Investigation the effects of vitreous humor on proliferation and dedifferentiation of differentiated NTERA2 cells

Mammals have a limited capacity to regenerate their tissues and organs. One of the mechanisms associated with natural regeneration is dedifferentiation. Several small molecules such as vitamin C and growth factors could improve reprogramming efficiency. In this study, the NTERA2-D1 (NT2) cells were induced towards differentiation (NT2-RA) with 10-5 M retinoic acid (RA) for three days and then subjected to various amounts of vitreous humor (VH). Results show that the growth rate of these cells was reduced, while this rate was partly restored upon treatment with VH (NT2-RA-VH). Cell cycle analysis with PI method also showed that the numbers of cells at the S phase of the cell cycle in these cells were increased. The levels of SSEA3 and TRA-1-81 antigens in NT2-RA were dropped but they increased in NT2- RA-VH to a level similar to the NT2 cells. The level of SSEA1 had an opposite pattern. Expression of OCT4 gene dropped after RA treatment, but it was recovered in NT2-RA-VH cells. In conclusion, we suggest VH as a potent mixture for improving the cellular reprogramming leading to dedifferentiation.

Using High-Pressure Technology to Develop Antioxidant-Rich Extracts from Bravo de Esmolfe Apple Residues

Bravo de Esmolfe (BE) is a traditional Portuguese apple highly appreciated by consumers due to its peculiar flavor and aroma. This apple contains higher concentration of phenolic compounds than other cultivars and is thus considered a rich source of antioxidants. Its sensorial and functional properties have attracted farmers’ associations to increase BE production. However, a large quantity of apples is wasted due to storage/transportation procedures that impact BE’s quality attributes. In this work, we applied high-pressure extraction methodologies to generate antioxidant-rich fractions from BE residues aiming at adding high value to these agro-food by-products. We performed a first extraction step using supercritical CO2, followed by a second extraction step where different CO2 + ethanol mixtures (10–100% v/v) were tested. All experiments were carried out at 25 MPa and 50 °C. Extracts were characterized in terms of global yield, phenolic content and antioxidant activity using chemical (ORAC, HOSC, HORAC) and cell-based assays (CAA). We demonstrated that, although the pressurized 100% ethanol condition promoted the highest recovery of phenolic compounds (509 ± 8 mg GAE/100 g BE residues), the extract obtained with 40% ethanol presented the highest CAA (1.50 ± 0.24 µmol QE/g dw) and ORAC (285 ± 16 µmol TEAC/g dw), as well as HOSC and HORAC values, which correlated with its content of epicatechin and procyanidin B2. Noteworthy, this fraction inhibited free radical production in human neurospheroids derived from NT2 cells, a robust 3D cell model for neuroprotective testing.

Neuronal Differentiation of NT2 Cells in Monolayer and Spheroid Cultures

Metabolism and differentiation of cultured cells are influenced by changes in cellular morphology. In this study, we investigated the differences in cell proliferation and neuronal differentiation of NT2 cells in monolayer (2D) and spheroid (3D) cultures. In the monolayer culture, the cells adhered and extended on a tissue culture plate. For the spheroid culture, we fabricated a microwell chip comprising 195 circular microwells (600 ìm in diameter) on a cutture plate, and the surface was modified with polyethylene glycol to promote spheroid formation. The cells were aggregated in each microwell and formed spheroids within 24 hours of culture, and the spheroid morphology was maintained thoughout the culture period. Although the cell proliferation ability in monolayer culture was higher than that in spheroid culture, the neuronal differentiation ability of NT2 spheroid culture was higher than that in monolayer culture. Furthermore, the neuronal differentiation of NT2 spheroids was dramatically enhanced by retinoic acid treatment. These results indicate that NT2 cell properties are influenced by differences in cell morphologies, and that spheroid culture is a promising technique to induce neuronal differentiation.

Hsa-miR-6165 downregulates insulin-like growth factor-1 receptor (IGF-1R) expression and enhances apoptosis in SW480 cells

MicroRNAs are small non-coding RNAs that are implicated in various biological processes. Hsa-miR-6165 (miR-6165), located in the p75NTR gene, is known to induce apoptosis in human cell lines, but its mechanism of action is not fully understood yet. Here, we predicted the insulin-like growth factor 1 receptor (IGF-1R) gene as a bona fide target for miR-6165. The overexpression of miR-6165 in SW480 cells resulted in significant downregulation of IGF-1R expression as detected by real time quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Also, it resulted in reduced transcript levels of AKT2, AKT3, PI3KR3, PI3KR5, CCND1, c-MYC and P21 genes detected by RT-qPCR analysis. In addition, a direct interaction between miR-6165 and a 3′UTR sequence of the IGF-1R gene was verified through a dual luciferase assay. Furthermore, miR-6165 and IGF-1R showed opposite patterns of expression during the neural differentiation process of NT2 cells. Annexin V analysis and MTT assay showed that miR-6165 overexpression was followed by increased apoptosis and reduced the viability rate of SW480 cells. Moreover, a lower expression level of miR-6165 was detected in high-grade colorectal tumors compared with low-grade tumors. Taken together, the results of our study suggest a tumor suppressive role of miR-6165 in colorectal cancer, which seems to take place by regulating IGF-1R gene expression.