Search

Dental pulp stem cells (DPSCs) derived from discarded orthodontic teeth are easily obtained and have become a promising source for mesenchymal stem cell-based therapy. However, the pulp tissue is limited, and long-term culture induces cell senescence. Hypoxic culture was expected to be suitable for DPSC expansion, but the results have been contradictory. The aim of this study was to verify the effect of hypoxic culture on human DPSCs (hDPSCs). hDPSCs were isolated and cultured in normoxic (ambient O 2 concentration) and hypoxic (5% O 2) environments from passage 3 (P3) to P6. The biological characteristics of the cells at P4 (short-term culture) and P6 (long-term culture) were evaluated, including the expression of surface markers, cellular proliferation activity, cellular senescence, and spontaneous and induced differentiation. The results showed that the morphology, phenotype, and proliferation activity of hDPSCs were not affected by hypoxic culture. Long-term normoxic culture of hDPSCs induced cell stemness loss and cell senescence, while hypoxic culture could alleviate these effects. The expression of the stemness markers STRO-1 and OCT4 was increased and the number of senescent cells and the expression of the senescence-related genes P53 and TGF-β were reduced by long-term hypoxic culture. Spontaneous osteogenic and adipogenic differentiation did not occur during long-term normoxic culture. However, hypoxic culture suppressed the expression of the osteogenic markers ALP and RUNX-2 and the adipogenic markers PPAR-γ and FABP4. The induced osteogenic and adipogenic differentiation was apparently reduced by hypoxic culture as well. Our findings indicate that long-term hypoxia culture is beneficial to the maintenance of hDPSCs' biological characteristics and provide some insights into their large-scale expansion. [Display omitted] • Hypoxia didn't influence hDPSCs' proliferation. • Hypoxia inhibits the spontaneous stemness decrease and senescence increase of hDPSCs. • hDPSCs cultured in normoxia did not undergo spontaneous osteogenic-odontoblast differentiation. • Hypoxia suppressed the osteogenic and adipogenic differentiation of hDPSCs. • The beneficial effect of hypoxia on the state of hDPSCs was passage dependent. [ABSTRACT FROM AUTHOR]

Background: Mesothelial cell proliferation and migration play important roles in reducing formation of postoperative peritoneal adhesions. Hepatocyte growth factor (HGF) is a multifunctional cytokine that stimulates proliferation and migration of various cell types, including mesothelial cells. Methods: We investigated the effect of adenovirus-mediated HGF gene expression on the proliferation and migration of mesothelial cells and evaluated its preventive effects on postoperative formation of peritoneal adhesions. Rat mesothelial cells were isolated and characterized by expression of cytokeratin and vimentin. Results: Immunohistochemical staining showed that these cells expressed c-Met, the receptor for HGF. Adenoviral-mediated HGF gene transfer into mesothelial cells resulted in high expression of HGF and enhanced migration. To evaluate the preventive effects of adenoviral-mediated HGF gene transfer on the formation of postoperative peritoneal adhesion, we employed a rat model of cecum abrasion-induced adhesion formation in which 80% of the rats developed postoperative peritoneal adhesions. Local application of recombinant adenovirus carrying the HGF gene reduced adhesion formation in 16 of 20 control rats compared with 7 of 20 treated rats in this model. Conclusions: These results suggest that adenoviral-mediated HGF gene transfer may be a novel strategy for preventing postoperative peritoneal adhesions. [Copyright &y& Elsevier]

Mesenchymal stem cells could differentiate into cardiomyocytes in vitro and have been shown to reconstitute the impaired myocardium in vivo. Hepatocyte growth factor, a recognized angiogenic factor and endothelial cell chemoattractant, has been applied in the treatment of myocardial ischemia. In this study, we used a ligation model of proximal left anterior descending coronary artery of rats to evaluate the effect of mesenchymal stem cells overexpressing hepatocyte growth factor in the treatment of myocardial ischemia. Bone marrow-derived mesenchymal stem cells were isolated, expanded, characterized, and infected with adenovirus carrying human hepatocyte growth factor cDNA (Ad-HGF). Mesenchymal stem cells infected by Ad-HGF released soluble HGF protein at a high level, which was maintained at least for 2 weeks. Implantation of mesenchymal stem cells overexpressing hepatocyte growth factor into left anterior descending risk areas improved the functions of impaired myocardium, including diminishing the area of ischemia, increasing the number of capillaries, and reducing collagen content. By using the sry gene as a marker, we also demonstrated that the engrafted cells or their progeny incorporated into ischemic cardiac muscle. These results showed that treatment of myocardial ischemia with bone marrow-derived mesenchymal stem cells overexpressing hepatocyte growth factor could be a novel strategy that can both restore local blood flow and regenerate lost cardiomyocytes. [Copyright &y& Elsevier]

Co-transfer of immunomodulatory and antiproliferative genes may be the basis for new strategies to potentiate tumor regression. In this study, we evaluated the in vitro effect of the introduction of human wild-type p53, granulocyte-macrophage colony-stimulating factor (GM-CSF), and B7-1 genes via recombinant adenovirus on the growth and immunogenicity of Hep-2 or primary laryngeal cancer cells. By the introduction of wild-type p53 gene, the growth of Hep-2 cells was inhibited via enhanced apoptosis. By the introduction of GM-CSF and B7-1 genes, the immunogenicity of cancer cells was enhanced. Significant proliferation of tumor infiltrating lymphocytes (TILs) and tumor-specific cytotoxicity of cytotoxic T lymphocytes (CTLs) were induced in vitro. Furthermore, the combinative effect of GM-CSF and B7-1 was even more evident than that of any one of them singly. These results suggest that the co-transfer of human wild-type p53, GM-CSF and B7-1 genes into tumor cells via recombinant adenovirus may be further developed into a potential combination gene therapy strategy for cancer. [Copyright &y& Elsevier]

Abstract: Shp-2, a ubiquitously expressed protein tyrosine phosphatase containing two Src homology 2 domains, plays an important role in integrating signaling from the cell surface receptors to intracellular signaling mechanisms. Previous studies have demonstrated that the Shp-2 is involved in hepatocyte growth factor (HGF)-induced cell scattering. Here we report that Shp-2 is required for the HGF-induced activation of sphingosine kinase-1 (SPK1), a highly conserved lipid kinase that plays an important role in cell migration. Loss-of-function mutation of Shp-2 did not affect the expression of SPK1, but resulted in its inactivation and the blockage of HGF-induced migration in embryonic fibroblasts. Reintroduction of functional wild type (WT) Shp-2 into the mutant cells partially restored SPK1 activation, and overexpression of SPK1 in these mutant cells enhanced HGF-induced cell migration. Inhibition of expression or activity of SPK1 in WT cells markedly decreased intracellular S1P levels and HGF-induced cell migration. Furthermore, we found that Shp-2 co-immunoprecipitated with SPK1 and c-Met in embryonic fibroblasts. These studies suggest that Shp-2 is an SPK1-interacting protein and that it plays an indispensable role in HGF-induced SPK1 activation. [Copyright &y& Elsevier]

Objective: Adenoviral vectors (Ad) were widely used in gene therapy and study of gene function, but the commonly used serotype 5 adenovirus-based vectors (Ad5) could poorly transduce hematopoietic cells because of low expression of viral receptors on these cells. To overcome this limitation, we developed a retargeting adenovector with a chimeric fiber of Ad5 and Ad11p (Ad5F11p) and evaluated its gene transfer ability in hematopoietic cells. Materials and Methods: An Ad11p fiber pseudotyped Ad5 vector was generated by modifying the fiber gene of pAdEasy-1 backbone plasmid. Ad5F11p-GFP encoding enhanced green fluorescence protein (GFP) gene was transferred into human leukemic cell lines, primary leukemic cells, and CD34+ hematopoietic cells. The gene transduction efficiency was determined by fluorescence-activated cell sorting assay. Results: More than 90% of U937 or K562 cells could be infected by Ad5F11p-GFP at a moderate multiplicity of infection (MOI). Ad5F11p-GFP is also significantly more effective than control Ad5-GFP in infection of primary myeloid leukemic cells. At 200 MOI, GFP-positive percentages of Ad5F11p-GFP transduced myeloid leukemic cells range from 10.58% to 92.63% with a median of 28.65%. Ad5F11p-GFP could transduce about 50% human hematopoietic stem/progenitor (CD34+) cells, while Ad5-GFP could transduce <15% at 200 MOI. CD46 was reported to be the receptor of Ad11p. Our data suggest that CD46 participates in the process of Ad5F11p-GFP infection but is not the unique molecule determining its gene transfer efficiency of host cells. Conclusion: We established a retargeting adenovector system, which could infect hematopoietic cells effectively and would benefit research work on Ad tropism. [Copyright &y& Elsevier]

We previously found that dibutyl phthalate (DBP) had a pharmacological activity in eliminating tumor cells and could be used as a purging agent in autologous bone marrow transplantation. In this study, we show that DBP can induce apoptosis in MO7e and U937 leukemia cell lines. Treatment of these cells with DBP up-regulates cellular activity of caspase-3/CPP32 and causes apoptosis rapidly as determined by cell viability and dUTP nick end labeling assay. Activation of caspase-3/CPP32 and cleavage of poly(ADP–ribose) polymerase were determined in DBP-induced apopotosis of leukemia cells. However, DBP treatment did not induce the expression of fas. Two caspase inhibitors, z-VAD-fmk and N-acetyl-Asp-Glu-Val-Asp-aldehyde partly blocked the cell death of MO7e cells induced by DBP. These results suggest that fas-independent activation of caspase-3 protease plays important roles in the purging effect of DBP on leukemia cells. [Copyright &y& Elsevier]