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 (

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder in children, is an X-linked recessive muscle disease characterized by the absence of dystrophin at the sarcolemma of muscle fibers. We examined a putative endometrial progenitor obtained from endometrial tissue samples to determine whether these cells repair muscular degeneration in a murine mdx model of DMD. Implanted cells conferred human dystrophin in degenerated muscle of immunodeficient mdx mice. We then examined menstrual blood-derived cells to determine whether primarily cultured nontransformed cells also repair dystrophied muscle. In vivo transfer of menstrual blood-derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of dystrophin. Labeling of implanted cells with enhanced green fluorescent protein and differential staining of human and murine nuclei suggest that human dystrophin expression is due to cell fusion between host myocytes and implanted cells. In vitro analysis revealed that endometrial progenitor cells and menstrual blood-derived cells can efficiently transdifferentiate into myoblasts/myocytes, fuse to C2C12 murine myoblasts by in vitro coculturing, and start to express dystrophin after fusion. These results demonstrate that the endometrial progenitor cells and menstrual blood-derived cells can transfer dystrophin into dystrophied myocytes through cell fusion and transdifferentiation in vitro and in vivo.

ABSTRACT

Spiropyran is a photoresponsive molecule, and nonionic spiropyran is reversibly changed by UV irradiation to a hydrophilic polar, zwitterionic merocyanine isomer, and back again by visible light irradiation. A copolymer of nitrobenzospiropyran and methyl methacrylate, poly(NSP-co-MMA) was used as a material with a photosensitive surface. UV irradiation of the photosensitive surface of poly(NSP-co-MMA)-coated glass plates decreased the water contact angles (11 +/- 1 degrees ) and increased diameter of a water drop relative to the unexposed surface. Light-induced detachment of platelets and mesenchymal stem (KUSA-A1) cells on poly(NSP-co-MMA)-coated glass plates was observed upon simple- and patterned-light irradiation, whereas no light-induced detachment of platelets and mesenchymal stem cells was observed on poly(methyl methacrylate)-coated glass plates. This is a result of the change from a closed nonpolar spiropyran to the polar zwitterionic merocyanine isomer induced by UV irradiation. Light-induced detachment of fibrinogen adsorbed on poly(NSP-co-MMA) coated glass plates was also observed in this investigation.

Human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) are expected to serve as an excellent alternative to bone marrow-derived human mesenchymal stem cells. However, it is difficult to study them because of their limited life span. To overcome this problem, we attempted to produce a strain of UCBMSCs with a long life span and to investigate whether the strain could maintain phenotypes in vitro. UCBMSCs were infected with retrovirus carrying the human telomerase reverse transcriptase (hTERT) to prolong their life span. The UCBMSCs underwent 30 population doublings (PDs) and stopped dividing at PD 37. The UCBMSCs newly established with hTERT (UCBTERTs) proliferated for >120 PDs. The p16 INK4a /RB braking pathway leading to senescence can be inhibited by introduction of Bmi-1, a polycomb-group gene, and human papillomavirus type 16 E7, but the extension of the life span of the UCBMSCs with hTERT did not require inhibition of the p16 INK4a /RB pathway. The characteristics of the UCBTERTs remained unchanged during the prolongation of life span. UCBTERTs provide a powerful model for further study of cellular senescence and for future application to cell-based therapy by using umbilical cord blood cells. INTRODUCTIONHuman mesenchymal stem cells (hMSCs) can be a useful source of cells for transplantation for several reasons: they have the ability to proliferate and differentiate into mesodermal tissues, and they entail no ethical or immunological problems (Caplan, 1991;Prockop, 1997;Caplan and Bruder, 2001). hMSCs have been studied extensively over the past 3 decades, and numerous independent research groups have successfully isolated hMSCs from a variety of sources, most commonly, from the bone marrow (Owen, 1988;Umezawa et al., 1992;Jaiswal et al., 1997;Makino et al., 1999;Pittenger et al., 1999;Sekiya et al., 2004). Umbilical cord blood (UCB) contains circulating stem/progenitor cells, and the cells contained in UCB are known to be distinct from those contained in bone marrow and adult peripheral blood (Mayani and Lansdorp, 1998). Isolation, characterization, and differentiation of clonally expanded hMSCs derived from UCB (UCBMSCs) have been reported (Goodwin et al., 2001;Lee et al., 2004), and UCBMSCs have been found to have multipotency, and the immunophenotype of the clonally expanded cells is consistent with that reported for bone marrow mesenchymal stem cells. Even now, most UCB is regarded as medical waste in the delivery rooms. Aspirating bone marrow from patients is, however, an invasive procedure, and the proliferation and differentiation capacity of hMSCs decreases with the donor age (D'Ippolito et al., 1999). Therefore, the applications of UCB should be further expanded.UCBMSCs will be useful sources for cell transplantation, however, it is difficult to study and apply them because of their limited life span. One of the reasons for this is that normal human cells undergo a limited number of cell division in culture and then enter a nondividing state called "senescence" (Hayflick, 1976;Campisi...

Human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) are expected to be an excellent source of cells for transplantation. In addition, the stem cell plasticity of human UCBMSCs, which can transdifferentiate into hepatocytes, has been reported. However, the mechanisms involved remain to be clarified. To identify the genes and/or signals that are important in specifying the hepatic fate of human UCBMSCs, we analyzed gene expression profiles during the hepatic differentiation of UCBMSCs with human telomerase reverse transcriptase, UCBMSCs immortalized by infection with a retrovirus carrying telomerase reverse transcriptase, but whose differentiation potential remains unchanged. Efficient differentiation was induced by 5-azacytidine (5-aza)/hepatocyte growth factor (HGF)/oncostatin M (OSM)/fibroblast growth factor 2 (FGF2) treatment in terms of function as well as protein expression: 2.5-fold increase in albumin, 4-fold increase in CCAAT enhancer-binding protein α, 1.5-fold increase in cytochrome p450 1A1/2, and 8-fold increase in periodic acid-Schiff staining. Consequently, we found that the expression of Wnt/β-catenin-related genes downregulated, and the translocation of β-catenin was observed along the cell membrane and in the cytoplasm, although some β-catenin was still in the nucleus. Downregulation of Wnt/β-catenin signals in the cells by Fz8-small interference RNA treatment, which was analyzed with a Tcf4 promoter-luciferase assay, resulted in similar hepatic differentiation to that observed with 5-azacytidine/HGF/OSM/FGF2. In addition, the subcellular distribution of β-catenin was similar to that of cells treated with 5-azacytidine/HGF/OSM/FGF2. In conclusion, the suppression of Wnt/β-catenin signaling induced the hepatic differentiation of UCBMSCs, suggesting that Wnt/β-catenin signals play an important role in the hepatic fate specification of human UCBMSCs.

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 ¼ 8) or vehicle (n ¼ 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 (Po0.05), 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 (Po0.05). The MSC administration significantly promoted neovascularization in the border areas compared with the controls (Po0.0005), 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 (Po0.05, 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 (Po0.0001). 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.

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