Mesenchymal stem cells (MSCs) have emerged as a promising source of multipotent cells for various cell-based therapies due to their unique properties, and formation of 3D MSC aggregates has been explored as a potential strategy to enhance therapeutic efficacy. In this study, poly(lactic-co-glycolic acid) (PLGA) microparticles modified with human E-cadherin fusion protein (hE-cad-PLGA microparticles) have been fabricated and integrated with human MSCs to form 3D cell aggregates. The results show that, compared with the plain PLGA, the hE-cad-PLGA microparticles distribute within the aggregates more evenly and further result in a more significant improvement of cellular proliferation and secretion of a series of bioactive factors due to the synergistic effects from the bioactive E-cadherin fragments and the PLGA microparticles. Meanwhile, the hE-cad-PLGA microparticles incorporated in the aggregates upregulate the phosphorylation of epidermal growth factor receptors and activate the AKT and ERK1/2 signaling pathways in the MSCs. Additionally, the E-cadherin/ß-catenin cellular membrane complex in the MSCs is markedly stimulated by the hE-cad-PLGA microparticles. Therefore, engineering 3D cell aggregates with hE-cad-PLGA microparticles can be a promising method for ex vivo multipotent stem-cell expansion with enhanced biological functions and may offer a novel route to expand multipotent stem-cell-based clinical applications.

Construction of a Defined Biomimetic Matrix for Long-Term Maintenance of Mouse Induced Pluripotent Stem Cells.

The existing in vitro culture systems often use undefined and animal-derived components for the culture of pluripotent stem cells. Artificial bioengineered peptides have the potential to become alternatives to these components of extracellular matrix (ECM). Integrins and cadherins are two cell adhesion proteins important for stem cell self-renewal, differentiation, and phenotype stability. In the present study, we sought to mimic the physico-biochemical properties of natural ECMs that allow self-renewal of mouse induced pluripotent stem cells (iPSCs). We develop a genetically engineered ECM protein (ERE-CBP) that contains (i) an integrin binding peptide sequence (RGD/R), (ii) an E-/N-cadherin binding peptide sequence (SWELYYPLRANL/CBP), and (iii) 12 repeats of APGVGV elastin-like polypeptides (ELPs/E).While ELPs allow efficient coating by binding to nontreated hydrophobic tissue culture plates, RGD/R and CBP support integrin- and cadherin-dependent cell attachment, respectively. Mouse iPSCs on this composite matrix exhibit a more compact phenotype compared to cells on control gelatin substrate. We also demonstrated that the ERE-CBP supports proliferation and long-term self-renewal of mouse iPSCs for up to 17 passages without GSK3ß (CHIR99021) and Erk (PD0325901) inhibitors. Overall, our engineered ECM protein, which is cost-effective to produce in prokaryotic origin and flexible to modify with other cell adhesion peptides or growth factors, provides a novel approach for expansion of mouse iPSCs in vitro.

Sugar-carrying Polystyrenes Facilitate Harvesting of APCs from MLRs: Possible Application of Sugar-carrying Polystyrenes to Immunotherapy.

Antigen-presenting cells (APCs) play a pivotal role in cancer immunotherapy. APCs in conventionally used flasks are harvested by enzymatic digestion or cell scraping for application to cancer immunotherapy. However, these methods may impair functional molecules expressed on the APC surface and reduce their effects in cancer immunotherapy. Recently, we found that APCs could be harvested by shaking at 4°C in flasks coated with poly[N-p-vinylbenzyl-O-2-acetoamide-2-deoxy-ß-D-glucopyranosyl-(1→4)-2-acetoamide-2-deoxy-ß-D-gluconamide] (PVGlcNAc) or a copolymer consisting of sulfonylurea (SU) linked to poly[N-p-vinyl-benzyl-4-O-ß-D-galactopyranosyl-D-gluconamide] [P(VLA-co-SU)]. In the present study, we compared the functions of cytotoxic T-lymphocytes (CTLs) induced by APCs generated in PVGlcNAc- or P(VLA-co-SU)-coated flasks and conventional flasks. APCs from PVGlcNAc- or P(VLA-co-SU)-coated flasks showed higher expression of cluster of differentiation (CD)80/86, CD11c, and major histocompatibility complex class II alloantigen I-A(d), and higher cytotoxicity than APCs from conventional flasks. These results suggest that the use of PVGlcNAc- or P(VLA-co-SU)-coated flasks is optimal for harvesting APCs. The generated APCs also have a higher antigen-presenting ability compared to those generated in conventional flasks. Our results may contribute to the development of effective cancer immunotherapies.

Innovative delivery of siRNA to solid tumors by super carbonate apatite.

RNA interference (RNAi) technology is currently being tested in clinical trials for a limited number of diseases. However, systemic delivery of small interfering RNA (siRNA) to solid tumors has not yet been achieved in clinics. Here, we introduce an in vivo pH-sensitive delivery system for siRNA using super carbonate apatite (sCA) nanoparticles, which is the smallest class of nanocarrier. These carriers consist simply of inorganic ions and accumulate specifically in tumors, yet they cause no serious adverse events in mice and monkeys. Intravenously administered sCA-siRNA abundantly accumulated in the cytoplasm of tumor cells at 4 h, indicating quick achievement of endosomal escape. sCA-survivin-siRNA induced apoptosis in HT29 tumors and significantly inhibited in vivo tumor growth of HCT116, to a greater extent than two other in vivo delivery reagents. With innovative in vivo delivery efficiency, sCA could be a useful nanoparticle for the therapy of solid tumors.

AFM characterization of chemically treated corneal cells.

We present a characterization of chemically treated cells using atomic force microscopy (AFM) which can observe changes in morphology and elasticity of cells. Since AFM has the significant advantage that it does not require fixation of samples, the method is simple and can capture various properties of living cells. In this study, corneal epithelial and endothelial cells were examined. The topography images of the corneal cells without glutaraldehyde (GA) fixation were successfully obtained. The images showed a natural three-dimensional shape of these cells, which scanning electron microscope (SEM) images could not provide. The AFM images of GA-fixed cells were taken and compared with a SEM image reported in the literature. Our results show that longer time for GA fixation makes the surface of the corneal endothelial tissue stiffer. Also, longer treatment results in relatively large structural variation in samples. Combined with conventional histochemical methods, this approach helps us gain an overall understanding of the influence of such chemical treatment.

Artificial biomimicking matrix modifications of nanofibrous scaffolds by hE-cadherin-Fc fusion protein to promote human mesenchymal stem cells adhesion and proliferation.

Extracellular matrix (ECM) plays a fundamental role in regulating cell attachment, proliferation, migration and differentiation. Both synthetic and biologically derived materials have been explored as an ECM in regenerative medicine and tissue engineering. To biomimick the extracellular matrix, we combined the advantages of the biological properties of nanofibrous scaffolds and the fusion protein to apply for the culture of human mesenchymal stem cells in vitro. In this study, we fabricated well random-oriented/aligned nanofibrous scaffolds with PCL, modified with hE-cadherin-Fc fusion protein and studied the synergistic effect of the scaffolds. The random-oriented/aligned architecture was observed in the nanofibrous scaffolds by SEM. XPS and WCA measurements evidenced that hE-cadherin-Fc was successfully modified on the PCL nanofibrous scaffolds and hydrophilicity of the scaffolds was well improved after fusion protein coating. The hE-cadherin-Fc modified markedly promoted the adhesion and proliferation of hMSCs and guided hMSCs to a spindlier morphology compared with unmodified nanofibrous scaffolds. Furthermore, hMSCs on the hE-cadherin-Fc-coated nanofibrous scaffolds also had differentiation potential. These results suggested that the combination of PCL nanofibrous scaffolds and hE-cadherin-Fc fusion protein may be a promising artificial ECM for the behavior of hMSCs in vitro.

Bile acids increase levels of microRNAs 221 and 222, leading to degradation of CDX2 during esophageal carcinogenesis.

BACKGROUND & AIMS: Bile reflux contributes to development of Barrett's esophagus (BE) and could be involved in its progression to esophageal adenocarcinoma (EAC). We investigated whether bile acids affect levels or functions of microRNAs (MIRs) 221 and 222, which bind to the 3'-UTR of p27Kip1 messenger RNA to inhibit its translation. Reduced p27Kip1 increases degradation of the transcription factor CDX2; levels of CDX2 have been reported to decrease during progression of BE to EAC. METHODS: We used quantitative reverse transcriptase polymerase chain reaction to compare levels of MIRs 221 and 222 and immunohistochemistry to compare levels of p27Kip1 and CDX2 proteins in areas of BE and EAC from each of 11 patients. We examined the effects of bile acid exposure on levels of MIRs 221 and 222 and CDX2 in EAC cells. We investigated the effects of inhibitors of MIRs 221 and 222 on growth of human EAC xenograft tumors in NOD/SCID/IL-2Rγ(null) mice. RESULTS: Levels of MIRs 221 and 222 increased and levels of p27Kip1 and CDX2 decreased in areas of EAC vs BE. Levels of MIRs 221 and 222 increased, along with activity of nuclear bile acid receptor/farnesoid X receptor (FXR), when cultured cells were exposed to bile acids. Incubation of cells with bile acids increased degradation of CDX2; this process was reduced when cells were also incubated with proteasome inhibitors. Overexpression of MIRs 221 and 222 reduced levels of p27Kip1 and CDX2, and knockdown of these MIRs increased levels of these proteins in cultured cells. Inhibitors of MIRs 221 and 222 increased levels of p27Kip1 and CDX2 in EAC cells and reduced growth of xenograft tumors in NOD/SCID/IL-2Rγ(null) mice. CONCLUSIONS: We observed increased levels of MIRs 221 and 222 in human EAC tissues, compared with areas of BE from the same patient. We found that exposure of esophageal cells to bile acids activates FXR and increases levels of MIRs 221 and 222, reducing levels of p27Kip1 and promoting degradation of CDX2 by the proteasome. Our work opened the perspective of therapeutically targeting this pathway either via FXR antagonists or inhibitors of MIRs as a treatment option for BE and EAC.

Fabrication and intracellular delivery of doxorubicin/carbonate apatite nanocomposites: effect on growth retardation of established colon tumor.

In continuing search for effective treatments of cancer, the emerging model aims at efficient intracellular delivery of therapeutics into tumor cells in order to increase the drug concentration. However, the implementation of this strategy suffers from inefficient cellular uptake and drug resistance. Therefore, pH-sensitive nanosystems have recently been developed to target slightly acidic extracellular pH environment of solid tumors. The pH targeting approach is regarded as a more general strategy than conventional specific tumor cell surface targeting approaches, because the acidic tumor microclimate is most common in solid tumors. When nanosystems are combined with triggered release mechanisms in endosomal or lysosomal acidic pH along with endosomolytic capability, the nanocarriers demonstrated to overcome multidrug resistance of various tumors. Here, novel pH sensitive carbonate apatite has been fabricated to efficiently deliver anticancer drug Doxorubicin (DOX) to cancer cells, by virtue of its pH sensitivity being quite unstable under an acidic condition in endosomes and the desirable size of the resulting apatite-DOX for efficient cellular uptake as revealed by scanning electron microscopy. Florescence microscopy and flow cytometry analyses demonstrated significant uptake of drug (92%) when complexed with apatite nanoparticles. In vitro chemosensitivity assay revealed that apatite-DOX nanoparticles executed high cytotoxicity in several human cancer cell lines compared to free drugs and consequently apatite-DOX-facilitated enhanced tumor inhibitory effect was observed in colorectal tumor model within BALB/cA nude mice, thereby shedding light on their potential applications in cancer therapy.

hE-cadherin-Fc fusion protein coated surface enhances the adhesion and proliferation of human mesenchymal stem cells.

A fusion protein consisting of human E-cadherin extracellular domain and the immunoglobulin G Fc region (hE-cadherin-Fc) was prepared and used as a cell-cell adhesion biomimicking matrix for the in vitro expansion of human mesenchymal stem cells (hMSCs) for use in regenerative medicine. The hE-cadherin-Fc was stably immobilized onto a polystyrene plate due to the hydrophobicity of the Fc domain, enhancing the surface wettability and topography of the plate. The hE-cadherin-Fc matrix markedly promoted the cell adhesion and proliferation of hMSCs compared with the tissue culture-treated plate (TC-PS) and the gelatin-coated plate. Furthermore, the expanded hMSCs on the hE-cadherin-Fc were positive for CD105, similar to those from the gelatin. Additionally, the expression of E-cadherin and ß-catenin in the hMSCs was improved on the hE-cadherin-Fc matrix, suggesting that the interactions of the hE-cadherin-Fc matrix with the hMSCs were substitutes for the cell-cell adhesion junctions during the initial culture stage in the absence of intercellular interactions. The hE-cadherin-Fc was shown to be a promising artificial ECM for the in vitro expansion of hMSCs.

Dynamic behaviors of vimentin induced by interaction with GlcNAc molecules.

The cytoskeleton protein vimentin is dramatically altered following pathological events such as fibrosis and tumorigenesis. Vimentin binds to multivalent N-acetylglucosamine (GlcNAc) molecules at the cell surface and interacts with O-linked ß-GlcNAc proteins. Moreover, dying cells can be engulfed by neighboring cells through surface interactions between vimentin and many O-GlcNAc proteins in cell debris. Here, we show that vimentin was altered by its interaction with GlcNAc-bearing molecules such as GlcNAc-bearing polymers. The interaction with GlcNAc-bearing polymers promoted the cell surface recruitment of vimentin followed by the phosphorylation of vimentin serine 71 and the increase in tetrameric vimentin disassembled from vimentin filaments in HeLa cells. Moreover, it was found that GlcNAc-bearing polymers and O-GlcNAc proteins from dying cells promoted vimentin expression and cell migration in the Madin-Darby canine kidney and Michigan Cancer Foundation-7 cells. These results suggest that interactions between surface vimentin and GlcNAc molecules, including the O-GlcNAc proteins from dying cells, may play a pivotal role in vimentin expression and the migration of cancer cells. We propose new mechanisms of vimentin expression in cancer cells.

Preparation and characterization of a VEGF-Fc fusion protein matrix for enhancing HUVEC growth.

To enhance vascularization of hydrophobic implants in vivo, a VEGF-Fc fusion protein consisting of vascular endothelial growth factor (VEGF) fused to the immunoglobulin G Fc domain was prepared as an artificial extracellular matrix (ECM). VEGF-Fc was stably immobilized on a polystyrene plate due to the hydrophobicity of the Fc domain, and significantly enhanced the adhesion of human umbilical vein endothelial cells (HUVECs). Additionally, the use of VEGF-Fc as an ECM markedly promoted the proliferation of HUVECs longer than 72 h and induced the reorganization of actin filaments into larger stress fibers within these cells. The VEGF-Fc fusion protein may be a promising artificial ECM for enhancing endothelial cell growth.

Knockdown of PLC-gamma-2 and calmodulin 1 genes sensitizes human cervical adenocarcinoma cells to doxorubicin and paclitaxel.

BACKGROUND: RNA interference (RNAi) is a powerful approach in functional genomics to selectively silence messenger mRNA (mRNA) expression and can be employed to rapidly develop potential novel drugs against a complex disease like cancer. However, naked siRNA being anionic is unable to cross the anionic cell membrane through passive diffusion and therefore, delivery of siRNA remains a major hurdle to overcome before the potential of siRNA technology can fully be exploited in cancer. pH-sensitive carbonate apatite has recently been developed as an efficient tool to deliver siRNA into the mammalian cells by virtue of its high affinity interaction with the siRNA and the desirable size distribution of the resulting siRNA-apatite complex for effective cellular endocytosis. Moreover, internalized siRNA was found to escape from the endosomes in a time-dependent manner and efficiently silence gene expression. RESULTS: Here we show that carbonate apatite-mediated delivery of siRNA against PLC-gamma-2 (PLCG2) and calmodulin 1 (CALM1) genes has led to the sensitization of a human cervical cancer cell line to doxorubicin- and paclitaxel depending on the dosage of the individual drug whereas no such enhancement in cell death was observed with cisplatin irrespective of the dosage following intracellular delivery of the siRNAs. CONCLUSION: Thus, PLCG2 and CALM1 genes are two potential targets for gene knockdown in doxorubicin and paclitaxel-based chemotherapy of cervical cancer.

Local mechanical stimulation of Mardin-Darby canine kidney cell sheets on temperature-responsive hydrogel.

Collective motion of cell sheets plays a role not only in development and repair, but also in devastating diseases such as cancer. However, unlike single-cell motility, collective motion of cell sheets involves complex cell-cell communication during migration; therefore, its mechanism is largely unknown. To elucidate propagation of signaling transduced by cell-cell interaction, we designed a hydrogel substrate that can cause local mechanical stretching of cell sheets. Poly (N-isopropyl acrylamide) (PNIPAAm) hydrogel is a temperature-responsive polymer gel whose volume changes isotropically in response to temperature changes below 37 °C. We designed a combined hydrogel substrate consisting of collagen-immobilized PNIPAAm as the local stimulation side and polyacrylamide (PAAm) as the non-stimulation side to assess propagation of mechanical transduction. Mardin-Darby canine kidney (MDCK) cells adhered to the collagen-immobilized PNIPAAm gel increased it area and were flattened as the gel swelled with temperature decrease. E-cadherin in these cells became undetectable in some domains, and actin stress fibers were more clearly observed at the cell base. In contrast, E-cadherin in cells adhered to the collagen-immobilized PAAm side was equally stained as that in cells adhered to the collagen-immobilized PAAm side even after temperature decrease. ERK1/2 MAPK activation of cells on the non-stimulated substrate occurred after partial stretching of the cell sheet suggesting the propagation of signaling. These results indicate that a change in the balance of mechanical tension induced by partial stretching of cell sheets leads to activation and propagation of the cell signaling.

Engulfment and clearance of apoptotic cells based on a GlcNAc-binding lectin-like property of surface vimentin.

The clearance of apoptotic cells is important to maintain tissue homeostasis. The engulfment of apoptotic cells is performed by professional phagocytes, such as macrophages, and also by non-professional phagocytes, such as mesenchymal cells. Here, we show that vimentin, a cytoskeletal protein, functions as an engulfment receptor on neighboring phagocytes, which recognize O-linked ß-N-acetylglucosamine (O-GlcNAc)-modified proteins from apoptotic cells as "eat me" ligands. Previously, we reported that vimentin possesses a GlcNAc-binding lectin-like property on cell surface. However, the physiological relevance of the surface localization and GlcNAc-binding property of vimentin remained unclear. In the present study, we observed that O-GlcNAc proteins from apoptotic cells interacted with the surface vimentin of neighboring phagocytes and that this interaction induced serine 71-phosphorylation and recruitment of vimentin to the cell surface of the neighboring phagocytes. Moreover, tetrameric vimentin that was disassembled by serine 71-phosphorylation possessed a GlcNAc-binding activity and was localized to the cell surface. We demonstrated our findings in vimentin-expressing common cell lines such as HeLa cells. Furthermore, during normal developmental processes, the phagocytic engulfment and clearance of apoptotic footplate cells in mouse embryos was mediated by the interaction of surface vimentin with O-GlcNAc proteins. Our results suggest a common mechanism for the clearance of apoptotic cells, through the interaction of surface vimentin with O-GlcNAc-modified proteins.

Cardiac differentiation of embryonic stem cells by substrate immobilization of insulin-like growth factor binding protein 4 with elastin-like polypeptides.

The establishment of cardiomyocyte differentiation of embryonic stem cells (ESCs) is a useful strategy for cardiovascular regenerative medicine. Here, we report a strategy for cardiomyocyte differentiation of ESCs using substrate immobilization of insulin-like growth factor binding protein 4 (IGFBP4) with elastin-like polypeptides. Recently, IGFBP4 was reported to promote cardiomyocyte differentiation of ESCs through inhibition of the Wnt/ß-catenin signaling. However, high amounts of IGFBP4 (approximately 1 µg/mL) were required to inhibit the Wnt/ß-catenin signaling and induce differentiation to cardiomyocytes. We report herein induction of cardiomyocyte differentiation using IGFBP4-immobilized substrates. IGFBP4-immobilized substrates were created by fusion with elastin-like polypeptides. IGFBP4 was stably immobilized to polystyrene dishes through fusion of elastin-like polypeptides. Cardiomyocyte differentiation of ESCs was effectively promoted by strong and continuous inhibition of Wnt/ß-catenin signaling with IGFBP4-immobilized substrates. These results demonstrated that IGFBP4 could be immobilized using fusion of elastin-like polypeptides. Our results also demonstrate that substrate immobilization of IGFBP4 is a powerful tool for differentiation of ESCs into cardiomyocytes. These findings suggest that substrate immobilization of soluble factors is a useful technique for differentiation of ESCs in regenerative medicine and tissue engineering.

Targeting N-acetylglucosamine-bearing polymer-coated liposomes to vascular smooth muscle cells.

The targeted delivery of anti-inflammatory agents has great therapeutic potential for treating restenosis following percutaneous coronary intervention. To develop a drug delivery system targeted to injured blood vessels, we examined whether N-acetylglucosamine (GlcNAc)-bearing polymer-coated liposomes (GlcNAc-Ls) are specifically taken up by vascular smooth muscle cells (VSMCs). Flow cytometric analysis revealed that GlcNAc-Ls were taken up by VSMCs in vitro. Furthermore, GlcNAc-Ls were intravenously administered to mice that had undergone wire-mediated vascular injury. GlcNAc-Ls markedly accumulated at the intramural site of the injured vessel walls but not at the contralateral (uninjured) vessel walls. These results demonstrated that GlcNAc-Ls can be specifically taken up by VSMCs both in vitro and in vivo. We propose a novel strategy of using GlcNAc-Ls that has potential for application in drug delivery targeted to injured blood vessels.

Gene delivery system based on highly specific recognition of surface-vimentin with N-acetylglucosamine immobilized polyethylenimine.

Gene and drug-delivery systems that use immobilization of carbohydrates are useful for the specific targeting of lectin-expressing tissues. Here, we report that N-acetylglucosamine (GlcNAc) with polyethylenimine (GlcNAc-PEI) specifically interacted with vimentin-expressing cells such as 293FT and HeLa cells. Recently, the intermediate filaments vimentin and desmin have been reported to have GlcNAc-binding lectin-like properties on the cell surface. Therefore, GlcNAc-conjugated agents can be targeted to vimentin- and desmin-expressing cells and tissues. Vimentin-expressing 293FT and HeLa cells were efficiently transfected with green fluorescent protein and luciferase genes by using GlcNAc-PEI; the expression of these genes in vimentin-knockdown cells were low. Confocal microscopic analysis showed that GlcNAc-PEI complexes interacted with vimentin on the cell surface of HeLa cells. These results demonstrate that GlcNAc-PEI/DNA complexes were specifically taken up by 293FT and HeLa cells via vimentin. We suggest that this gene-delivery system could be used to target various vimentin-expressing cells such as fibroblasts and tumor cells.