A Comparative Study of the Bone Marrow- and Umbilical Cord-Derived Mesenchymal Stem Cells (MSCs) Efficiency on Generating MSC-Educated Macrophages (MEMs).

BACKGROUND: Mesenchymal stem cells (MSCs) have gained much more attention in cell therapy and regenerative medicine due to their immunosuppressive effects. MSCs have interaction with other immune cells, such as macrophages (MQs). Bone marrow (BM)-derived MSCs can educate MQs toward MSC-educated MQs (MEMs) which possess an anti-inflammatory immunophenotype. Given this and based on the important limitations of BM collection, we hypothesized whether co-culture of MQs with umbilical cord (UC)-derived MSCs can result in the MEM phenotype. METHODS: First, isolated monocytes cultured for five days to obtain M0 MQs. Then, they were co-cultured with either BM- or UC-MSCs under direct and indirect conditions. After three days of co-culture, MEM-specific surface markers, as well as the gene expression of inflammatory and anti-inflammatory cytokines, were evaluated. RESULTS: Surface expression of CD163/CD206, as specific markers for M2 MQs, increased in MEMs after co-culture with BM- and UC-derived MSCs, while CD80/CD86 expression (specific markers for M1 MQs) didn't change significantly. The mRNA expressions of PDL-1 as well as anti-inflammatory cytokines, including IL-6, IL-10, and TGFß also increased in MEMs after co-culture of UC-MSCs compared to control MQs (p <.05), while the expression of IL-12 was significantly decreased (p<.001). CONCLUSIONS: To the best of our knowledge, this study shows for the first time that the co-culture of MQs with UC-derived MSCs efficiently contributes to the generation of MEMs even greater than BM-MSCs; shedding light on the promising potential of UC as an alternative source to educate MQs in vitro.

TLR4 blockade using TAK-242 suppresses ovarian and breast cancer cells invasion through the inhibition of extracellular matrix degradation and epithelial-mesenchymal transition.

Numerous links exist between inflammation and tumor development. Toll-like receptor 4 (TLR4) expression by tumor cells can be a contributing factor that promotes tumor cell proliferation, survival, migration, and metastasis. In this study, we explored the impact of TLR4 inhibition using TAK-242, a specific inhibitor of TLR4, on the invasion properties of ovarian (A2780CP, 2008C13, SKOV3, and A2780S) and breast (MCF7, SKBR3, MDA-MB-231, and BT-474) cancer cell lines. Six out of eight cell lines expressed TLR4 and its downstream mediators (MyD88, NF-ĸB1, and RELB), indicating that these cell lines could be proper candidates for the TLR4 inhibition. TAK-242 induced a cytotoxic effect on all tested cell lines; however, a different cell sensitivity pattern was noticeable. Interestingly, in the TLR4-expressing cell lines, there was a significant correlation between the TLR4/MyD88 expressions and the cancer cell response to TAK-242: the higher the expression, the higher the IC50. To the best of our knowledge, no study has addressed the effects of TAK-242 on invasive abilities of cancer cells and our study suggests for the first time that TAK-242 could considerably decrease invasion properties of ovarian and breast cancer cell lines. We found that not only did TAK-242 reduce the enzymatic activity of MMP2 and MMP9, but also down-regulated gene expressions of epithelial-mesenchymal transition (EMT)-related genes. In sum, it seems that targeting TLR4 using TAK-242 possesses novel promising potential in cancer treatment strategies and may prevent invasion in patients suffering from ovarian and breast cancers, especially in those with over-expression of TLR4.

Over expression of circulating miR-155 predicts prognosis in diffuse large B-cell lymphoma.

INTRODUCTION: The expression patterns of microRNAs in plasma are involved in potential biomarkers for several diseases. The goal of this study was to explore the expression level of miR-155 in diffuse large B-cell lymphoma (DLBCL) and its clinical significance. MATERIALS AND METHODS: We used qRT-PCR to assess the peripheral blood plasma of 40 DLBCL patients for the expression of miRNA-155. The median of miR-155 expression divided the DLBCL patients into miR-155 low-expression (miR-155low) and miR-155 high-expression (miR-155high) groups. RESULTS AND DISCUSSION: We found that plasma miR-155 expression was significantly up-regulated in patients with DLBCL (median expression value: 4.29, range: 1.52-27.86) compared to healthy individuals (median expression value: 2.14, range: 0.29-10.56, P < 0.002). Moreover, DLBCL cases with an elevated level of miR-155 had shorter overall survival (median 9 vs. 13 months, P = 0.043) than those with a lower miR-155 expression.

Delivery of plasmid encoding interleukin-12 gene into hepatocytes by conjugated polyethylenimine-based nanoparticles.

In the present investigation, polyethylenimine (PEI) was conjugated with succinic anhydride at four substitution degrees and the efficiency of the modified PEI derivatives in transferring the plasmid encoding interleukin-12 gene was evaluated. The results revealed that the conjugated PEI derivatives enhanced the transfection efficiency by up to 3-fold relative to unmodified PEI, with the highest increase occurring at conjugation degrees around 30%. The results demonstrated the ability of the modified PEI derivatives in condensation of the plasmid into the nanoparticles in the size range of approximately 100 nm. Also, the PEI derivatives exhibited substantial decrease in cell-induced toxicity.

Tetraiodothyroacetic acid-conjugated polyethylenimine for integrin receptor mediated delivery of the plasmid encoding IL-12 gene.

Targeted delivery by polymer-based nanoparticles has been considered as an efficient approach to transfer genetic materials into cells. Considering the over expression of integrin αVß3 receptor on tumor cells and the presence of the binding site for tetraiodothyroacetic acid (tetrac) on integrin receptor, we hypothesized that the conjugation of tetrac to polyethylenimine (PEI) might be an effective strategy for pDNA delivery into the cells over-expressing integrins on their surfaces. In order to test the hypothesis, tetrac conjugated PEI/plasmid DNA complexes were prepared and their ability in the delivery of plasmid encoding IL-12 gene was investigated. Moreover, the conjugates were characterized with respect to plasmid DNA condensation ability, particle size and zeta potential as well as cell-induced toxicity and plasmid protection against DNase degradation. The results demonstrated that tetrac conjugated derivatives of PEI were able to condense the plasmid and protect it against enzyme degradation. The results of dynamic light scattering (DLS) and atomic force microscopy (AFM) revealed that the formed nanoparticles were in the size range of 85-125nm. The highest level of IL-12 gene expression was achieved by terac-conjugated PEIs at the carrier to plasmid ratio of 8 where they could increase the level of gene expression up to 4 fold in the cell lines over-expressing integrin αVß3 receptor whereas no increase in the level of IL-12 expression in the cell lines lacking integrin receptors was observed. Also, the results of the competitive inhibition of the receptors demonstrated the specificity of transfection for the cells over expressing αvß3 receptor. On the other hand, tetrac conjugation of PEI significantly reduced the polymer-induced apoptotic effects. The results obtained in this investigation suggest the potential of tetrac as a small molecule mimicking the binding properties of integrin binding peptides (e.g., RGD) for targeted gene delivery.

The Evaluation of Nerve Growth Factor Over Expression on Neural Lineage Specific Genes in Human Mesenchymal Stem Cells.

OBJECTIVE: Treatment and repair of neurodegenerative diseases such as brain tumors, spinal cord injuries, and functional disorders, including Alzheimer's disease, are challenging problems. A common treatment approach for such disorders involves the use of mesenchymal stem cells (MSCs) as an alternative cell source to replace injured cells. However, use of these cells in hosts may potentially cause adverse outcomes such as tumorigenesis and uncontrolled differentiation. In attempt to generate mesenchymal derived neural cells, we have infected MSCs with recombinant lentiviruses that expressed nerve growth factor (NGF) and assessed their neural lineage genes. MATERIALS AND METHODS: In this experimental study, we cloned the NGF gene sequence into a helper dependent lentiviral vector that contained the green fluorescent protein (GFP) gene. The recombinant vector was amplified in DH5 bacterial cells. Recombinant viruses were generated in the human embryonic kidney 293 (HEK-293) packaging cell line with the helper vectors and analyzed under fluorescent microscopy. Bone marrow mesenchymal cells were infected by recombinant viruses for three days followed by assessment of neural differentiation. We evaluated expression of NGF through measurement of the NGF protein in culture medium by ELISA; neural specific genes were quantified by real-time polymerase chain reaction (PCR). RESULTS: We observed neural morphological changes after three days. Quantitative PCR showed that expressions of NESTIN, glial derived neurotrophic factor (GDNF), glial fibrillary acidic protein (GFAP) and Microtubule-associated protein 2 (MAP2) genes increased following induction of NGF overexpression, whereas expressions of endogenous NGF and brain derived neural growth factor (BDNF) genes reduced. CONCLUSION: Ectopic expression of NGF can induce neurogenesis in MSCs. Direct injection of MSCs may cause tumorigenesis and an undesirable outcome. Therefore an alternative choice to overcome this obstacle may be the utilization of differentiated neural stem cells.

Enhanced Delivery of Plasmid Encoding Interleukin-12 Gene by Diethylene Triamine Penta-Acetic Acid (DTPA)-Conjugated PEI Nanoparticles.

Recombinant therapeutic proteins have been considered as an efficient category of medications used for the treatment of various diseases. Despite their effectiveness, there are some reports on the systemic adverse effects of recombinant therapeutic proteins limiting their wide clinical applications. Among different cytokines used for cancer immunotherapy, interleukin-12 (IL-12) has shown great ability as a powerful antitumor and antiangiogenic agent. However, significant toxic reactions following the systemic administration of IL-12 have led researchers to seek for alternative approaches such as the delivery and local expression of the IL-12 gene inside the tumor tissues. In order to transfer the plasmid encoding IL-12 gene, the most extensively investigated polycationic polymer, polyethylenimine (PEI), was modified by diethylene triamine penta-acetic acid (DTPA) to modulate the hydrophobic-hydrophilic balance of the polymer as well as its toxicity. DTPA-conjugated PEI derivatives were able to form complexes in the size range around 100-180 nm with great condensation ability and protection of the plasmid against enzymatic degradation. The highest gene transfer ability was achieved by the DTPA-conjugated PEI at the conjugation degree of 0.1 % where the level of IL-12 production increased up to twofold compared with that of the unmodified PEI. Results of the present study demonstrated that modulation of the surface positive charge of PEI along with the improvement of the polymer hydrophobic balance could be considered as a successful strategy to develop safe and powerful nanocarriers.