BackgroundThe development of a suitable extracellular matrix (ECM) scaffold is the first step in vascular tissue engineering (VTE). Synthetic vascular grafts are available as an alternative to autologous vessels in large-diameter arteries (>8 mm) and medium-diameter arteries (6–8 mm). In small-diameter vessels (<6 mm), synthetic vascular grafts are of limited use due to poor patency rates. Compared with a vascular prosthesis, natural tissue ECM has valuable advantages. Despite considerable progress in recent years, identifying an optimal protocol to create a scaffold for use in small-diameter (<6 mm) fully natural tissue-engineered vascular grafts (TEVG), remains elusive. Although reports on different decellularization techniques have been numerous, combination of and comparison between these methods are scarce; therefore, we have compared five different decellularization protocols for making small-diameter (<6 mm) ECM scaffolds and evaluated their characteristics relative to those of fresh vascular controls.ResultsThe protocols differed in the choice of enzymatic digestion solvent, the use of non-ionic detergent, the durations of the individual steps, and UV crosslinking. Due to their small diameter and ready availability, rabbit arteria carotis were used as the source of the ECM scaffolds. The scaffolds were subcutaneously implanted in rats and the results were evaluated using various microscopy and immunostaining techniques.ConclusionsOur findings showed that a 2 h digestion time with 1× EDTA, replacing non-ionic detergent with double-distilled water for rinsing and the application of UV crosslinking gave rise to an ECM scaffold with the highest biocompatibility, lowest cytotoxicity and best mechanical properties for use in vivo or in situ pre-clinical research in VTE in comparison.Electronic supplementary materialThe online version of this article (doi:10.1186/s12938-017-0344-9) contains supplementary material, which is available to authorized users.

Mesenchymal stem cells (MSCs) negatively modulate immune properties. Induced pluripotent stem cells (iPSCs)-derived MSCs are alternative source of MSCs. However, the effects of iPSC-MSCs on T cells phenotypes in vivo remain unclear. We established an iPSC-MSC-transplanted host versus graft reaction mouse model using subcapsular kidney injection. Th1, Th2, regulatory T cells (Treg), and Th17 phenotypes and their cytokines were investigated in vivo and in vitro. The role of caspases and the soluble factors involved in the effects of MSCs were examined. We found that iPSC-MSC grafts led to more cell survival and less infiltration of inflammatory cells in mice. iPSC-MSC transplantation inhibited T cell proliferation, decreased Th1 and Th2 phenotypes and cytokines, upregulated Th17 and Treg subsets. Moreover, iPSC-MSCs inhibited the cleavage of caspases 3 and 8 and inhibition of caspases downregulated Th1, Th2 responses and upregulated Th17, Treg responses. Soluble factors were determined using protein array and TGF-b1/2/3, IL-10, and MCP-1 were found to be highly expressed in iPSC-MSCs. The administration of the soluble factors decreased Th1/2 response, upregulated Treg response and inhibited the cleavage of caspases. Our results demonstrate that iPSC-MSCs regulate T cell responses as a result of a combined action of the above soluble factors secreted by iPSC-MSCs. These factors suppress T cell responses by inhibiting the cleavage of caspases. These data provide a novel immunomodulatory mechanism for the underlying iPSC-MSC-based immunomodulatory effects on T cell responses. STEM CELLS 2017;35:1719-1732 SIGNIFICANCE STATEMENTWe established an iPSC-MSC-transplanted host versus graft reaction mouse model using subcapsular kidney injection and demonstrated an underlying mechanism for the immunosuppressive effect of iPSC-MSCs through inhibition of the cleavage of caspases, which was inhibited by a series of paracrine factors secreted by iPSC-MSCs. Inhibition of caspases suppressed T cell responses, decreased Th1 and Th2 frequency, and increased CD4 1 CD25 1 Foxp3 1 regulatory T cells. These data indicate a novel mechanism for the immunomodulation of MSC via regulation of caspases.

Multidrug resistance (MDR) of tumor cells attenuates the efficacy of anticancer drugs and has become the main reason for chemotherapy failure. It is indispensable to establish an effective way to reverse multidrug resistance. Our previous work has shown that downregulation of the ERK/MAPK signaling pathway activity can reverse the drug resistance of resistant cells. Furthermore, the effect of signal transduction is strongly associated with lipid rafts. The drug resistance is reversed successfully after lipid rafts are destroyed by heptakis(2,6-di-O-methyl)-β-cyclodextrin (MβCD). However, reversal of the drug resistance is not associated with downregulation of ERK1/2 expression. Cell membrane permeability may increase when lipid rafts are destroyed by MβCD, causing the reversal of drug resistance due to an increased drug accumulation in cytoplasm. To minimize the influence of MβCD on the cell membrane structure, we selected flotillin, a marker protein of lipid rafts, as the target molecule to further investigate the mechanism of changes in drug resistance after destruction of lipid rafts. The effect of flotillin on the reversal of drug resistance was examined using RNA interference (RNAi) in a retroviral system in human drug resistant strains of colorectal cancer cell line HCT-15. The results demonstrate that flotillin-1 downregulation by RNAi (Flot1-RNAi) reduced the drug resistance, caused cell cycle arrest and decreased the expression of ERK1/2; however, apoptosis was not significantly affected. Knockdown of flotillin-2 by RNAi (Flot2-RNAi) had effects similar to those of Flot1-RNAi except that the effects on ERK1/2 expression and apoptosis were different. Screening of multiple pathways indicated that the PI3K/Akt signaling pathway was closely related. This experiment demonstrates an association between PI3K and drug resistance through the activation of PI3K and suggests that PI3K may play a key role during the development of resistance in CRC. The results reveal that the levels of IRS-1 and PI3K proteins in the Flot1-RNAi and Flot2-RNAi groups were significantly downregulated. Knockdown of flotillins by RNAi reduced the resistance of HCT-15/ADM cells; and the results on Akt pathway indicate a decrease in resistance after lipid raft destruction. These data confirm that knockdown of flotillin reduces the resistance of HCT-15/ADM cells and the mechanism may be relevant to the PI3K/Akt pathway. Additionally, flotillin may be used as a potential target for chemotherapy in the treatment of colorectal cancer.

The aim of this study was to evaluate and determine the potential mechanisms of As₂O₃ in accelerated rejection mediated by alloreactive CD4⁺ memory T cells. Vascularized heterotopic cardiac transplantation from C57BL/6 mice to nude mice (pre-transferred CD4⁺ memory T cells) was performed on Day 0, and As₂O₃ was administered to recipient mice from Day 0 to 10. As a result, As₂O₃ could reduce the proliferation of allo-primed CD4⁺ memory T cells in vitro in MLR and the baseline rate of proliferation was restored by the addition of exogenous IL-2. In vivo, compared with the control[+] group, the mean survival time of cardiac allografts in the As₂O₃ group was prolonged from 5.8 ± 0.7 to 14.2 ± 2.5 days. Five days after transplantation, the relative gene expression of IL-2, IFN-γ and Foxp3 was reduced in the grafts by As₂O₃ treatment, but the expression of IL-10 and TGF-β was increased. Correspondingly, the proportions of CD4⁺ T cells, CD4⁺ memory T cells and regulatory T cells (Tregs), both in recipient spleens and lymph nodes, were lowered. These results indicate the potential of As2O3 as a novel immunosuppressant targeting CD4⁺ memory T cells.

Dendritic cells (DCs) have the tolerogenic potential to regulate adaptive immunity and induce allografts acceptance. Here we investigated whether blockade of the CD40 pathway could enhance the immune tolerance induced by DC2.4 cells modified to express Jagged-1 (JAG1-DC) in heart transplantation. Results showed that JAG1-DC treatment combined with anti-CD40L monoclonal antibody (mAb) administration significantly prolonged cardiac allograft survival in mice, with long-term survival (>110 days) of 50% of the allografts in the recipients. The therapy specifically inhibited the immune response, induced alloantigen-specific T-cell hyporesponsiveness, upregulated transforming growth factor-β synthesis and increased the population of regulatory T cells (Tregs) driven by Jagged-1-Notch activation. These results highlight the potential application of gene therapy to induce alloantigen-specific Tregs effectively by providing the Jagged-1 stimulation.