Taro Uyama

Novel Cardiac Precursor‐Like Cells from Human Menstrual Blood‐Derived Mesenchymal Cells

Stem cell therapy can help repair damaged heart tissue. Yet many of the suitable cells currently identified for human use are difficult to obtain and involve invasive procedures. In our search for novel stem cells with a higher cardiomyogenic potential than those available from bone marrow, we discovered that potent cardiac precursor‐like cells can be harvested from human menstrual blood. This represents a new, noninvasive, and potent source of cardiac stem cell therapeutic material. We demonstrate that menstrual blood‐derived mesenchymal cells (MMCs) began beating spontaneously after induction, exhibiting cardiomyocyte‐specific action potentials. Cardiac troponin‐I‐positive cardiomyocytes accounted for 27%–32% of the MMCs in vitro. The MMCs proliferated, on average, 28 generations without affecting cardiomyogenic transdifferentiation ability, and expressed mRNA of GATA‐4 before cardiomyogenic induction. Hypothesizing that the majority of cardiomyogenic cells in MMCs originated from detached uterine endometrial glands, we established monoclonal endometrial gland‐derived mesenchymal cells (EMCs), 76%–97% of which transdifferentiated into cardiac cells in vitro. Both EMCs and MMCs were positive for CD29, CD105 and negative for CD34, CD45. EMCs engrafted onto a recipients heart using a novel 3‐dimensional EMC cell sheet manipulation transdifferentiated into cardiac tissue layer in vivo. Transplanted MMCs also significantly restored impaired cardiac function, decreasing the myocardial infarction (MI) area in the nude rat model, with tissue of MMC‐derived cardiomyocytes observed in the MI area in vivo. Thus, MMCs appear to be a potential novel, easily accessible source of material for cardiac stem cell‐based therapy.

The Significant Cardiomyogenic Potential of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells In Vitro

We tested the cardiomyogenic potential of the human umbilical cord blood‐derived mesenchymal stem cells (UCBMSCs). Both the number and function of stem cells may be depressed in senile patients with severe coronary risk factors. Therefore, stem cells obtained from such patients may not function well. For this reason, UCBMSCs are potentially a new cell source for stem cell‐based therapy, since such cells can be obtained from younger populations and are being routinely utilized for clinical patients. The human UCBMSCs (5 × 103 per cm2) were cocultured with fetal murine cardiomyocytes ([CM] 1 × 105 per cm2). On day 5 of cocultivation, approximately half of the green fluorescent protein (GFP)‐labeled UCBMSCs contracted rhythmically and synchronously, suggesting the presence of electrical communication between the UCBMSCs. The fractional shortening of the contracted UCBMSCs was 6.5% ± 0.7% (n = 20). The UCBMSC‐derived cardiomyocytes stained positive for cardiac troponin‐I (clear striation +) and connexin 43 (diffuse dot‐like staining at the margin of the cell) by the immunocytochemical method. Cardiac troponin‐I positive cardiomyocytes accounted for 45% ± 3% of GFP‐labeled UCBMSCs. The cardiomyocyte‐specific long action potential duration (186 ± 12 milliseconds) was recorded with a glass microelectrode from the GFP‐labeled UCBMSCs. CM were observed in UCBMSCs, which were cocultivated in the same dish with mouse cardiomyocytes separated by a collagen membrane. Cell fusion, therefore, was not a major cause of CM in the UCBMSCs. Approximately half of the human UCBMSCs were successfully transdifferentiated into cardiomyocytes in vitro. UCBMSCs can be a promising cellular source for cardiac stem cell‐based therapy.

Molecular biological approaches to the accumulation and reduction of vanadium by ascidians

Abstract About 90 years ago, Henze discovered high levels of vanadium in the blood (coelomic) cells of an ascidian collected from the Bay of Naples. His discovery attracted the interdisciplinary attention of chemists, physiologists, and biochemists. Two decades ago, we quantified the vanadium levels in several ascidian tissues definitively using neutron-activation analysis and revealed that some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, corresponding to about 107 times that found in seawater. Vanadium accumulated is reduced to the +3 oxidation state via the +4 oxidation state and stored in vacuoles of vanadocytes (vanadium-containing blood cells) where high levels of protons and sulfate are also contained. To investigate this unusual phenomenon, we isolated several proteins and genes that are expressed in vanadocytes. To date, three types of vanadium-binding protein, designated as Vanabins, have been isolated, with molecular masses of 12.5, 15, and 16 kDa, along with the cDNAs encoding these proteins. In addition, four types of enzyme related to the pentose phosphate pathway that produces NADPH were revealed to be located in vanadocytes. The pentose phosphate pathway participates in the reduction of V(V) to V(IV). The cDNA for each of the vacuolar-type H+ATPase (VATPase) A, B, C, and D subunits, which are located on the vacuolar membranes of vanadocytes, has been isolated and analyzed. VATPase generates a proton-motive force, and is thought to provide the energy for vanadium accumulation. To clarify the entire mechanism involved in the accumulation and reduction, much more genes and proteins expressed in the blood cells need to be systematically identified. Thus, we have performed an expressed sequence tag (EST) analysis of blood cells and have established the functional assay system to elucidate the functions of genes and proteins obtained from ascidian blood cells.

Coronary vein infusion of multipotent stromal cells from bone marrow preserves cardiac function in swine ischemic cardiomyopathy via enhanced neovascularization

Few reports have examined the effects of adult bone marrow multipotent stromal cells (MSCs) on large animals, and no useful method has been established for MSC implantation. In this study, we investigate the effects of MSC infusion from the coronary vein in a swine model of chronic myocardial infarction (MI). MI was induced in domestic swine by placing beads in the left coronary artery. Bone marrow cells were aspirated and then cultured to isolate the MSCs. At 4 weeks after MI, MSCs labeled with dye (n=8) or vehicle (n=5) were infused retrogradely from the anterior interventricular vein without any complications. Left ventriculography (LVG) was performed just before and at 4 weeks after cell infusion. The ejection fraction (EF) assessed by LVG significantly decreased from baseline up to a follow-up at 4 weeks in the control group (P<0.05), whereas the cardiac function was preserved in the MSC group. The difference in the EF between baseline and follow-up was significantly greater in the MSC group than in the control group (P<0.05). The MSC administration significantly promoted neovascularization in the border areas compared with the controls (P<0.0005), though it had no affect on cardiac fibrosis. A few MSCs expressed von Willebrand factor in a differentiation assay, but none of them expressed troponin T. In quantitative gene expression analysis, basic fibroblast growth factor and vascular endothelial growth factor (VEGF) levels were significantly higher in the MSC-treated hearts than in the controls (P<0.05, respectively). Immunohistochemical staining revealed VEGF production in the engrafted MSCs. In vitro experiment demonstrated that MSCs significantly stimulated endothelial capillary network formation compared with the VEGF protein (P<0.0001). MSC infusion via the coronary vein prevented the progression of cardiac dysfunction in chronic MI. This favorable effect appeared to derive not from cell differentiation, but from enhanced neovascularization by angiogenic factors secreted from the MSCs.

Mesenchymal to embryonic incomplete transition of human cells by chimeric OCT4/3 (POU5F1) with physiological co-activator EWS

POU5F1 (more commonly known as OCT4/3) is one of the stem cell markers, and affects direction of differentiation in embryonic stem cells. To investigate whether cells of mesenchymal origin acquire embryonic phenotypes, we generated human cells of mesodermal origin with overexpression of the chimeric OCT4/3 gene with physiological co-activator EWS (product of the EWSR1 gene), which is driven by the potent EWS promoter by translocation. The cells expressed embryonic stem cell genes such as NANOG, lost mesenchymal phenotypes, and exhibited embryonal stem cell-like alveolar structures when implanted into the subcutaneous tissue of immunodeficient mice. Hierarchical analysis by microchip analysis and cell surface analysis revealed that the cells are subcategorized into the group of human embryonic stem cells and embryonal carcinoma cells. These results imply that cells of mesenchymal origin can be traced back to cells of embryonic phenotype by the OCT4/3 gene in collaboration with the potent cis-regulatory element and the fused co-activator. The cells generated in this study with overexpression of chimeric OCT4/3 provide us with insight into cell plasticity involving OCT4/3 that is essential for embryonic cell maintenance, and the complexity required for changing cellular identity.

Scanning X-ray Microscopy of Living and Freeze-Dried Blood Cells in Two Vanadium-Rich Ascidian Species, Phallusia mammillata and Ascidia sydneiensis samea

Abstract Some ascidians (sea squirts) accumulate the transitional metal vanadium in their blood cells at concentrations of up to 350 mM, about 107 times its concentration found in seawater. There are approximately 10 different types of blood cell in ascidians. The identity of the true vanadium-containing blood cell (vanadocyte) is controversial and little is known about the subcellular distribution of vanadium. A scanning x-ray microscope installed at the ID21 beamline of the European Synchrotron Radiation Facility to visualize vanadium in ascidian blood cells. Without fixation, freezing or staining realized the visualization of vanadium localized in living signet ring cells and vacuolated amoebocytes of two vanadium-rich ascidian species, Phallusia mammillata and Ascidia sydneiensis samea. A combination of transmission and fluorescence images of signet ring cells suggested that in both species the vacuoles contain vanadium.

Direct reduction from vanadium(V) to vanadium(IV) by NADPH in the presence of EDTA. A consideration of the reduction and accumulation of vanadium in the ascidian blood cells

Abstract Vanadium(V) species are reduced to vanadium(IV) directly by NADPH in the presence of EDTA. At neutral pH, vanadium(V)–EDTA complex, [VO 2 (EDTA)] 3− (20 mM) was almost completely reduced to [VO(EDTA)] 2− directly by NADPH (200 mM) after 15 h under aerobic and anaerobic conditions. The reduction is markedly accelerated at low pH. At pH 3, the vanadium(V) complex (20 mM) was reduced by NADPH (20 mM) within 1 h. Oxygen had little effect, but inhibited the reduction to some extent at low pH. At low pH, simple vanadium(V) species were also partly reduced without the assistance of EDTA, resulting in the formation of mixed-valence (V(IV)–V(V)) polynuclear species. The accumulation and reduction of vanadium in the vanadocytes of ascidians are discussed based on the present results.

Exclusive Expression of Transketolase in the Vanadocytes of the Vanadium-Rich Ascidian, Ascidia sydneiensis samea

Ascidians, especially those belonging to the Ascidiidae, are known to accumulate extremely high levels of vanadium in vanadocytes, one type of blood (coelomic) cell. Vanadium, which exists in the +5 oxidation state in seawater, is accumulated in the vanadocytes and reduced to the +3 oxidation state. We have been trying to characterize all of the polypeptides specific to vanadocytes and to specify the proteins that participate in the accumulation and reduction of vanadium. To date, we have localized three enzymes in vanadocytes: 6-phosphogluconate dehydrogenase (6-PGDH: EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6PDH: EC 1.1.1.49), and glycogen phosphorylase (GP: EC 2.4.1.1), all of which are involved in the pentose phosphate pathway. In the current study, we cloned a cDNA for transketolase, an essential and rate-limiting enzyme in the non-oxidative part of the pentose phosphate pathway, from vanadocytes. The cDNA encoded a protein of 624 amino acids, which showed 61.8% identity to the human adult-type transketolase gene product. By immunocytochemistry and immunoblot analyses, the transketolase was revealed to be a protein that was expressed only in vanadocytes and not in any of the more than ten other types of blood cell. This finding, taken together with the localized expression of the other three enzymes, strongly supports the hypothesis that the pentose phosphate pathway functions exclusively in vanadocytes.

Hepatic differentiation of human bone marrow-derived UE7T-13 cells: Effects of cytokines and CCN family gene expression.

Aim:  Bone marrow‐derived mesenchymal stem cells (MSC) are expected to be an excellent source of cells for transplantation. We aimed to study the culture conditions and involved genes to differentiate MSC into hepatocytes.

Glucose-6-Phosphate Dehydrogenase in the Pentose Phosphate Pathway Is Localized in Vanadocytes of the Vanadium-Rich Ascidian, Ascidia sydneiensis samea.

Abstract Ascidians are sessile marine animals known to accumulate high levels of vanadium selectively in vanadium-containing blood cells (vanadocytes). Almost all the vanadium accumulated in the vacuoles of vanadocytes is reduced to the +3 oxidation state via the +4 oxidation state, although vanadium is dissolved in the +5 oxidation state in sea water. Some of the reducing agents that participate in the reduction have been proposed. By chemical study, vanadium in the +5 oxidation state was reported to be reduced to the +4 oxidation state in the presence of NADPH. The present study revealed the existence of glucose-6-phosphodehydrogenase (G6PDH), the first enzyme to produce NADPH in the pentose phosphate pathway, in vanadocytes of a vanadium-rich ascidian. The results suggested that G6PDH conjugates the reduction of vanadium from the +5 through to the +4 oxidation state in vanadocytes of ascidians.