IGF-1 enhances cell proliferation and survival during early differentiation of mesenchymal stem cells to neural progenitor-like cells.

BACKGROUND: There has been increasing interest recently in the plasticity of mesenchymal stem cells (MSCs) and their potential to differentiate into neural lineages. To unravel the roles and effects of different growth factors in the differentiation of MSCs into neural lineages, we have differentiated MSCs into neural lineages using different combinations of growth factors. Based on previous studies of the roles of insulin-like growth factor 1 (IGF-1) in neural stem cell isolation in the laboratory, we hypothesized that IGF-1 can enhance proliferation and reduce apoptosis in neural progenitor-like cells (NPCs) during differentiation of MSCs into NCPs.We induced MSCs differentiation under four different combinations of growth factors: (A) EGF + bFGF, (B) EGF + bFGF + IGF-1, (C) EGF + bFGF + LIF, (D) EGF + bFGF + BDNF, and (E) without growth factors, as a negative control. The neurospheres formed were characterized by immunofluorescence staining against nestin, and the expression was measured by flow cytometry. Cell proliferation and apoptosis were also studied by MTS and Annexin V assay, respectively, at three different time intervals (24 hr, 3 days, and 5 days). The neurospheres formed in the four groups were then terminally differentiated into neuron and glial cells. RESULTS: The four derived NPCs showed a significantly higher expression of nestin than was shown by the negative control. Among the groups treated with growth factors, NPCs treated with IGF-1 showed the highest expression of nestin. Furthermore, NPCs derived using IGF-1 exhibited the highest cell proliferation and cell survival among the treated groups. The NPCs derived from IGF-1 treatment also resulted in a better yield after the terminal differentiation into neurons and glial cells than that of the other treated groups. CONCLUSIONS: Our results suggested that IGF-1 has a crucial role in the differentiation of MSCs into neuronal lineage by enhancing the proliferation and reducing the apoptosis in the NPCs. This information will be beneficial in the long run for improving both cell-based and cell-free therapy for neurodegenerative diseases.

IGF-1 acts as controlling switch for long-term proliferation and maintenance of EGF/FGF-responsive striatal neural stem cells.

BACKGROUND: Long-term maintenance of neural stem cells in vitro is crucial for their stage specific roles in neurogenesis. To have an in-depth understanding of optimal conditional microenvironmental niche for long-term maintenance of neural stem cells (NSCs), we imposed different combinatorial treatment of growth factors to EGF/FGF-responsive cells. We hypothesized, that IGF-1-treatment can provide an optimal niche for long-term maintenance and proliferation of EGF/FGF-responsive NSCs. OBJECTIVE: This study was performed to investigate the cellular morphology and growth of rat embryonic striatal tissue derived-NSCs in long-term culture under the influence of different combinatorial effects of certain growth factors, such as EGF, bFGF, LIF and IGF-1. METHODS: The NSCs were harvested and cultured from striatal tissue of 18 days old rat embryos. We have generated neurospheres from these NSCs and cultured them till passage 7 (28 days in vitro) under four different conditional microenvironments: (A) without growth factor, (B) EGF/bFGF, (C) EGF/bFGF/LIF, (D) EGF/bFGF/IGF-1 and (E) EGF/bFGF/LIF/IGF-1. Isolated NSCs were characterised by Immunoflouroscence for nestin expression. The cell growth and proliferation was evaluated at different time intervals (P1, P3, P5 & P7), assessing the metabolic activity based cell proliferation. Apoptosis was studied in each of these groups by In situ cell death assay. RESULTS: Our results demonstrated certain important findings relevant to long-term culture and maintenance of striatal NSC-derived neurospheres. This suggested that IGF-1 can induce enhanced cell proliferation during early stages of neurogenesis, impose long-term maintenance (up to passage 7) to cultured NSCs and enhance survival efficiency in vitro, in the presence of EGF and FGF. CONCLUSIONS: Our findings support the hypothesis that the enforcement of IGF-1 treatment to the EGF/FGF-responsive NSCs, can lead to enhanced cell proliferation during early stages of neurogenesis, and an extended life span in vitro. This information will be beneficial for improving future therapeutic implication of NSCs, by addressing improved in vitro production of NSCs.

Cytotoxicity evaluation of vancomycin and its complex with beta-cyclodextrin on human glial cell line.

The possible cytotoxic effects of vancomycin and its complex with beta-cyclodextrin (ß-CD) on human glial cell line (CRL 8621) were studied accordingly by means of MTS assay. The cultured cells were incubated with various concentrations of vancomycin, ß-CD as well as ß-CD/vancomycin complex ranging from 4.69 to 300 ug/ml. A linear dose-dependency cytotoxicity followed by hermetic-like biphasic dose-dependence was observed after incubation period of 72 hours. In general, significant increase (p<0.001) of cell proliferation was observed at lower concentrations: <18.75 µg/ml for cells treated with ß-CD and their complex while < 9.38 µg/ml for cells treated with vancomycin. In contrary, regardless of the treatments given, significant (p<0.001) reduce in cell survival was found at higher concentrations >150 µg/ml. In particular, 50 % inhibitory in vitro was achieved at the concentrations of 115.95 µg/ml (for ß-CD), 116.48 µg/ml (for vancomycin) and 115.44 µg/ml (for ß-CD/vancomycin complex).

Fibrinolytic activity and dose-dependent effect of incubating human blood clots in caffeic acid phenethyl ester: in vitro assays.

Background. Caffeic acid phenethyl ester (CAPE) has been reported to possess time-dependent fibrinolytic activity by in vitro assay. This study is aimed at investigating fibrinolytic dose-dependent activity of CAPE using in vitro assays. Methods. Standardized human whole blood (WB) clots were incubated in either blank controls or different concentrations of CAPE (3.75, 7.50, 15.00, 22.50, and 30.00 mM). After 3 hours, D-dimer (DD) levels and WB clot weights were measured for each concentration. Thromboelastography (TEG) parameters were recorded following CAPE incubation, and fibrin morphology was examined under a confocal microscope. Results. Overall, mean DD (µg/mL) levels were significantly different across samples incubated with different CAPE concentrations, and the median pre- and postincubation WB clot weights (grams) were significantly decreased for each CAPE concentration. Fibrin removal was observed microscopically and indicated dose-dependent effects. Based on the TEG test, the Ly30 fibrinolytic parameter was significantly different between samples incubated with two different CAPE concentrations (15.0 and 22.50 mM). The 50% effective dose (ED50) of CAPE (based on DD) was 1.99 mg/mL. Conclusions. This study suggests that CAPE possesses fibrinolytic activity following in vitro incubation and that it has dose-dependent activities. Therefore, further investigation into CAPE as a potential alternative thrombolytic agent should be conducted.

Newcastle disease virus interaction in targeted therapy against proliferation and invasion pathways of glioblastoma multiforme.

Glioblastoma multiforme (GBM), or grade IV glioma, is one of the most lethal forms of human brain cancer. Current bioscience has begun to depict more clearly the signalling pathways that are responsible for high-grade glioma initiation, migration, and invasion, opening the door for molecular-based targeted therapy. As such, the application of viruses such as Newcastle disease virus (NDV) as a novel biological bullet to specifically target aberrant signalling in GBM has brought new hope. The abnormal proliferation and aggressive invasion behaviour of GBM is reported to be associated with aberrant Rac1 protein signalling. NDV interacts with Rac1 upon viral entry, syncytium induction, and actin reorganization of the infected cell as part of the replication process. Ultimately, intracellular stress leads the infected glioma cell to undergo cell death. In this review, we describe the characteristics of malignant glioma and the aberrant genetics that drive its aggressive phenotype, and we focus on the use of oncolytic NDV in GBM-targeted therapy and the interaction of NDV in GBM signalling that leads to inhibition of GBM proliferation and invasion, and subsequently, cell death.

In vitro whole blood clot lysis for fibrinolytic activity study using d-dimer and confocal microscopy.

This study aimed to evaluate in vitro whole blood (WB) clot lysis method for the assessment of fibrinolytic activity. Standardized unresected (uncut) retracted WB clot was incubated in pool platelet poor plasma (PPP) for varying incubation times and in streptokinase (SK) at different concentrations. The fibrinolytic activity was assessed by D-dimer (DD), confocal microscopy, and clot weight. DD was measured photometrically by immunoturbidimetric method. There was a significant difference in mean DD levels according to SK concentrations (P = 0.007). The mean DD ± SD according to the SK concentrations of 5, 30, 50, and 100 IU/mL was: 0.69 ± 0.12, 0.78 ± 0.14, 1.04 ± 0.14 and 2.40 ± 1.09 µ g/mL. There were no significant changes of clot weight at different SK concentrations. Gradual loss and increased branching of fibrin in both PPP and SK were observed. Quantitation of DD and morphology of fibrin loss as observed by the imaging features are in keeping with fibrinolytic activity. Combination of DD levels and confocal microscopic features was successfully applied to evaluate the in vitro WB clot lysis method described here.

Viability reduction and Rac1 gene downregulation of heterogeneous ex-vivo glioma acute slice infected by the oncolytic Newcastle disease virus strain V4UPM.

Oncolytic viruses have been extensively evaluated for anticancer therapy because this virus preferentially infects cancer cells without interfering with normal cells. Newcastle Disease Virus (NDV) is an avian virus and one of the intensively studied oncolytic viruses affecting many types of cancer including glioma. Nevertheless, the capability of NDV infection on heterogeneous glioma tissue in a cerebrospinal fluid atmosphere has never been reported. Recently, Rac1 is reported to be required for efficient NDV replication in human cancer cells and established a link between tumourigenesis and sensitivity to NDV. Rac1 is a member of the Rho GTPases involved in the regulation of the cell migration and cell-cycle progression. Rac1 knockdown leads to significant inhibition of viral replication. In this work, we demonstrated that NDV treatment led to significant reduction of tumour tissue viability of freshly isolated heterogeneous human brain tumour slice, known as an ex vivo glioma acute slice (EGAS). Analysis of gene expression indicated that reduced tissue viability was associated with downregulation of Rac1. However, the viability reduction was not persistent. We conclude that NDV treatment induced EGAS viability suppression, but subsequent downregulation of Rac1 gene may reduce the NDV replication and lead to regrowth of EGAS tissue.