Takaaki Kanemaru

Pigment cell organization in the hypodermis of zebrafish

Zebrafish have a characteristic horizontal‐stripe pigment pattern made by a specific distribution of three types of pigment cells: melanophores, xanthophores, and iridophores. This pattern is a valuable model to investigate how the spatial patterns form during animal development. Although recent findings suggest that the interactions among the pigment cells play a key role, the particular details of these interactions have not yet been clarified. In this report, we performed transmission electron microscopic study to show the distribution, conformation, and how the cells contact with each other in the hypodermis. We found that the pigment cells form complex but ordered, layered structures in both stripe and interstripe regions. The order of the layered structures is kept strictly all through the hypodermal regions. Our study will provide basic information to investigate the mechanism of pigment pattern formation in zebrafish. Developmental Dynamics 227:497–503, 2003.

Hydroxyapatite‐coating on titanium arc sprayed titanium implants

We developed a new titanium spray technique using an inert gas shielded arc spray (titanium arc spray). Hydroxyapatite (HA)-coating can be applied to the implant without any surface pore obstruction after the rough surface is made by this technique. Scanning electron microscopy (SEM) of various porous implant surfaces after HA-coating revealed that the bead and fiber metal-coated implants had either a pore obstruction or an uneven HA-coating. On the other hand, the titanium arc sprayed implant demonstrated an even HA-coating all the way to the bottom of the surface pore. In the first set of animal experiments (Exp. 1), the interfacial shear strength to bone of four kinds of cylindrical Ti-6A1-4V (Ti) implants were compared using a canine transcortical push-out model 4 and 12 weeks after implantation. The implant surfaces were roughened by titanium arc spray (group A-C) and sand blasting (group D) to four different degrees (roughness average, Ra = group A: 56.1, B: 44.9, C: 28.3, D: 3.7 microns). The interfacial shear strength increased in a surface roughness-dependent manner at both time periods. However, the roughest implants (group A) showed some failed regions in the sprayed layers after pushout test. In the second set of animal experiments (Exp. 2), four kinds of Ti implants; HA-coated smooth Ti (sHA) with Ra of 3.4 microns, bead-coated Ti (Beads), titanium arc sprayed Ti (Ti-spray) with Ra of 38.1 microns and HA-coated Ti-spray (HA + Ti-spray) with Ra of 28.3 microns were compared using the same model as that in Exp. 1. The interfacial shear strength of HA + Ti-spray was significantly greater than that of sHA and Beads at both time periods, and that of Ti-spray at 4 weeks. Although a histological examination revealed that HA-coating enhanced bone ingrowth, sHA showed the lowest shear strength at both time periods. SEM after pushout test showed that sHA consistently demonstrated some regional failure at the HA-implant substrate interface. HA + Ti-spray had many failed regions either at the HA-bone interface or within the bone tissue rather than at the HA-implant substrate interface. These results suggested that the HA-coated smooth surfaced implants had a mechanical weakness at the HA-substrate interface. Therefore, HA should be coated on the rough surfaced implants to avoid a detachment of the HA-coating layer from the substrate and thus obtain a mechanical anchoring strength to bone. HA-coating on this new type of surface morphology may thus lead to a solution to the problems of conventional HA-coated and porous-coated implants.

Human cord blood--derived cells generate insulin-producing cells in vivo.

Here we report the capacity of human cord blood (CB)–derived cells to generate insulin‐producing cells. To investigate in vivo capacity of human CB–derived cells, T cell–depleted mononuclear cells were intravenously transplanted into nonobese diabetic/severe combined immunodeficient/β2‐microglobulinnull mice within 48 hours of birth. At 1–2 months post‐transplantation, immunofluorescence staining for insulin and fluorescence in situ hybridization (FISH) analysis using a human chromosome probe indicated that human CB–derived cells generated insulin‐producing cells at a frequency of 0.65% ± 0.64% in xenogeneic hosts. Reverse transcription–polymerase chain reaction analysis confirmed the transcription of human insulin in the pancreatic tissue of the recipient mice. To clarify the mechanism underlying CB‐derived insulin‐producing cells, double FISH analysis using species‐specific probes was performed. Almost equal proportions of human chromosome+ murine chromosome− insulin+ cells and human chromosome+ murine chromosome+ insulin+ cells were present in recipient pancreatic islets. Taken together, human CB contains progenitor cells, which can generate insulin‐producing cells in recipient pancreatic tissues across a xenogeneic histocompatibility barrier by fusion‐dependent and ‐independent mechanisms.

Purified human hematopoietic stem cells contribute to the generation of cardiomyocytes through cell fusion

To obtain insights into the cardiomyogenic potential of hematopoietic tissue, we intravenously (i.v.) injected purified hematopoietic stem/progenitor cells into newborn recipients that may fully potentiate the developmental plasticity of stem cells. Transplantation of mouse bone marrow (BM) lineage antigen‐negative (Lin−) cells resulted in the generation of the cells that displayed cardiomyocyte‐specific antigenic profiles and contractile function when transplanted into syngeneic newborn recipients. To clarify the mechanism underlying the cardiomyogenic potential, green fluorescent protein (GFP)‐labeled BM Lin−ScaI+ hematopoietic progenitors were transplanted into neonatal mice constitutively expressing cyan fluorescence protein (CFP). Lambda image acquisition and linear unmixing analysis using confocal microscopy successfully separated GFP and CFP, and revealed that donor GFP+ cardiomyocytes coexpressed host‐derived CFP. We further reconstituted human hemopoietic‐ and immune systems in mice by injecting human cord blood (CB)‐derived Lin−CD34+CD38− hematopoietic stem cells (HSCs) into neonatal T cell−B cell−NK cell− immune‐deficient NOD/SCID/IL2rγnull mice. Fluoroescence in situ hybridization analysis of recipient cardiac tissues demonstrated that human and murine chromosomes were colocalized in the same cardiomyocytes, indicating that cell fusion occurred between human hematopoietic progeny and mouse cardiomyocytes. These syngeneic‐ and xenogeneic neonatal transplantations provide compelling evidence that hematopoietic stem/progenitor cells contribute to the postnatal generation of cardiomyocytes through cell fusion, not through transdifferentiation.— Ishikawa, F., Shimazu, H., Shultz, L. D., Fukata, M., Nakamura, R., Lyons, B., Shimoda, K., Shimoda, S., Kanemaru, T., Nakamura, K‐i., Ito, H., Kaji, Y., Perry, A. C. F., Harada, M. Purified human hematopoietic stem cells contribute to the generation of cardiomyocytes through cell fusion. FASEB J. 20, E11–E17 (2006)

Contribution of bone marrow-derived hematopoietic stem/progenitor cells to the generation of donor-marker⁺ cardiomyocytes in vivo.

Background Definite identification of the cell types and the mechanism relevant to cardiomyogenesis is essential for effective cardiac regenerative medicine. We aimed to identify the cell populations that can generate cardiomyocytes and to clarify whether generation of donor-marker+ cardiomyocytes requires cell fusion between BM-derived cells and recipient cardiomyocytes. Methodology/Principal Findings Purified BM stem/progenitor cells from green fluorescence protein (GFP) mice were transplanted into C57BL/6 mice or cyan fluorescence protein (CFP)-transgenic mice. Purified human hematopoietic stem cells (HSCs) from cord blood were transplanted into immune-compromised NOD/SCID/IL2rγnull mice. GFP+ cells in the cardiac tissue were analyzed for the antigenecity of a cardiomyocyte by confocal microscopy following immunofluorescence staining. GFP+ donor-derived cells, GFP+CFP+ fused cells, and CFP+ recipient-derived cells were distinguished by linear unmixing analysis. Hearts of xenogeneic recipients were evaluated for the expression of human cardiomyocyte genes by real-time quantitative polymerase chain reaction. In C57BL/6 recipients, Lin−/lowCD45+ hematopoietic cells generated greater number of GFP+ cardiomyocytes than Lin−/lowCD45− mesenchymal cells (37.0+/−23.9 vs 0.00+/−0.00 GFP+ cardiomyocytes per a recipient, P = 0.0095). The number of transplanted purified HSCs (Lin−/lowSca-1+ or Lin−Sca-1+c-Kit+ or CD34−Lin−Sca-1+c-Kit+) showed correlation to the number of GFP+ cardiomyocytes (P<0.05 in each cell fraction), and the incidence of GFP+ cardiomyocytes per injected cell dose was greatest in CD34−Lin−Sca-1+c-Kit+ recipients. Of the hematopoietic progenitors, total myeloid progenitors generated greater number of GFP+ cardiomyocytes than common lymphoid progenitors (12.8+/−10.7 vs 0.67+/−1.00 GFP+ cardiomyocytes per a recipient, P = 0.0021). In CFP recipients, all GFP+ cardiomyocytes examined coexpressed CFP. Human troponin C and myosin heavy chain 6 transcripts were detected in the cardiac tissue of some of the xenogeneic recipients. Conclusions/Significance Our results indicate that HSCs resulted in the generation of cardiomyocytes via myeloid intermediates by fusion-dependent mechanism. The use of myeloid derivatives as donor cells could potentially allow more effective cell-based therapy for cardiac repair.

The effect of hydroxyapatite coating on the bonding of bone to titanium implants in the femora of ovariectomised rats

We have studied the effect of hydroxyapatite (HA) coating in 15 ovariectomised and 15 normal rats which had had a sham procedure. Twenty-four weeks after operation, HA-coated implants were inserted into the intramedullary canal of the right femur and uncoated implants into the left femur. The prostheses were removed four weeks after implantation. Twelve specimens in each group had mechanical push-out tests. Sagittal sections of the other three were evaluated by SEM. The bone mineral density (BMD) of the dissected left tibia was measured by dual-energy x-ray absorptiometry. The difference in BMD between the control and ovariectomised tibiae was 35.01 mg/cm2 (95% CI, 26.60 to 43.42). The push-out strength of the HA-coated implants was higher than that of the uncoated implants in both groups (p < 0.0001), but the HA-coated implants of the ovariectomised group had a reduction in push-out strength of 40.3% compared with the control group (p < 0.0001). Our findings suggest that HA-coated implants may improve the fixation of a cementless total hip prosthesis but that the presence of osteoporosis may limit the magnitude of this benefit.

Leptospira idonii sp. nov., isolated from environmental water.

Strain Eri-1(T) was isolated from a water sample on the campus of Kyushu University, Fukuoka, Japan. The motility and morphology of the isolate were similar to those of members of the genus Leptospira, but the spiral structure of the isolate was sharper under dark-field microscopy. Cells were 10.6 ± 1.3 µm long and 0.2 µm in diameter, with a wavelength of 0.9 µm and an amplitude of 0.4 µm. Strain Eri-1(T) grew in Korthofs medium at both 13 and 30 °C, and also in the presence of 8-azaguanine. 16S rRNA gene-based phylogenetic analysis placed strain Eri-1(T) within the radiation of the genus Leptospira where it formed a unique lineage within the clade of the known saprophytic species of the genus Leptospira. The strain was not pathogenic to hamsters. Strain Eri-1(T) exhibited low levels (11.2-12.6 %) of similarity by DNA-DNA hybridization to the three most closely related species of the genus Leptospira. The DNA G+C content of the genome of strain Eri-1(T) was 42.5 ± 0.1 mol%. These results suggest that strain Eri-1(T) represents a novel species of the genus Leptospira, for which the name Leptospira idonii sp. nov. is proposed. The type strain is Eri-1(T) ( = DSM 26084(T) = JCM 18486(T)).

Human cord blood- and bone marrow-derived CD34+ cells regenerate gastrointestinal epithelial cells

In the present study, we aimed to clarify the capacity of human cord blood‐ and bone marrow‐ derived progenitor cells to generate gastrointestinal epithelial cells in clinical and experimental transplantation settings. First, in a clinical transplantation setting, gastrointestinal tissues derived from female pediatric or juvenile recipients of allogeneic sex‐mismatched bone marrow and cord blood transplantation were examined for the presence of donor‐derived epithelial cells. Gastrointestinal specimens of allogeneic recipients included Y chromosome+ cytokeratin+ epithelial cells at a frequency of 0.4–1.9%. To further determine the capacity of purified human progenitor cells, human cord blood‐ or bone marrow‐derived CD34+ cells were transplanted into newborn NOD/SCID/β2‐microglobulinnull mice as an experimental transplantation assay. When gastrointestinal tissues derived from recipient mice were subjected to FISH and immunofluorescence analyses, human epithelial cells were identified at a frequency of 0.24– 0.58% at 3 months posttransplantation. Finally, double FISH analyses using species‐specific probes revealed that human chromosome+ epithelial cells did not possess any murine chromosomes, indicating that donor‐derived epithelial cells were not generated only by cell fusion. On the basis of these findings, it is concluded that purified human cord blood and bone marrow CD34+ progenitor cells can generate gastrointestinal epithelial cells across allogeneic and xenogeneic histocompatibility barriers.

Dual Modulation of Airway Smooth Muscle Contraction by Th2 Cytokines via Matrix Metalloproteinase-1 Production

Altered contractility of airway smooth muscle (SM) is one of the main causes of allergic asthma, in which the predominance of Th2 over Th1 cytokines plays a central role. In the present study, we examine the effects of Th2 cytokines on airway SM contraction. Treatment with a low concentration of IL-4 (0.2 ng/ml) for 6 h augmented, whereas higher concentrations (2–20 ng/ml) inhibited, agonist-induced contractions of collagen gels containing bovine tracheal SM cells. Another Th2 cytokine (IL-13) showed an augmentation of gel contraction in the concentration range of 20–200 ng/ml. IL-4 and IL-13 increased mRNA expression and protein secretion of matrix metalloproteinase (MMP)-1, but these cytokines did not affect Ca2+-mobilizing properties and phosphorylation levels of myosin L chain in bovine tracheal SM cells. These changes were sensitive to wortmannin, an inhibitor of PI3K, but not to leflunomide, an inhibitor of STAT6. Scanning electron microscope observation revealed that collagen fibers twining around SM cells were completely dissolved in 20 ng/ml IL-4-treated gels and reorganized into basket-like structure in 20 ng/ml IL-13-treated gels. Exogenous application of high and low concentrations of MMP-1 also induced the inhibition and augmentation of gel contraction, respectively. Furthermore, nonselective MMP inhibitor galardin suppressed the effects of IL-4 and IL-13 on gel contraction, and MMP-1-targeted small-interfering RNA reversed the inhibitory effects of IL-4 on gel contraction to the augmentation. This indicates that Th2 cytokines modulate airway contraction without affecting cellular contractility but by secreting MMP-1 from the SM cells via PI3K activation and changing cell-to-matrix interactions.

Recombinant mitochondrial transcription factor A protein inhibits nuclear factor of activated T cells signaling and attenuates pathological hypertrophy of cardiac myocytes.

The overexpression of mitochondrial transcription factor A (TFAM) attenuates the decrease in mtDNA copy number after myocardial infarction, ameliorates pathological hypertrophy, and markedly improves survival. However, non-transgenic strategy to increase mtDNA for the treatment of pathological hypertrophy remains unknown. We produced recombinant human TFAM protein (rhTFAM). rhTFAM rapidly entered into mitochondria of cultured cardiac myocytes. rhTFAM increased mtDNA and abolished the activation of nuclear factor of activated T cells (NFAT), which is well known to activate pathological hypertrophy. rhTFAM attenuated subsequent morphological hypertrophy of myocytes as well. rhTFAM would be an attractive molecule in attenuating cardiac pathological hypertrophy.