Neural differentiation of brain-derived neurotrophic factor-expressing human umbilical cord blood-derived mesenchymal stem cells in culture via TrkB-mediated ERK and ß-catenin phosphorylation and following transplantation into the developing brain.

The ability of mesenchymal stem cells (MSCs) to differentiate into neural cells makes them potential replacement therapeutic candidates in neurological diseases. Presently, overexpression of brain-derived neurotrophic factor (BDNF), which is crucial in the regulation of neural progenitor cell differentiation and maturation during development, was sufficient to convert the mesodermal cell fate of human umbilical cord blood-derived MSCs (hUCB-MSCs) into a neuronal fate in culture, in the absence of specialized induction chemicals. BDNF overexpressing hUCB-MSCs (MSCs-BDNF) yielded an increased number of neuron-like cells and, surprisingly, increased the expression of neuronal phenotype markers in a time-dependent manner compared with control hUCB-MSCs. In addition, MSCs-BDNF exhibited a decreased labeling for MSCs-related antigens such as CD44, CD73, and CD90, and decreased potential to differentiate into mesodermal lineages. Phosphorylation of the receptor tyrosine kinase B (TrkB), which is a receptor of BDNF, was increased significantly in MSC-BDNF. BDNF overexpression also increased the phosphorylation of ß-catenin and extracellular signal-regulated kinases (ERKs). Inhibition of TrkB availability by treatment with the TrkB-specific inhibitor K252a blocked the BDNF-stimulated phosphorylation of ß-catenin and ERKs, indicating the involvement of both the ß-catenin and ERKs signals in the BDNF-stimulated and TrkB-mediated neural differentiation of hUCB-MSCs. Reduction of ß-catenin availability using small interfering RNA-mediated gene silencing inhibited ERKs phosphorylation. However, ß-catenin activation was maintained. In addition, inhibition of ß-catenin and ERKs expression levels abrogated the BDNF-stimulated upregulation of neuronal phenotype markers. Furthermore, MSC-BDNF survived and migrated more extensively when grafted into the lateral ventricles of neonatal mouse brain, and differentiated significantly into neurons in the olfactory bulb and periventricular astrocytes. These results indicate that BDNF induces the neural differentiation of hUCB-MSCs in culture via the TrkB-mediated phosphorylation of ERKs and ß-catenin and following transplantation into the developing brain.

Microporation is a valuable transfection method for efficient gene delivery into human umbilical cord blood-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are an attractive source of adult stem cells for therapeutic application in clinical study. Genetic modification of MSCs with beneficial genes makes them more effective for therapeutic use. However, it is difficult to transduce genes into MSCs by common transfection methods, especially nonviral methods. In this study, we applied microporation technology as a novel electroporation technique to introduce enhanced green fluorescent protein (EGFP) and brain-derived neurotropfic factor (BDNF) plasmid DNA into human umbilical cord blood-derived MSCs (hUCB-MSCs) with significant efficiency, and investigated the stem cell potentiality of engineered MSCs through their phenotypes, proliferative capacity, ability to differentiate into multiple lineages, and migration ability towards malignant glioma cells. RESULTS: Using microporation with EGFP as a reporter gene, hUCB-MSCs were transfected with higher efficiency (83%) and only minimal cell damage than when conventional liposome-based reagent (<20%) or established electroporation methods were used (30-40%). More importantly, microporation did not affect the immunophenotype of hUCB-MSCs, their proliferation activity, ability to differentiate into mesodermal and ectodermal lineages, or migration ability towards cancer cells. In addition, the BDNF gene could be successfully transfected into hUCB-MSCs, and BDNF expression remained fairly constant for the first 2 weeks in vitro and in vivo. Moreover, microporation of BDNF gene into hUCB-MSCs promoted their in vitro differentiation into neural cells. CONCLUSION: Taken together, the present data demonstrates the value of microporation as an efficient means of transfection of MSCs without changing their multiple properties. Gene delivery by microporation may enhance the feasibility of transgenic stem cell therapy.

MK886-induced apoptosis depends on the 5-LO expression level in human malignant glioma cells.

Mounting evidence suggests that lipoxygenase (LO)-catalyzed products may play a key role in the development and progression of human cancers. In this study, we analyzed the effects of a 5-LO inhibitor, which inhibits the conversion of arachidonic acid to leukotrienes, on cell proliferation and apoptosis in human malignant glioma cells, including 5-LO-expressing cells U-87MG, A172 and 5-LO non-expressing cell U373. Growth of U-87MG and A172 cells, but not that of U373 cells, was inhibited in a dose-dependent manner by treatment with MK886. Similarly, specific 5-LO silencing by small interfering RNA reduced the growth of U-87MG and A172 cells. MK886 treatment reduced 5-LO activity independently of 5-LO-activating protein (FLAP) in human malignant glioma cells. MK886 treatment also induced cell apoptosis, measured by DNA fragmentation and nuclear condensation, in U-87MG and A172 cells but there were no signs in U373 cells. Moreover, this treatment reduced ERKs phosphorylation and anti-apoptotic molecule Bcl-2 expression, and increased Bax expression in U-87MG and A172 cells. In summary, our results show there is a link between the 5-LO expression status and the extent of MK886-inhibited cell proliferation and apoptosis. Taken together, this study suggest that 5-LO is a possible target for treating patients with gliomas, and 5-LO inhibition might be potent therapy for patients with 5-LO-expressing malignant gliomas.

The merlin tumor suppressor interacts with Ral guanine nucleotide dissociation stimulator and inhibits its activity.

Neurofibromatosis type 2 (NF2) is the most commonly mutated gene in benign tumors of the human nervous system such as schwannomas and meningiomas. The NF2 gene encodes a protein called schwannomin or merlin, which is involved in regulating cell growth and proliferation through protein-protein interactions with various cellular proteins. In order to better understand the mechanism by which merlin exerts its function, yeast two-hybrid screening was performed and Ral guanine nucleotide dissociation stimulator (RalGDS), a downstream molecule of Ras, was identified as a merlin-binding protein. The direct interaction between merlin and RalGDS was confirmed both in vitro and in the NIH3T3 cells. The domain analyses revealed that the broad C-terminal region of merlin (aa 141-595) is necessary for the interaction with the C-terminal Ras-binding domain (RBD) of RalGDS. Functional studies showed that merlin inhibits the RalGDS-induced RalA activation, the colony formation and the cell migration in mammalian cells. These results suggest that merlin can function as a tumor suppressor by inhibiting the RalGDS-mediated oncogenic signals.

MAP, a protein interacting with a tumor suppressor, merlin, through the run domain.

Merlin (or schwannomin) is a tumor suppressor encoded by the neurofibromatosis type 2 gene. Many studies have suggested that merlin is involved in the regulation of cell growth and proliferation through interactions with various cellular proteins. To better understand the function of merlin, we tried to identify the proteins that bind to merlin using the yeast two-hybrid screening. Characterization of the positive clones revealed a protein of 749 amino acids named merlin-associated protein (MAP), which showed wide tissue distribution in Northern blot analysis. Sequence analysis revealed that MAP is a potential homologue of a yeast check-point protein, BUB2, and contains TBC, SH3, and RUN domains, thereby implicating its role in the Ras-like GTPase signal pathways. MAP and merlin were directly associated in vitro and in vivo, and colocalized in NIH3T3 cells. The RUN domain of MAP and the C-terminus of merlin appeared to be responsible for their interaction. MAP decreased the AP-1-dependent promoter activity additively with merlin in NIH3T3 cells. In addition, merlin and MAP synergistically reduced the colony formation of NIH3T3 cells. These results suggest that MAP may play a cooperative role in the merlin-mediated growth suppression of cells.

Merlin, a tumor suppressor, interacts with transactivation-responsive RNA-binding protein and inhibits its oncogenic activity.

The neurofibromatosis type 2 gene-encoded protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of membrane-cytoskeleton-associated proteins. Recent studies suggest that the loss of neurofibromatosis type 2 function contributes to tumor development and metastasis. Although the cellular functions of merlin as a tumor suppressor are relatively well characterized, the cellular mechanism whereby merlin controls cell proliferation from membrane locations is still poorly understood. During our efforts to find potential merlin modulators through protein-protein interactions, we identified transactivation-responsive RNA-binding protein (TRBP) as a merlin-binding protein in a yeast two-hybrid screen. The interaction between TRBP and merlin was confirmed by glutathione S-transferase pull-down assays, co-immunoprecipitation, and co-localization experiments. The carboxyl-terminal regions of each protein were responsible for their interaction. Cells overexpressing TRBP showed enhanced cell growth in cell proliferation assays and also exhibited transformed phenotypes, such as anchorage-independent cell growth and tumor development in mouse xenografts. Merlin efficiently inhibited these oncogenic activities of TRBP in our experiments. These results provide the first clue to the functional interaction between TRBP and merlin and suggest a novel mechanism for the tumor suppressor function of merlin both in vitro and in vivo.

Lymphokine activated killer cells from umbilical cord blood show higher antitumor effect against anaplastic astrocytoma cell line (U87) and medulloblastoma cell line (TE671) than lymphokine activated killer cells from peripheral blood.

OBJECTS: The aims of this study were to assess the cytotoxic capability of lymphokine-activated killer (LAK) cells from umbilical cord blood (UCB), to compare them with those of peripheral blood (PB)-derived cells against anaplastic astrocytoma cell line (U87) and medulloblastoma cell line (TE671), and to identify which mechanism and genes were involved in cytotoxicity. METHODS: The effector cells were generated by interleukin-2 from UCB and PB. The antitumor property of effector cells against the target cells (U87, TE671) were estimated using a visual survival cell assay. The mixed target and effector (UCB) cells were analyzed for whether DNA fragmentation was present or not. Reverse transcription polymerase chain reaction analysis was then performed to estimate the statement of the perforin and FasL genes in activated and inactivated cells from UCB. RESULTS: The higher in vitro antitumor properties of the LAK cells from UCB were observed in comparison to the LAK cells from PB against the U87 and the TE671 ( p<0.05). Apoptosis may be one of the lysis mechanisms of target cells by the LAK cells from UCB. The contributing genes could be FasL and perforin. CONCLUSIONS: This study suggests that UCB may be used as a source of LAK cells in adults and children suffering from anaplastic astrocytoma or medulloblastoma.