Gene expression levels of ß4-galactosyltransferase 5 correlate with the tumorigenic potentials of B16-F10 mouse melanoma cells.

Our previous studies showed that mouse ß4-galactosyltransferase 5 (ß4GalT5) is a lactosylceramide (Lac-Cer) synthase, and that its gene expression increases by 2- to 3-fold upon malignant transformation of cells. In the present study, we examined whether or not the tumorigenic and metastatic potentials of B16-F10 mouse melanoma cells can be suppressed by reducing the expression of the ß4GalT5 gene. We isolated a stable clone named E5 whose ß4GalT5 gene expression level was reduced to 35% that of a control clone C1 by transfection of its antisense cDNA. Thin-layer chromatography analysis of glycosphingolipids showed that the amounts of Lac-Cer and ganglioside GM3 are significantly less in clone E5 than in clone C1. Clone C1 and E5 cells were each transplanted subcutaneously or injected intravenously into C57BL/6 mice, and the sizes of tumors and numbers of colonies formed in the lungs were determined. The average tumor size and average number of colonies formed with clone E5 were decreased to 44 and 49%, respectively, of those formed with clone C1. Furthermore, the numbers and sizes of colonies formed in the soft agarose gels, and the volumes of tumors formed in athymic mice with fibroblasts from wild type, heterozygous and homozygous ß4GalT5-knockout mouse embryos upon transformation with the polyoma virus oncogene correlated with the ß4GalT5 gene dosage. These results strongly indicate that the amounts of Lac-Cer synthesized by ß4GalT5 correlate with the tumorigenic potentials of malignantly transformed cells.

Treatment of human mesenchymal stem cells with angiotensin receptor blocker improved efficiency of cardiomyogenic transdifferentiation and improved cardiac function via angiogenesis.

To improve the modest efficacy of mesenchymal stem cell (MSC) transplantation, the treatment of human MSCs with angiotensin receptor blockers (ARBs) was investigated. MSCs were cultured with or without the medium containing 3 µmol/l of ARBs before cardiomyogenic induction. After cardiomyogenic induction in vitro, cardiomyogenic transdifferentiation efficiency (CTE) was calculated by immunocytochemistry using anticardiac troponin-I antibody. In the nude rat chronic myocardial infarction model, we injected MSCs pretreated with candesartan (A-BM; n = 18) or injected MSCs without pretreatment of candesartan (BM; n = 25), each having survived for 2 weeks. The left ventricular function, as measured by echocardiogram, was compared with cardiomyogenic transdifferentiation in vivo, as determined by immunohistochemistry. Pretreatment with ARBs significantly increased the CTE in vitro (10.1 ± 0.8 n = 12 vs. 4.6 ± 0.3% n = 25, p < .05). Transplantation of candesartan-pretreated MSCs significantly improved the change in left ventricular ejection fraction (BM; -7.2 ± 2.0 vs. A-BM; 3.3 ± 2.3%). Immunohistochemistry revealed significant improvement of cardiomyogenic transdifferentiation in A-BM in vivo (BM; 0 ± 0 vs. A-BM; 0.014 ± 0.006%). Transplantation of ARB-pretreated MSCs significantly improved cardiac function and can be a promising cardiac stem cell source from which to expect cardiomyogenesis.

Pretreatment of human mesenchymal stem cells with pioglitazone improved efficiency of cardiomyogenic transdifferentiation and cardiac function.

The efficacy of transplantation of default human marrow-derived mesenchymal stem cells (MSCs) was modest. In this study, our challenge was to improve the efficacy of MSC transplantation in vivo by pretreatment of MSCs with pioglitazone. MSCs were cultured with or without medium containing 1 µM of pioglitazone before cardiomyogenic induction. After cardiomyogenic induction in vitro, cardiomyogenic transdifferentiation efficiency (CTE) was calculated by immunocytochemistry using anti-cardiac troponin-I antibody. For the in vivo experiments, myocardial infarction (MI) at the anterior left ventricle was made in nude rats. Two weeks after MI, MSCs pretreated with pioglitazone (p-BM; n = 30) or without pioglitazone (BM; n = 17) were injected, and then survived for 2 weeks. We compared left ventricular function by echocardiogram and immunohistochemistry to observe cardiomyogenic transdifferentiation in vivo. Pretreatment with pioglitazone significantly increased the CTE in vitro (1.9% ± 0.2% n = 47 vs. 39.5% ± 4.7% n = 13, p < .05). Transplantation of pioglitazone pretreated MSCs significantly improved change in left ventricular % fractional shortening (BM; -4.8% ± 2.1%, vs. p-BM; 5.2% ± 1.5%). Immunohistochemistry revealed significant improvement of cardiomyogenic transdifferentiation in p-BM in vivo (BM; 0% ± 0% n = 5, vs. p-BM; 0.077% ± 0.041% n = 5). Transplantation of pioglitazone-pretreated MSCs significantly improved cardiac function and can be a promising cardiac stem cell source to expect cardiomyogenesis.

Xenografted human amniotic membrane-derived mesenchymal stem cells are immunologically tolerated and transdifferentiated into cardiomyocytes.

RATIONALE: Amniotic membrane is known to have the ability to transdifferentiate into multiple organs and is expected to stimulate a reduced immunologic reaction. OBJECTIVE: Determine whether human amniotic membrane-derived mesenchymal cells (hAMCs) can be an ideal allograftable stem cell source for cardiac regenerative medicine. METHODS AND RESULTS: We established hAMCs. After cardiomyogenic induction in vitro, hAMCs beat spontaneously, and the calculated cardiomyogenic transdifferentiation efficiency was 33%. Transplantation of hAMCs 2 weeks after myocardial infarction improved impaired left ventricular fractional shortening measured by echocardiogram (34+/-2% [n=8] to 39+/-2% [n=11]; P<0.05) and decreased myocardial fibrosis area (18+/-1% [n=9] to 13+/-1% [n=10]; P<0.05), significantly. Furthermore hAMCs transplanted into the infarcted myocardium of Wistar rats were transdifferentiated into cardiomyocytes in situ and survived for more than 4 weeks after the transplantation without using any immunosuppressant. Immunologic tolerance was caused by the hAMC-derived HLA-G expression, lack of MHC expression of hAMCs, and activation of FOXP3-positive regulatory T cells. Administration of IL-10 or progesterone, which is known to play an important role in feto-maternal tolerance during pregnancy, markedly increased HLA-G expression in hAMCs in vitro and, surprisingly, also increased cardiomyogenic transdifferentiation efficiency in vitro and in vivo. CONCLUSIONS: Because hAMCs have a high ability to transdifferentiate into cardiomyocytes and to acquire immunologic tolerance in vivo, they can be a promising cellular source for allograftable stem cells for cardiac regenerative medicine.

Serum-independent cardiomyogenic transdifferentiation in human endometrium-derived mesenchymal cells.

Media with high concentrations of serum are commonly used to induce cardiomyogenic transdifferentiation in mesenchymal stem cells; however, serum contains numerous unknown growth factors and interferes with definition of specific cardiomyogenic transdifferentiation factors secreted from feeder cells. In the present study, we determined whether the transdifferentiation of human mesenchymal cells can be observed in a FBS-free medium. The efficiency of transdifferentiation was observed in 10% FBS-containing standard medium (10%FBS) and in FBS-free medium containing insulin and thyroxin (FBS-free). In the present study, we used human uterine endometrium-derived mesenchymal cells (EMC100, EMC214) and menstrual blood-derived mesenchymal cells (MMCs). After cardiomyogenic transdifferentiation, the efficiency and physiological properties of cardiomyogenesis (fractional shortening of the cell [%FS] and action potential [AP]) were evaluated. The efficiency of transdifferentiation in EMC100 and in MMCs increased 36%* and 163%* (*P < 0.05), respectively. The %FS in EMCs increased to 103%*. AP-duration more than 250 ms with a marked plateau was only observed in FBS-free (3/19), and not in 10% FBS (0/41). The cardiomyogenic transdifferentiation of human mesenchymal cells can be observed in the FBS-free medium. Phenotypes of generated cardiomyocytes were significantly more physiological in FBS-free than in 10% FBS.

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 recipient's 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.

Tyrosine kinase inhibitor, methyl 2,5-dihydromethylcinnimate, induces PML nuclear body formation and apoptosis in tumor cells.

Promyelocytic leukemia (PML) nuclear bodies (PML-NBs) are the nuclear structure consisting of various proteins such as PML, SUMO-1, and p53. PML-NBs are implicated in the regulation of tumor suppression, antiviral responses, and apoptosis. In this study, we searched for bioactive metabolites that would promote the formation of PML-NBs in tumor cells. As a result, methyl 2,5-dihydromethylcinnimate (2,5-MeC), a tyrosine kinase inhibitor, enhanced expression and/or stability of PML proteins and induced PML-NB formation in p53 null H1299 cells established from non-small cell lung cancer (NSCLC) and wild-type p53-expressing U2OS cells derived from osteosarcoma. Furthermore, it enhanced apoptosis by exogenously expressed wild type p53 and the expression of p53-responsive genes, such as PUMA and p21, in H1299 cells. 2,5-MeC also activated endogenous p53 and induced apoptosis in U2OS cells. The results suggest that 2,5-MeC is likely to be a promising candidate drug for the clinical treatment of terminal cancer-expressing wild-type p53.

'Working' cardiomyocytes exhibiting plateau action potentials from human placenta-derived extraembryonic mesodermal cells.

The clinical application of cell transplantation for severe heart failure is a promising strategy to improve impaired cardiac function. Recently, an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and embryonic stem cells, have become important candidates for cell sources for cardiac repair. In the present study, we focused on the placenta as a cell source. Cells from the chorionic plate in the fetal portion of the human placenta were obtained after delivery by the primary culture method, and the cells generated in this study had the Y sex chromosome, indicating that the cells were derived from the fetus. The cells potentially expressed 'working' cardiomyocyte-specific genes such as cardiac myosin heavy chain 7beta, atrial myosin light chain, cardiac alpha-actin by gene chip analysis, and Csx/Nkx2.5, GATA4 by RT-PCR, cardiac troponin-I and connexin 43 by immunohistochemistry. These cells were able to differentiate into cardiomyocytes. Cardiac troponin-I and connexin 43 displayed a discontinuous pattern of localization at intercellular contact sites after cardiomyogenic differentiation, suggesting that the chorionic mesoderm contained a large number of cells with cardiomyogenic potential. The cells began spontaneously beating 3 days after co-cultivation with murine fetal cardiomyocytes and the frequency of beating cells reached a maximum on day 10. The contraction of the cardiomyocytes was rhythmical and synchronous, suggesting the presence of electrical communication between the cells. Placenta-derived human fetal cells may be useful for patients who cannot supply bone marrow cells but want to receive stem cell-based cardiac 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 x 10(3) per cm(2)) were cocultured with fetal murine cardiomyocytes ([CM] 1 x 10(5) per cm(2)). 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. Disclosure of potential conflicts of interest is found at the end of this article.

Membranous osteogenesis system modeled with KUSA-A1 mature osteoblasts.

Several stromal cells were established from murine bone marrow cultures. One of the KUSA subclones, KUSA-A1 cells, displays osteogenic characteristics in vitro and in vivo. The calcium deposition, osteocalcin release, and parathyroid hormone (PTH) responsiveness of KUSA-A1 cells indicate that they are mature osteoblasts or osteocytes. Bone had formed in subcutaneous tissue 1 week after subcutaneous injection of cells into immunodeficient mice. The osteogenesis by KUSA-A1 was not mediated by chondrogenesis and thus was considered to be membranous ossification. These unique characteristics of KUSA-A1 cells provide an opportunity to analyze the process of membranous ossification in detail.

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.

Induction of apoptosis in leukemic cells by homovanillic acid derivative, capsaicin, through oxidative stress: implication of phosphorylation of p53 at Ser-15 residue by reactive oxygen species.

Capsaicin (N-vanillyl-8-methyl-1-nonenamide) is a homovanillic acid derivative found in pungent fruits. Several investigators have reported the ability of capsaicin to inhibit events associated with the promotion of cancer. However, the effects of capsaicin on human leukemic cells have never been investigated. We investigated the effects of capsaicin on leukemic cells in vitro and in vivo and further examined the molecular mechanisms of capsaicin-induced apoptosis in myeloid leukemic cells. Capsaicin suppressed the growth of leukemic cells, but not normal bone marrow mononuclear cells, via induction of G(0)-G(1) phase cell cycle arrest and apoptosis. Capsaicin-induced apoptosis was in association with the elevation of intracellular reactive oxygen species production. Interestingly, capsaicin-sensitive leukemic cells were possessed of wild-type p53, resulting in the phosphorylation of p53 at the Ser-15 residue by the treatment of capsaicin. Abrogation of p53 expression by the antisense oligonucleotides significantly attenuated capsaicin-induced cell cycle arrest and apoptosis. Pretreatment with the antioxidant N-acetyl-L-cystein and catalase, but not superoxide dismutase, completely inhibited capsaicin-induced apoptosis by inhibiting phosphorylation of Ser-15 residue of p53. Moreover, capsaicin effectively inhibited tumor growth and induced apoptosis in vivo using NOD/SCID mice with no toxic effects. We conclude that capsaicin has potential as a novel therapeutic agent for the treatment of leukemia.

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.

Overexpression of phosphatidylinositol synthase enhances growth and G1 progression in NIH3T3 cells.

Phosphatidylinositol (PI) turnover is thought to play an important role in the regulation of cell growth. PI synthase (PIS, cytidine diphosphate (CDP)-diacylglycerol (DG): myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) acts at the last step in the de novo biosynthesis of PI by catalyzing the condensation of CDP-DG and myo-inositol. To study the physiological role of PIS, we established murine NIH3T3 fibroblasts that stably overexpress PIS, by transfection with PIS cDNA (NIH-PIS cells). In immunofluorescence assays, the constitutively overexpressed PIS was found to be localized in the endoplasmic reticulum, as previously reported for the native enzyme activity. NIH-PIS cells showed an increase in PI synthesis in vitro and in vivo, as well as increased cellular levels of PI-4,5-P2 and PI-3,4,5-P3. They also displayed a decrease in their doubling time and accelerated G1 progression. Overexpression of PIS increased cellular levels of the cyclin D1 and E proteins and Akt kinase activity in serum-stimulated quiescent NIH3T3 cells. Moreover, PIS overexpression potentiated the colony formation of NIH3T3 cells in soft agar. These results suggest that PIS accelerates G1 progression and stimulates growth by increasing cellular levels of cyclins D1 and E.