Radiation induces acute and subacute vascular regression in a three-dimensional microvasculature model.

Radiation treatment is one of the most frequently used therapies in patients with cancer, employed in approximately half of all patients. However, the use of radiation therapy is limited by acute or chronic adverse effects and the failure to consider the tumor microenvironment. Blood vessels substantially contribute to radiation responses in both normal and tumor tissues. The present study employed a three-dimensional (3D) microvasculature-on-a-chip that mimics physiological blood vessels to determine the effect of radiation on blood vessels. This model represents radiation-induced pathophysiological effects on blood vessels in terms of cellular damage and structural and functional changes. DNA double-strand breaks (DSBs), apoptosis, and cell viability indicate cellular damage. Radiation-induced damage leads to a reduction in vascular structures, such as vascular area, branch length, branch number, junction number, and branch diameter; this phenomenon occurs in the mature vascular network and during neovascularization. Additionally, vasculature regression was demonstrated by staining the basement membrane and microfilaments. Radiation exposure could increase the blockage and permeability of the vascular network, indicating that radiation alters the function of blood vessels. Radiation suppressed blood vessel recovery and induced a loss of angiogenic ability, resulting in a network of irradiated vessels that failed to recover, deteriorating gradually. These findings demonstrate that this model is valuable for assessing radiation-induced vascular dysfunction and acute and chronic effects and can potentially improve radiotherapy efficiency.

Three-Dimensional In Vitro Lymphangiogenesis Model in Tumor Microenvironment.

Lymphangiogenesis is a stage of new lymphatic vessel formation in development and pathology, such as inflammation and tumor metastasis. Physiologically relevant models of lymphatic vessels have been in demand because studies on lymphatic vessels are required for understanding the mechanism of tumor metastasis. In this study, a new three-dimensional lymphangiogenesis model in a tumor microenvironment is proposed, using a newly designed macrofluidic platform. It is verified that controllable biochemical and biomechanical cues, which contribute to lymphangiogenesis, can be applied in this platform. In particular, this model demonstrates that a reconstituted lymphatic vessel has an in vivo-like lymphatic vessel in both physical and biochemical aspects. Since biomechanical stress with a biochemical factor influences robust directional lymphatic sprouting, whether our model closely approximates in vivo, the initial lymphatics in terms of the morphological and genetic signatures is investigated. Furthermore, attempting an incorporation with a tumor spheroid, this study successfully develops a complex tumor microenvironment model for use in lymphangiogenesis and reveals the microenvironment factors that contribute to tumor metastasis. As a first attempt at a coculture model, this reconstituted model is a novel system with a fully three-dimensional structure and can be a powerful tool for pathological drug screening or disease model.

Gamma irradiation exposure for collapsed cell junctions and reduced angiogenesis of 3-D in vitro blood vessels.

During radiotherapy, microenvironments neighboring the tumor are also exposed to gamma irradiation; this results in unexpected side effects. Blood vessels can serve as microenvironments for tumors and they play an important role in providing nutrients to tumors. This is mostly related to tumor progression, metastasis, and relapse after therapy. Many studies have been performed to obtain a better understanding of tumor vasculature after radiotherapy with in vitro models. However, compared to 3-D models, 2-D in vitro endothelial monolayers cannot physiologically reflect in vivo blood vessels. We previously remodeled the extracellular matrix (ECM) hydrogel that enhanced the tight barrier formation of 3-D blood vessels and the vascular endothelial growth factor (VEGF) gradient induced angiogenesis in a microfluidic device. In this study, the blood vessel model is further introduced to understand how gamma irradiation affects the endothelial monolayer. After the gamma irradiation exposure, we observed a collapsed endothelial barrier and a reduced angiogenic potential. Changes in the cell behaviors of the tip and stalk cells were also detected in the angiogenesis model after irradiation, which is difficult to observe in 2-D monolayer models. Therefore, the 3-D in vitro blood vessel model can be used to understand radiation-induced endothelial injuries.

Simulation and Experimental Study of Ion Concentration Polarization Induced Electroconvective Vortex and Particle Movement.

Ion concentration polarization (ICP) has been widely applied in microfluidic systems in pre-concentration, particle separation, and desalination applications. General ICP microfluidic systems have three components (i.e., source, ion-exchange, and buffer), which allow selective ion transport. Recently developed trials to eliminate one of the three components to simplify the system have suffered from decreased performance by the accumulation of unwanted ions. In this paper, we presented a new ICP microfluidic system with only an ion-exchange membrane-coated channel. Numerical investigation on hydrodynamic flow and electric fields with a series of coupled governing equations enabled a strong correlation to experimental investigations on electroconvective vortices and the trajectory of charged particles. This study has significant implications for the development and optimization of ICP microfluidic and electrochemical systems for biomarker concentration and separation to improve sensing reliability and detection limits in analytic chemistry.

Hydrogel-incorporating unit in a well: 3D cell culture for high-throughput analysis.

The microfluidic 3D cell culture system has been an attractive model because it mimics the tissue and disease model, thereby expanding our ability to control the local cellular microenvironment. However, these systems still have limited value as quantitative assay tools due to the difficulties associated with the manipulation and maintenance of microfluidic cells, and their lack of compatibility with the high-throughput screening (HTS) analysis system. In this study, we suggest a microchannel-free, 3D cell culture system that has a hydrogel-incorporating unit integrated with a multi-well plate (24- to 96-well plate), which can provide better reproducibility in biological experiments. This plate was devised considering the design constraints imposed by various cell biology applications as well as by high-throughput analysis where the physical dimensions of the micro-features in the hydrogel-incorporating units were altered. We also demonstrated that the developed plate is potentially applicable to a variety of quantitative biochemical assays for qRT-PCR, Western blotting, and microplate-reader-based assays, such as ELISA, viability assay, and high content-screening (HCS) as well as the co-culture for biological studies. Human neural progenitor cells (hNPCs) that produce pathogenic Aß species for modeling Alzheimer's disease (AD) were three-dimensionally cultured, and the efficacy of the inhibitors of Aß production was assessed by ELISA in order to demonstrate the performance of this plate.

In vitro nasal mucosa gland-like structure formation on a chip.

The emergence of microfluidic epithelial models using diverse types of cells within a physiologically relevant microenvironment has the potential to be a powerful tool for preclinical drug screening and pathophysiological studies. However, to date, few studies have reported the development of a complicated in vitro human nasal epithelial model. The aim of this study was to produce an in vitro human nasal mucosa model for reliable drug screening and clinical applications. Here, we integrated and optimized several culture conditions such as cell type, airway culture conditions, and hydrogel scaffolds into a microfluidic chip to construct an advanced in vitro human nasal mucosa model. We observed that the inducing factors for nasal gland-like structures were secreted from activated human dermal microvascular endothelial cells. Furthermore, our in vitro nasal mucosa presented different appearance and characteristics under hypoxic conditions. Morphological and functional similarities between in vivo nasal mucosa and our model indicated its utilization as a reliable research model for nasal diseases including allergic rhinitis, chronic sinusitis, and nasal polyposis.

Spheroid Formation of Hepatocarcinoma Cells in Microwells: Experiments and Monte Carlo Simulations.

The formation of spherical aggregates during the growth of cell population has long been observed under various conditions. We observed the formation of such aggregates during proliferation of Huh-7.5 cells, a human hepatocarcinoma cell line, in a microfabricated low-adhesion microwell system (SpheroFilm; formed of mass-producible silicone elastomer) on the length scales up to 500 µm. The cell proliferation was also tracked with immunofluorescence staining of F-actin and cell proliferation marker Ki-67. Meanwhile, our complementary 3D Monte Carlo simulations, taking cell diffusion and division, cell-cell and cell-scaffold adhesion, and gravity into account, illustrate the role of these factors in the formation of spheroids. Taken together, our experimental and simulation results provide an integrative view of the process of spheroid formation for Huh-7.5 cells.

Effect of mesenchymal stem cells and extracts derived from the placenta on trophoblast invasion and immune responses.

Tightly regulated trophoblast invasion and immunomodulation at the feto-maternal interface is important during implantation and fetal development. Although trophoblasts as a pregnancy-specific cell has been reported to be a key factor capable of regulating certain events during implantation, however, its regulatory mechanisms are still unclear. In this study, we analyzed the effects of chorionic plate-derived mesenchymal stem cells (CP-MSCs) and human placenta extract (hPE) isolated from human normal placentas on trophoblasts invasion and immune responses. We investigated the effects of CP-MSCs, hPE treatment, and their combination on trophoblasts invasion and on T-cells suppression through human leukocyte antigen-G (HLA-G) expression. Trophoblasts invasion was significantly increased by co-culture of CP-MSCs or by hPE treatment (P<0.05), and enhanced by the combination of CP-MSCs and hPE treatment (P<0.05). The proliferation of T-cells was decreased by co-culture of CP-MSCs and hPE treatment, whereas the population of regulatory T-cells was increased (P<0.05). Also, the dynamics alterations of multiple cytokines were observed in the culture supernatants of trophoblasts and T-cells depending on CP-MSCs co-culture and hPE treatment. Interestingly, the concentration of soluble HLA-G was increased by CP-MSCs co-culture, by hPE treatment and by combination of them on trophoblasts and activated T-cells (P<0.05). These findings suggested that CP-MSCs and hPE can regulate trophoblasts invasion and T-cell by alteration of HLA-G expression. These results will provide understandings of trophoblasts invasion and the immunological network at the feto-maternal interface during pregnancy and contribute to the foundation of a new treatment strategy for pregnancy disorders.

Altered expression of norepinephrine transporter and norepinephrine in human placenta cause pre-eclampsia through regulated trophoblast invasion.

OBJECTIVE: We investigated the norepinephrine transporter (NET) expression in normal and pre-eclamptic placentas and analyzed the invasion activity of trophoblastic cells based on norepinephrine (NE)-NET regulation. METHODS: NET and NE expression levels were examined by western blot and enzyme-linked immunosorbent assay, respectively. Trophoblast invasion activity, depending on NE-NET regulation, was determined by NET-small interfering RNA (siRNA) and NET transfection into the human extravillous trophoblast cells with or without NE treatment and invasion rates were analyzed by zymography and an invasion assay. RESULTS: NET mRNA was expressed at a low level in pre-eclamptic placentas compared with normal placentas and NE concentration in maternal plasma increased significantly in pre-eclamptic women compared to normal pregnant women (p<0.05). NET gene upregulation and NE treatment stimulated trophoblast cell invasion up to 2.5-fold (p<0.05) by stimulating matrix metalloproteinase-9 activity via the phosphoinositol-3-kinase/AKT signaling pathway, whereas NET-siRNA with NE treatment reduced invasion rates. CONCLUSION: NET expression is reduced by inadequate regulation of NE levels during placental development. This suggests that a complementary balance between NET and NE regulates trophoblast cell invasion activities during placental development.

Hypoxia-induced downregulation of XIAP in trophoblasts mediates apoptosis via interaction with IMUP-2: implications for placental development during pre-eclampsia.

The regulation of trophoblast apoptosis is essential for normal placentation, and increased placental trophoblast cell apoptosis is the cause of pathologies such as intrauterine growth retardation (IUGR) and pre-eclampsia. X-linked inhibitor of apoptosis (XIAP) is expressed in trophoblasts, but little is known about the role of XIAP in placental development. In the present study, the function of XIAP in the placenta and in HTR-8/SVneo trophoblasts under hypoxic conditions was examined. In addition, the correlation between XIAP and immortalization-upregulated protein-2 (IMUP-2) was demonstrated in HTR-8/SVneo trophoblasts under hypoxia, based on a previous study showing that increased IMUP-2 induces trophoblast apoptosis and pre-eclampsia. XIAP was downregulated in pre-eclamptic placentas (P < 0.05). In HTR-8/SVneo trophoblasts, XIAP expression was decreased and the expression of apoptosis-related genes was increased in response to hypoxia. Ectopic expression of hypoxia inducible factor (HIF)-1α in HRT-8 SV/neo cells induced the nuclear translocation of XIAP and alterations of XIAP protein stability. Furthermore, hypoxia induced nuclear translocated XIAP co-localized with upregulated IMUP-2 in trophoblast nuclei, and the interaction between XIAP and IMUP-2 induced apoptosis in HRT-8 SV/neo cells. The present results suggest that hypoxia-induced down-regulation of XIAP mediates apoptosis in trophoblasts through interaction with increased IMUP-2, and that this mechanism underlies the pathogenesis of pre-eclampsia.

Dynamic alterations in integrin α4 expression by hypoxia are involved in trophoblast invasion during early implantation.

Implantation of the blastocyst into the maternal endometrium is mediated by a population of well-differentiated primary cells of the placenta known as trophoblasts, which grow in an invasive and destructive fashion similar to tumor cells. Interactions between the endometrium and trophoblasts are regulated by a coordinated interplay of extracellular matrix (ECM) proteins secreted by the invading extravillous trophoblasts. Integrins act as adhesion receptors and mediate both cell-ECM and cell-cell interactions. However, the correlation between integrin expression and trophoblast invasion under hypoxia is unclear. Here, we analyzed the expression of integrins in HTR-8/SVneo trophoblast cells exposed to hypoxic conditions in order to demonstrate an association between invasion activity and integrin expression in trophoblasts. Trophoblasts were examined by microarray analysis, RT-PCR, western blotting, and zymography after 1% hypoxic treatment, and cell invasion was estimated. The dynamic expression of integrins and human matrix metalloproteinases (MMPs) was observed under hypoxic conditions. The invasiveness of trophoblasts cultured under 1% hypoxic conditions was significantly greater than that of trophoblasts cultured under normoxic conditions through alterations in MMP-2 and -9 (P < 0.05). Notably, integrin α4 expression during early hypoxia was negatively regulated by hypoxia-inducible factor-1alpha (HIF-1alpha) expression in trophoblasts. The downregulation of integrin α4 expression by siRNA treatment controlled trophoblast invasion activity (P < 0.05). Taken together, we suggest that dynamic changes in integrins, including those in integrin α4 expression by hypoxia, play a regulatory role in trophoblast invasion. These findings expand our understanding of the potential roles of integrin α4 in implantation.

Placenta extract promote liver regeneration in CCl4-injured liver rat model.

The human placenta is an organ for fetus development and abundant reservoir of various bioactive molecules. Interest to human placenta extract (hPE) is growing, and application with trial of hPE is widening in oriental medicine including in liver diseases. However, underlying mechanisms for therapeutic effects are still unclear. Here, we investigated therapeutic effects of hPE in carbon tetrachloride (CCl(4))-injured rat liver model in vivo and in damaged rat hepatic cells exposed to CCl(4) in vitro. In addition, regulation of inflammatory responses by treatment of hPE was investigated. Serum levels of GOT/AST and GPT/ALT were significantly reduced (P<0.05), and uptake/excretion of indocyanine green in serum was significantly induced at 3 weeks after intravenous hPE administration in CCl(4)-injured rat model (P<0.05). Expression of type I collagen (Col I) and α-smooth muscle actin (α-SMA) was decreased, whereas that of matrix metalloproteinase-9 (MMP-9) was increased resulting in improvement of score for fibrotic grade in hPE group. Also, albumin, proliferation activities and molecules associated with liver regeneration (e.g. interleukin-6, gp130, ATP binding cassette transporters, cyclin A) were more increased in hPE administration group than Non-hPE group. hPE administration suppressed activated T-cell proliferation via increasing anti-inflammatory cytokines and decreasing pro-inflammatory cytokines. These results suggest that hPE could be effective for liver disease through reduction of fibrosis, induction of liver regeneration, and regulation of inflammatory responses. These findings are important for understanding the roles of hPE and provide evidences for therapeutic effects of hPE in hepatic diseases which could lead to potential clinical applications.

Anti-fibrotic effect of chorionic plate-derived mesenchymal stem cells isolated from human placenta in a rat model of CCl(4)-injured liver: potential application to the treatment of hepatic diseases.

Translational studies have explored the therapeutic effects of stem cells, raising hopes for the treatment of numerous diseases. Here, we evaluated the therapeutic effect of chorionic plate-derived mesenchymal stem cells (CP-MSCs) isolated from human placenta and transplanted into rats with carbon tetrachloride (CCl(4))-injured livers. CP-MSCs were analyzed for hepatocyte-specific gene expression, indocyanine green (ICG) uptake, glycogen storage, and urea production following hepatogenic differentiation. PKH26-labeled CP-MSCs were directly transplanted into the livers of rats that had been exposed to CCl(4) (1.6 g/kg, twice per week for 9 weeks). Blood and liver tissue were analyzed at 1, 2, and 3 weeks post-transplantation. The expression of type I collagen (Col I) and matrix metalloproteinases (MMPs) was analyzed in rat T-HSC/Cl-6 hepatic stellate cells co-cultured with CP-MSCs following exposure to TGF-ß. The expression levels of α-smooth muscle actin (α-SMA) and Col I were lower in transplanted (TP) rats than in non-transplanted (Non-TP) animals (P < 0.05), whereas the expression levels of albumin and MMP-9 were increased. TP rats exhibited significantly higher uptake/excretion of ICG than non-TP rats (P < 0.005). In addition, collagen synthesis in T-HSC/Cl-6 cells exposed to TGF-ß was decreased by co-culture with CP-MSCs, which triggered the activation of MMP-2 and MMP-9. These results contribute to our understanding of the potential pathophysiological roles of CP-MSCs, including anti-fibrotic effects in liver disease, and provide a foundation for the development of new cell therapy-based strategies for the treatment of difficult-to-treat liver diseases.

Characterization of Fetal Tissue-derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have unique immunologic properties that may someday prove useful in cell-based therapy for various degenerative diseases. Its potential is limited, however, by several factors, including the rarity of these cells and difficulty in isolating them. To evaluate their potential as new sources for cell therapy, we isolated MSCs from human fetal tissue (hfMSC) derived from spontaneous abortus (8∼10 weeks) then studied their cell cycle and cell surface marker expression using a fluorescence-activated cell sorter (FACS), as well as the expression of differentiation markers using real-time polymerase chain reaction (RT-PCR). The hfMSCs were able to undergo PCR up to 20 times without displaying significant changes in morphology or expression of various stemness markers (Nanog and human telomerase reverse transcriptase [hAFP]), including germ layer markers (hNF68, alpha-cardiac actin, and hAFP). Also, teratomas were not seen in mice with severe combined immunodeficiency syndrome (SCID) that received a transplantation of hfMSCs with hTERT activity. The FACS analysis revealed that the majority of hfMSCs express mesenchymal markers CD13, CD44, CD71, CD90, CD105, CD253a, and HLA-ABC, but did not express CD31, CD34, CD38, CD45, and HLA-DR. Interestingly, hfMSCs derived from the cell membrane during early passages were negative for both HLA-ABC and HLA-DR, although HLA-ABC expression was detected during later passages (>20 passages). We found that hfMSCs could be differentiated into an osteogenic lineage; this was indicated by modulation of osteoblast markers specific for mRNA. We conclude that hfMSCs could be used as a new source of cells to treat patients with osteogenic diseases, as well as to understand the mechanisms of immunosuppression by MSCs.