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Biomedical subjects

Satoshi Gojo

Publications and source records attributed to Satoshi Gojo.

At least 19 recordsLinked to original sources
PubMed2006

Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 undergo the stochastic cardiomyogenic fate and behave like transient amplifying cells.

Bone marrow-derived stromal cells can give rise to cardiomyocytes as well as adipocytes, osteocytes, and chondrocytes in vitro. The existence of mesenchymal stem cells has been proposed, but it remains unclear if a single-cell-derived stem cell stochastically commits toward a cardiac lineage. By single-cell marking, we performed a follow-up study of individual cells during the differentiation of 9-15c mesenchymal stromal cells derived from bone marrow cells. Three types of cells, i.e., cardiac myoblasts, cardiac progenitors and multipotent stem cells were differentiated from a single cell, implying that cardiomyocytes are generated stochastically from a single-cell-derived stem cell. We also demonstrated that overexpression of Csx/Nkx2.5 and GATA4, precardiac mesodermal transcription factors, enhanced cardiomyogenic differentiation of 9-15c cells, and the frequency of cardiomyogenic differentiation was increased by co-culturing with fetal cardiomyocytes. Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 behaved like cardiac transient amplifying cells, and still retained their plasticity in vivo.

Animals↗
PubMed2006

Increased mobilization of c-kit+ Sca-1+ Lin- (KSL) cells and colony-forming units in spleen (CFU-S) following de novo formation of a stem cell niche depends on dynamic, but not stable, membranous ossification.

Stem cells are thought to inhabit in a unique microenvironment, known as "niche," in which they undergo asymmetric cell divisions that results in reproducing both stem cells and progenies to maintain various tissues throughout life. The cells of osteoblastic lineage have been identified as a key participant in regulating the number of hematopoietic stem cells (HSCs). HSCs receive their regulatory messages from the microenvironment in the bone marrow. This would account for a reason why the localization of hematopoiesis is usually restricted in the bone marrow. To clarify the above possibility we employed a cell implantation-based strategy with a unique osteoblast cell line (KUSA-A1) derived from a C3H/He mouse. The implantation of KUSA-A 1 cells resulted in the generation of ectopic bones in the subcutaneous tissues of the athymic BALB/c nu/nu mice. Subsequently the mice obtained a greater amount of the bone marrow than normal mice, and they showed an increased number of HSCs. These results indicate that the newly generated osteoblasts-derived ectopic bones are responsible for the increase in the number of the HSC population. Furthermore, the increased number of HSCs directly correlates with both the magnitude of dynamic osteogenic process and the size of the newly generated bone or "niche."

Animals↗
PubMed2005

Sonoporation using microbubble BR14 promotes pDNA/siRNA transduction to murine heart.

Naked plasmid DNA (pDNA) and short interfering RNA (siRNA) duplexes were transduced into adult murine heart by means of sonoporation using the third-generation microbubble, BR14. Plasmid DNAs carrying luciferase, beta-galactosidase (beta-gal), or enhanced green fluorescent protein (EGFP) reporter genes were mixed with BR14 and injected percutaneously into the left ventricular (LV) cavity of C57BL/6 mice while exposed to transthoracic ultrasound at 1MHz for 60s. Sonoporation at an output intensity of 2.0W/cm(2) and a 50% pulse duty ratio resulted in the highest luciferase expression in the heart. Histological examinations revealed significant expression of the beta-gal and EGFP reporters in the subendocardial myocardium of LV. Intraventricular co-injection of siRNA-GFP and BR14 with concomitant ultrasonic exposure resulted in substantial reduction in EGFP expression in the coronary artery in EGFP transgenic mice. The present method may be applicable to gain-of-function and loss-of-function genetic engineering in vivo of adult murine heart.

Animals↗
PubMed2004

Sequence-specific gene silencing in murine muscle induced by electroporation-mediated transfer of short interfering RNA.

BACKGROUND: Post-genomic biomedical research requires efficient techniques for functional analyses of poorly characterized genes in living organisms. Sequence-specific gene silencing in mammalian organs may provide valuable information on the physiological and pathological roles of predicted genes in mammalian systems. Here, we attempted targeted gene knockdown in vivo in murine skeletal muscle through the electroporation-mediated transfer of short interfering RNA (siRNA). METHODS: siRNA duplexes corresponding to the firefly luciferase (Luc), green fluorescent protein (GFP), or glyceraldehyde-3-phosphate dehydrogenase (GAPD) genes were delivered by electroporation into the tibial muscle of normal or enhanced GFP (EGFP) transgenic mice. Plasmid vectors carrying the Luc, hRluc or beta-galactosidase (beta-gal) reporter genes were also delivered. The Luc and hRluc activities in the muscle lysates were assayed. The EGFP and GAPD expression was detected by fluorescence microscopic observation and RT-PCR, respectively. RESULTS: When Luc-specific siRNA was co-delivered with the Luc expression vector into the tibial muscle, the reporter gene expression was markedly suppressed (less than 1% of the control level) for 5 days. As little as 0.05 micro g of siRNA almost completely blocked the reporter gene expression from 10 micro g of the plasmid. To examine whether siRNA can also suppress expression of an endogenous gene, transgenic mice carrying the EGFP gene received intramuscular transfection of a mixture of beta-gal plasmid and GFP-specific siRNA. beta-Gal-positive cells failed to express detectable levels of EGFP, while EGFP expression was not inhibited in control mice that received nonspecific siRNA. Expression of GAPD was also suppressed by the specific siRNA. CONCLUSIONS: The present system may provide a useful means of phenotypic analysis of genetic information in mammalian organs for basic research as well as therapeutic molecular targeting in the post-genomic era.

Animals↗
PubMed2004

Can the life span of human marrow stromal cells be prolonged by bmi-1, E6, E7, and/or telomerase without affecting cardiomyogenic differentiation?

BACKGROUND: Cell transplantation has recently been challenged to improve cardiac function of severe heart failure. Human mesenchymal stem cells (hMSCs) are multipotent cells that can be isolated from adult marrow stroma, but because of their limited life span, it is difficult to study them further. To overcome this problem, we attempted to prolong the life span of hMSCs and investigate whether the hMSCs modified with cell-cycle-associated genes can differentiate into cardiomyocytes in vitro. METHODS: We attempted to prolong the life span of hMSCs by infecting retrovirus encoding bmi-1, human papillomavirus E6 and E7, and/or human telomerase reverse transcriptase genes. To determine whether the hMSCs with an extended life span could differentiate into cardiomyocytes, 5-azacytidine-treated hMSCs were co-cultured with fetal cardiomyocytes in vitro. RESULT: The established hMSCs proliferated over 150 population doublings. On day 3 of co-cultivation, the hMSCs became elongated, like myotubes, began spontaneously beating, and acquired automaticity. Their rhythm clearly differed from that of the surrounding fetal mouse cardiomyocytes. The number of beating cardiomyocytes increased until 3 weeks. hMSCs clearly exhibited differentiated cardiomyocyte phenotypes in vitro as revealed by immunocytochemistry, RT-PCR, and action potential recording. CONCLUSIONS: The life span of hMSCs was prolonged without interfering with cardiomyogenic differentiation. hMSCs with an extended life span can be used to produce a good experimental model of cardiac cell transplantation and may serve as a highly useful cell source for cardiomyocytic transplantation.

Action Potentials↗
PubMed2004

[Aortic root replacement using an allograft for active infective endocarditis with periannular abscess: single center experience].

OBJECTIVES: Results of aortic root replacement using an allograft for active infective endocarditis in the aortic position and periannular abscess were studied. METHODS: Aortic root replacement using a cryopreserved aortic allograft was performed 13 times in 12 patients (9 men, 3 women, mean age 52.8 years) at Saitama Medical School. Allografts were obtained from the Tokyo University Tissue Bank. Infection affected the prosthetic valve in 6 patients and the native valve in 7. Causative organisms were Staphylococci in 7 patients, Streptococci in 2, and unknown in 4. New York Heart Association functional class was II in 2 patients, III in 3, and IV in 8. Six patients required inotropic support. Four were mechanically ventilated. Two were in a state of profound shock. Four had multiple organ failure. Surgery was conducted under hypothermic cardiopulmonary bypass and cardioplegic arrest. Allograft was sewn into place by monofilament continuous sutures. Carrel patch technique was used for coronary artery reconstruction. Five patients underwent concomitant procedures (replacement of the ascending aorta in 2 patients, coronary artery bypass grafting in 1, mitral valve repair in 1, pulmonary artery reconstruction in 1). RESULTS: Duration of surgery, cardiopulmonary bypass, and aortic clamping were 507 +/- 154, 307 +/- 111, and 189 +/- 49 min, respectively. Two patients required intraaortic balloon pumping, and one required percutaneous cardiopulmonary support. Two patients in a state of shock died(hospital mortality: 15.4%). Duration of mechanical ventilation was 2.9 +/- 3.7 days. Intensive care unit stay was 4.6 +/- 5.5 days, and hospital stay was 61 +/- 26 days. One patient died of cancer 18 months later. One patient required reoperation 11 months later. Including operative death cases, cumulative survival at 2 years was 66.5% and freedom from cardiac events was 72.7%. CONCLUSIONS: Aortic root replacement with an allograft yielded acceptable clinical results.

Abscess↗
PubMed2003

Intravascular insulin gene delivery as potential therapeutic intervention in diabetes mellitus.

We assessed therapeutic potential of intravascular insulin gene delivery in a diabetic murine model. The rat proinsulin-1 gene cDNA engineered to harbor furin consensus cleavage sequences was inserted into EBV-based plasmid vectors that contained CAG promoter or multimerized rat insulin promoter (RIP). Normal or streptozotocin (STZ)-induced diabetic mice were given an injection of the plasmids via the tail vein under high pressure. Transfection of the CAG-proinsulin construct markedly improved hyperglycemia of diabetic mice, accompanied by a considerable increase in serum insulin concentrations. Although the RIP-plasmid failed to reduce fasting blood glucose, the glucose tolerance test and RT-PCR analysis revealed that insulin production was regulated in the liver in a blood glucose level-dependent manner. The present results suggest a potential therapeutic means of controlling DM.

Animals↗
PubMed2003

Redifferentiation of dedifferentiated chondrocytes and chondrogenesis of human bone marrow stromal cells via chondrosphere formation with expression profiling by large-scale cDNA analysis.

Characterization of dedifferentiated chondrocytes (DECs) and mesenchymal stem cells capable of differentiating into chondrocytes is of biological and clinical interest. We isolated DECs and bone marrow stromal cells (BMSCs), H4-1 and H3-4, and demonstrated that the cells started to produce extracellular matrices, such as type II collagen and aggrecan, at an early stage of chondrosphere formation. Furthermore, cDNA sequencing of cDNA libraries constricted by the oligocapping method was performed to analyze difference in mRNA expression profiling between DECs and marrow stromal cells. Upon redifferentiation of DECs, cartilage-related extracellular matrix genes, such as those encoding leucine-rich small proteoglycans, cartilage oligomeric matrix protein, and chitinase 3-like 1 (cartilage glycoprotein-39), were highly expressed. Growth factors such as FGF7 and CTGF were detected at a high frequency in the growth stage of monolayer stromal cultures. By combining the expression profile and flow cytometry, we demonstrated that isolated stromal cells, defined by CD34(-), c-kit(-), and CD140alpha(- or low), have chondrogenic potential. The newly established human mesenchymal cells with expression profiling provide a powerful model for a study of chondrogenic differentiation and further understanding of cartilage regeneration in the means of redifferentiated DECs and BMSCs.

Bone Marrow Cells↗
PubMed2003

In vivo cardiovasculogenesis by direct injection of isolated adult mesenchymal stem cells.

The characterization of mesenchymal stem cells (MSCs) is of biological and clinical interest. We demonstrate that isolated MSCs, defined by CD34(low) c-kit(+) CD140a(+) Sca-1(high), are able to differentiate into cardiomyocytes, endothelial cells, and pericytes or smooth muscle cells by direct injection into adult heart. In skeletal muscle and lung, they also contributed to formation of the vasculature. MSCs did not transform into malignant cells or form excess extracellular matrix. This study suggests that MSCs may supply an ideal donor source of cardiovascular cells in patients with cardiopulmonary diseases.

Animals↗
PubMed2003

Xenogeneic thymus transplantation in a pig-to-baboon model.

BACKGROUND: We have tested whether fetal porcine thymic tissue transplantation can lead to tolerance across a discordant (pig-to-baboon) xenogeneic barrier. METHODS: Six baboons underwent a conditioning regimen with thymectomy, splenectomy, and anti-monkey CD3 antibody conjugated to a diphtheria toxin binding site mutant (FN18-CRM9). Porcine fetal or neonatal thymic tissue was transplanted into three baboons. Three control baboons received either no transplanted pig tissue (n=1) or adult pig lymph node (n=2). Cellular responses and skin xenografts were used to test for tolerance. RESULTS: After T-cell depletion and thymic transplantation, recovery of thymus-dependent naïve-type CD4 cells (CD4/CD45RA ) and in vitro xenogeneic hyporesponsiveness were observed. No sensitization of alpha-galactosyl antibody responses was observed. The thymic grafts survived up to 48 days. Porcine skin xenografts were performed in two of these animals with survival of 22 and 24 days. Only two of these animals were completely T-cell depleted, and both failed to recover thymus-dependent T cells (CD4/CD45RA ). In one animal, general in vitro hyporesponsiveness was observed, with subsequent death from infection. The second animal demonstrated delayed recovery of T cells and prolonged general hyporesponsiveness in vitro. Neither animal demonstrated prolongation of porcine skin grafts compared with allografts (both rejected by day 13). CONCLUSIONS: Porcine thymic tissue is able to induce xenogeneic hyporesponsiveness. More efficient thymic engraftment may allow this approach to induce xenograft tolerance.

Animals↗
PubMed2003

Use of isolated mature osteoblasts in abundance acts as desired-shaped bone regeneration in combination with a modified poly-DL-lactic-co-glycolic acid (PLGA)-collagen sponge.

Controlled regeneration of bone or cartilage has recently begun to facilitate a host of novel clinical treatments. An osteoblast line, which we isolated is able to form new bone matrix in vivo within 2 days and exhibits a mature osteoblast phenotype both in vitro and in vivo. Using these cells, we show that cuboidal bones can be generated into a predesigned shaped-bone with high-density bone trabeculae when used in combination with a modified poly-DL-lactic-co-glycolic acid (PLGA)-collagen sponge. PLGA coated with collagen gel serves as a good scaffold for osteoblasts. These results indicate that mature osteoblasts, in combination with a scaffold such as PLGA-collagen sponge, show promise for use in a custom-shaped bone regeneration tool for both basic research into osteogenesis and for development of therapeutic applications.

Absorbable Implants↗
PubMed2003

Electrochemo-gene therapy of cancer: intratumoral delivery of interleukin-12 gene and bleomycin synergistically induced therapeutic immunity and suppressed subcutaneous and metastatic melanomas in mice.

To treat established melanoma in mice, intratumoral transfer of bleomycin and/or an interleukin (IL)-12 expression vector was performed by means of electroporation. Although either bleomycin alone or the IL12 gene alone significantly suppressed the subcutaneous tumors, the combination therapy drastically improved the therapeutic outcome. Three of eight mice (37.5%) that received both bleomycin and the IL12 gene showed complete remission of the preestablished tumors and rejected subsequent rechallenge with the tumor cells. We also examined whether electrochemo-gene therapy for subcutaneous tumor mass induced suppression of pulmonary metastasis that had been established by intravenous inoculation of the melanoma cells. Although metastatic foci were significantly reduced in number in groups that were given IL12 gene alone or bleomycin plus IL12 gene, it was only the combination therapy that significantly prolonged the mean survival period of the tumor-bearing animals. Natural killer (NK) and cytotoxic T lymphocyte cytolytic activities were markedly enhanced in the mice that received the chemo-gene therapy, while IL12 gene therapy alone partially elevated the NK cytotoxicity. The present study suggests that the electroporation-mediated delivery of the IL12 gene and bleomycin synergistically elicits innate and adaptive anti-melanoma immune responses, resulting in marked suppression of the treated tumors as well as bystander metastatic lesions.

Animals↗
PubMed2003

Interleukin (IL)-21 and IL-15 genetic transfer synergistically augments therapeutic antitumor immunity and promotes regression of metastatic lymphoma.

IL-21 supports proliferation of mature T and B cells and facilitates expansion and maturation of natural killer (NK) cells in synergy with IL-15. However, the biological implications of IL-21 in vivo have not been fully elucidated. IL-21 and IL-15 expression plasmids were intravenously injected under high pressure into the tail veins of mice, which were subsequently challenged by an intravenous injection of RLmale1 lymphoma cells. The IL15 gene transfection significantly reduced the numbers of metastatic tumor foci in the liver. In contrast, when IL21 and IL15 genes were cotransfected, complete regression was achieved in 80% of the mice. The cytokine gene therapy was also performed in mice that had been intravenously inoculated with the tumor cells. Forty percent of mice that received a single injection of a mixture of cytokine genes successfully rejected the preestablished metastatic lymphoma and showed tumor-free survival for more than 300 days. IL-21 significantly elevated the cytotoxic T lymphocyte activity in the spleens of tumor-inoculated mice, while the two cytokines augmented NK killing activity in a synergistic manner. These results strongly suggest that the codelivery of IL-21 and IL-15 elicits powerful antitumor immune responses, resulting in marked therapeutic efficacy against metastatic tumors.

Animals↗
PubMed2003

Plasticity of mesenchymal stem cells--regenerative medicine for diseased hearts.

The phenomenon of regeneration is of growing interest to medical researchers. Until recently this was an area in which research in flatworms and newts predominated, but there is now a proliferation of research concerning regeneration in virtually all of the organs, not only the heart. One of the object is restoration of function to a failing heart through cell transplantation, and there have been many reports seeking donor sources of somatic stem cells, i.e.: stem cells in marrow or skeletal muscle and ES cells, beginning with those in embryonic myocardial cell transplant experiments. In particular, reports of mesenchymal stem cell differentiation into nerve cell, myocardial cell, skeletal muscle cell, and vascular endothelial cell series have drawn attention to cell plasticity, and clinical applications are awaited.

Animals↗
PubMed2003

Combined coronary artery bypass grafting and abdominal aortic aneurysm repair.

The purpose of this paper was to assess the results and feasibility of simultaneous coronary artery bypass grafting and abdominal aortic aneurysm repair. Twenty nine patients with a mean age of 65 years underwent simultaneous coronary artery bypass grafting and abdominal aortic aneurysm repair between June 1990 and March 2002. All patients had significant coronary artery disease and were considered as indicated for coronary artery bypass grafting. This was performed first in 28 patients and simultaneously with abdominal aortic aneurysm repair in one, with a mean number of grafts of 2.5, a mean aortic cross-clamp time of 40 minutes, and a mean bypass time of 115 minutes. Eight straight and 21 bifurcated grafts were employed. The total operating time averaged 400 minutes. The median postoperative hospital stay was 18 days. One patient died of stroke and mediastinitis, for a mortality rate of 3.5%. This experience suggests that combined coronary artery bypass grafting and abdominal aortic aneurysm repair is both safe and effective.

Adult↗
PubMed2003

Noncardioplegic surgery for ischemic mitral regurgitation.

Twenty-seven consecutive patients underwent surgery for ischemic mitral regurgitation (MR): papillary muscle rupture (1), papillary muscle dysfunction (11) and annular dilatation (15). The grade of MR was moderate or severe, and the ejection fraction (EF) was less than 30% in 8 patients (mean, 43%). Three cases were reoperation and 3 were emergencies. Under ventricular fibrillation (VF) and intermittent aortic cross-clamping at moderate hypothermia, coronary artery bypass grafting (CABG) was performed first, followed by the mitral procedure through a right-sided left atriotomy (repair 21, replacement 6) performed under VF with the heart perfused through the native coronary arteries and CABG grafts. Concomitant procedures were CABG (23), Dor's procedure (5), and tricuspid annuloplasty (3). In one reoperative case with cardiogenic shock, CABG was impossible because of dense adhesions and the patient died just after surgery (hospital mortality, 3.7%). Five patients required intra-aortic balloon pump (IABP) support intraoperatively, but none required prolonged (> or =7 days) inotropic support or IABP use, although the serum concentrations of creatine kinase and its myocardial fraction were elevated remarkably. Other morbid events were refractory ventricular arrhythmia in one case and stroke in another. Median duration of mechanical ventilation and intensive care unit stay was 8 h and 3 days, respectively. Mean EF at hospital discharge was 48%. The extended period of VF was not associated with unfavorable clinical outcomes. Noncardioplegic surgery for ischemic MR was carried out with acceptable mortality and morbidity, and can be a good alternative, especially when clamping the aorta is undesirable.

Aged↗
PubMed2003

Simultaneous abdominal aortic aneurysm repair during the on-pump coronary artery bypass grafting.

A case of simultaneous coronary artery bypass grafting (CABG) and abdominal aortic aneurysm (AAA) repair on cardiopulmonary bypass (CPB) is reported. A 74-year-old man was diagnosed with left main coronary disease and infrarenal AAA. Triple CABG and infrarenal AAA repair were performed simultaneously, by different surgeons, on CPB. The duration of CPB, aortic clamp time, and total operation time was 81 min, 33 min, and 245 min, respectively. The patient was extubated three hours after ICU admission and the postoperative course was uneventful. This method is useful for reduction of operation time, for blood salvage, and for adjustment of preload and afterload of the vulnerable heart during AAA repair.

Aged↗