Bongju Kim

Directed and Systematic Differentiation of Cardiovascular Cells From Mouse Induced Pluripotent Stem Cells

Background— Induced pluripotent stem (iPS) cells are a novel stem cell population induced from mouse and human adult somatic cells through reprogramming by transduction of defined transcription factors. However, detailed differentiation properties and the directional differentiation system of iPS cells have not been demonstrated. Methods and Results— Previously, we established a novel mouse embryonic stem (ES) cell differentiation system that can reproduce the early differentiation processes of cardiovascular cells. We applied our ES cell system to iPS cells and examined directional differentiation of mouse iPS cells to cardiovascular cells. Flk1 (also designated as vascular endothelial growth factor receptor-2)-expressing mesoderm cells were induced from iPS cells after ≈4-day culture for differentiation. Purified Flk1+ cells gave rise to endothelial cells and mural cells by addition of vascular endothelial growth factor and serum. Arterial, venous, and lymphatic endothelial cells were also successfully induced. Self-beating cardiomyocytes could be induced from Flk1+ cells by culture on OP9 stroma cells. Time course and efficiency of the differentiation were comparable to those of mouse ES cells. Occasionally, reexpression of transgene mRNAs, including c-myc, was observed in long-term differentiation cultures. Conclusions— Various cardiovascular cells can be systematically induced from iPS cells. The differentiation properties of iPS cells are almost completely identical to those of ES cells. This system would greatly contribute to a novel understanding of iPS cell biology and the development of novel cardiovascular regenerative medicine.

Requirement of Interaction between Mast Cells and Skin Dendritic Cells to Establish Contact Hypersensitivity

The role of mast cells (MCs) in contact hypersensitivity (CHS) remains controversial. This is due in part to the use of the MC-deficient Kit W/Wv mouse model, since Kit W/Wv mice congenitally lack other types of cells as a result of a point mutation in c-kit. A recent study indicated that the intronic enhancer (IE) for Il4 gene transcription is essential for MCs but not in other cell types. The aim of this study is to re-evaluate the roles of MCs in CHS using mice in which MCs can be conditionally and specifically depleted. Transgenic Mas-TRECK mice in which MCs are depleted conditionally were newly generated using cell-type specific gene regulation by IE. Using this mouse, CHS and FITC-induced cutaneous DC migration were analyzed. Chemotaxis assay and cytoplasmic Ca2+ imaging were performed by co-culture of bone marrow-derived MCs (BMMCs) and bone marrow-derived dendritic cells (BMDCs). In Mas-TRECK mice, CHS was attenuated when MCs were depleted during the sensitization phase. In addition, both maturation and migration of skin DCs were abrogated by MC depletion. Consistently, BMMCs enhanced maturation and chemotaxis of BMDC in ICAM-1 and TNF-α dependent manners Furthermore, stimulated BMDCs increased intracellular Ca2+ of MC upon direct interaction and up-regulated membrane-bound TNF-α on BMMCs. These results suggest that MCs enhance DC functions by interacting with DCs in the skin to establish the sensitization phase of CHS.

Basophils are required for the induction of Th2 immunity to haptens and peptide antigens

The relative contributions of basophils and dendritic cells in Th2 skewing to foreign antigen exposure remain unclear. Here we report the ability of basophils to induce Th2 polarization upon epicutaneous sensitization with different antigens using basophil conditionally depleted Bas TRECK transgenic mice. Basophils are responsible for Th2 skewing to haptens and peptide antigens, but not protein antigens in vivo. Consistent with this, basophils cannot take up or process ovalbumin protein in significant quantities, but present ovalbumin peptide to T cells for Th2 differentiation via major histocompatibility complex class II. Intriguingly, basophils promote Th2 skewing upon ovalbumin protein exposure in the presence of dendritic cells. Taken together, our results suggest that basophils alone are able to induce Th2 skewing with haptens and peptide antigens but require dendritic cells for the induction of Th2 for protein antigens upon epicutaneous immunization.

Cytoplasmic Na+‐dependent modulation of mitochondrial Ca2+ via electrogenic mitochondrial Na+–Ca2+ exchange

To clarify the role of mitochondrial Na+–Ca2+ exchange (NCXmito) in regulating mitochondrial Ca2+ (Ca2+mito) concentration at intact and depolarized mitochondrial membrane potential (ΔΨmito), we measured Ca2+mito and ΔΨmito using fluorescence probes Rhod‐2 and TMRE, respectively, in the permeabilized rat ventricular cells. Applying 300 nm cytoplasmic Ca2+ (Ca2+c) increased Ca2+mito and this increase was attenuated by cytoplasmic Na+ (Na+c) with an IC50 of 2.4 mm. To the contrary, when ΔΨmito was depolarized by FCCP, a mitochondrial uncoupler, Na+c enhanced the Ca2+c‐induced increase in Ca2+mito with an EC50 of about 4 mm. This increase was not significantly affected by ruthenium red or cyclosporin A. The inhibition of NCXmito by CGP‐37157 further increased Ca2+mito when ΔΨmito was intact, while it suppressed the Ca2+mito increase when ΔΨmito was depolarized, suggesting that ΔΨmito depolarization changed the exchange mode from forward to reverse. Furthermore, ΔΨmito depolarization significantly reduced the Ca2+mito decrease via forward mode, and augmented the Ca2+mito increase via reverse mode. When the respiratory chain was attenuated, the induction of the reverse mode of NCXmito hyperpolarized ΔΨmito, while ΔΨmito depolarized upon inducing the forward mode of NCXmito. Both changes in ΔΨmito were remarkably inhibited by CGP‐37157. The above experimental data indicated that NCXmito is voltage dependent and electrogenic. This notion was supported theoretically by computer simulation studies with an NCXmito model constructed based on present and previous studies, presuming a consecutive and electrogenic Na+–Ca2+ exchange and a depolarization‐induced increase in Na+ flux. It is concluded that Ca2+mito concentration is dynamically modulated by Na+c and ΔΨmito via electrogenic NCXmito.

The mitochondrial Na+-Ca2+ exchanger, NCLX, regulates automaticity of HL-1 cardiomyocytes

Mitochondrial Ca2+ is known to change dynamically, regulating mitochondrial as well as cellular functions such as energy metabolism and apoptosis. The NCLX gene encodes the mitochondrial Na+-Ca2+ exchanger (NCXmit), a Ca2+ extrusion system in mitochondria. Here we report that the NCLX regulates automaticity of the HL-1 cardiomyocytes. NCLX knockdown using siRNA resulted in the marked prolongation of the cycle length of spontaneous Ca2+ oscillation and action potential generation. The upstrokes of action potential and Ca2+ transient were markedly slower, and sarcoplasmic reticulum (SR) Ca2+ handling were compromised in the NCLX knockdown cells. Analyses using a mathematical model of HL-1 cardiomyocytes demonstrated that blocking NCXmit reduced the SR Ca2+ content to slow spontaneous SR Ca2+ leak, which is a trigger of automaticity. We propose that NCLX is a novel molecule to regulate automaticity of cardiomyocytes via modulating SR Ca2+ handling.

Stimulating effect of graphene oxide on myogenesis of C2C12 myoblasts on RGD peptide-decorated PLGA nanofiber matrices

BackgroundIn the field of biomedical engineering, many studies have focused on the possible applications of graphene and related nanomaterials due to their potential for use as scaffolds, coating materials and delivery carriers. On the other hand, electrospun nanofiber matrices composed of diverse biocompatible polymers have attracted tremendous attention for tissue engineering and regenerative medicine. However, their combination is intriguing and still challenging.ResultsIn the present study, we fabricated nanofiber matrices composed of M13 bacteriophage with RGD peptide displayed on its surface (RGD-M13 phage) and poly(lactic-co-glycolic acid, PLGA) and characterized their physicochemical properties. In addition, the effect of graphene oxide (GO) on the cellular behaviors of C2C12 myoblasts, which were cultured on PLGA decorated with RGD-M13 phage (RGD/PLGA) nanofiber matrices, was investigated. Our results revealed that the RGD/PLGA nanofiber matrices have suitable physicochemical properties as a tissue engineering scaffold and the growth of C2C12 myoblasts were significantly enhanced on the matrices. Moreover, the myogenic differentiation of C2C12 myoblasts was substantially stimulated when they were cultured on the RGD/PLGA matrices in the presence of GO.ConclusionIn conclusion, these findings propose that the combination of RGD/PLGA nanofiber matrices and GO can be used as a promising strategy for skeletal tissue engineering and regeneration.

Pivotal role of mitochondrial Na+–Ca2+ exchange in antigen receptor mediated Ca2+ signalling in DT40 and A20 B lymphocytes

Non‐technical summary  Elevation of cytoplasmic Ca2+ is one of the early responses of lymphocytes upon the antigen recognition by the surface receptor. We show here that the Ca2+ response is maintained by Ca2+ transfer from mitochondria to endoplasmic reticulum through mitochondrial Na+–Ca2+ exchange. The result helps us understanding how lymphocyte responses to antigen are organized at organelle level.

Preventive Effects of Epigallocatechin-3-O-Gallate against Replicative Senescence Associated with p53 Acetylation in Human Dermal Fibroblasts

Considering the various pharmacological activities of epigallocatechin-3-O-gallate (EGCG) including anticancer, and anti-inflammatory, antidiabetic, and so forth, relatively less attention has been paid to the antiaging effect of EGCG on primary cells. In this study, the preventive effects of EGCG against serial passage-induced senescence were investigated in primary cells including rat vascular smooth muscle cells (RVSMCs), human dermal fibroblasts (HDFs), and human articular chondrocytes (HACs). The involvement of Sirt1 and acetylated p53 was examined as an underlying mechanism for the senescence preventive activity of EGCG in HDFs. All cells were employed with the initial passage number (PN) between 3 and 7. For inducing senescence, the cells were serially passaged at the predetermined times and intervals in the absence or presence of EGCG (50 or 100 μM). Serial passage-induced senescence in RVSMCs and HACs was able to be significantly prevented at 50 μM EGCG, while in HDFs, 100 μM EGCG could significantly prevent senescence and recover their cell cycle progression close to the normal level. Furthermore, EGCG was found to prevent serial passage- and H2O2-induced senescence in HDFs by suppressing p53 acetylation, but the Sirt1 activity was unaffected. In addition, proliferating HDFs showed similar cellular uptake of FITC-conjugated EGCG into the cytoplasm with their senescent counterparts but different nuclear translocation of it from them, which would partly account for the differential responses to EGCG in proliferating versus senescent cells. Taking these results into consideration, it is suggested that EGCG may be exploited to craft strategies for the development of an antiaging or age-delaying agent.

Graphene oxide-coated guided bone regeneration membranes with enhanced osteogenesis: Spectroscopic analysis and animal study

ABSTRACT Guided bone regeneration (GBR) is a technique where a barrier membrane is placed over the bone defect to prevent cell growth from the connective tissue and epithelium. Titanium (Ti) has excellent mechanical properties and is one of the most frequently used materials in implant dentistry. This study examined how graphene oxide (GO)-coated Ti (GO-Ti) membranes can enhance the osteogenesis of MC3T3-E1 preosteoblasts and promote new bone formation for potential applications to GBR. The physicochemical properties of GO-Ti membranes were characterized by atomic force microscopy, Raman spectroscopy, X-ray diffraction, and contact angle measurements. The cellular behaviors of MC3T3-E1 preosteoblasts on GO-Ti membranes were examined by cell counting kit-8 and alkaline phosphatase (ALP) activity assays. The effects of GO-Ti membranes on bone regeneration were evaluated by implanting them into rat calvarial defects. GO was coated uniformly on Ti substrates, which allowed a decrease in surface roughness and contact. GO-Ti membranes stimulated significantly ALP activity without interfering with their proliferation. Furthermore, GO-Ti membranes enhanced new bone formation significantly in full-thickness calvarial defects without inflammatory responses. Therefore, this suggests that GO-Ti membranes can be applied effectively to GBR because these graphene-coated Ti membranes have potent effects on stimulating osteogenic differentiation and exhibit superior bioactivity.

Hyaluronic Acid/PLGA Core/Shell Fiber Matrices Loaded with EGCG Beneficial to Diabetic Wound Healing

During the last few decades, considerable research on diabetic wound healing strategies has been performed, but complete diabetic wound healing remains an unsolved problem, which constitutes an enormous biomedical burden. Herein, hyaluronic acid (HA)/poly(lactic-co-glycolic acid, PLGA) core/shell fiber matrices loaded with epigallocatechin-3-O-gallate (EGCG) (HA/PLGA-E) are fabricated by coaxial electrospinning. HA/PLGA-E core/shell fiber matrices are composed of randomly-oriented sub-micrometer fibers and have a 3D porous network structure. EGCG is uniformly dispersed in the shell and sustainedly released from the matrices in a stepwise manner by controlled diffusion and PLGA degradation over four weeks. EGCG does not adversely affect the thermomechanical properties of HA/PLGA-E matrices. The number of human dermal fibroblasts attached on HA/PLGA-E matrices is appreciably higher than that on HA/PLGA counterparts, while their proliferation is steadily retained on HA/PLGA-E matrices. The wound healing activity of HA/PLGA-E matrices is evaluated in streptozotocin-induced diabetic rats. After two weeks of surgical treatment, the wound areas are significantly reduced by the coverage with HA/PLGA-E matrices resulting from enhanced re-epithelialization/neovascularization and increased collagen deposition, compared with no treatment or HA/PLGA. In conclusion, the HA/PLGA-E matrices can be potentially exploited to craft strategies for the acceleration of diabetic wound healing and skin regeneration.