A proteomic time course through the differentiation of human induced pluripotent stem cells into hepatocyte-like cells.

Numerous in vitro models endeavour to mimic the characteristics of primary human hepatocytes for applications in regenerative medicine and pharmaceutical science. Mature hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (hiPSCs) are one such in vitro model. Due to insufficiencies in transcriptome to proteome correlation, characterising the proteome of HLCs is essential to provide a suitable framework for their continual optimization. Here we interrogated the proteome during stepwise differentiation of hiPSCs into HLCs over 40 days. Whole cell protein lysates were collected and analysed using stabled isotope labelled mass spectrometry based proteomics. Quantitative proteomics identified over 6,000 proteins in duplicate multiplexed labelling experiments across two different time course series. Inductive cues in differentiation promoted sequential acquisition of hepatocyte specific markers. Analysis of proteins classically assigned as hepatic markers demonstrated trends towards maximum relative abundance between differentiation day 30 and 32. Characterisation of abundant proteins in whole cells provided evidence of the time dependent transition towards proteins corresponding with the functional repertoire of the liver. This data highlights how far the proteome of undifferentiated precursors have progressed to acquire a hepatic phenotype and constructs a platform for optimisation and improved maturation of HLC differentiation

Effect of Sutherlandia frutescens and Hypoxis hemerocallidea extracts on inflammatory markers in vitro

Immunomodulating effects of Hypoxis hemerocallidea, Sutherlandia frutescens and standard compounds found in these plants were determined. Neither of the extracts, nor the standard compounds were cytotoxic to THP-1 and U937 macrophages. Antioxidant activity of H. hemerocallidea was equivalent to 0.2 mg/mL Trolox. Canavanine showed antioxidant activity comparable to that of curcumin (positive control). Curcumin (9.2 μg/mL) and beta-sitosterol (12.5 μg/mL) reduced IL-1β and IL-8 production significantly (p < 0.05) and decreased the production of TNF-α. Beta-sitosterol (25 μg/mL) and pinitol (50 μg/mL) significantly (p<0.01) decreased extracellular PGE2 levels in U937 macrophages by 12 % and 14 %, respectively. From the current results it would appear that the standard compounds present in the plants are far more effective in modulating the immune system than the extracts.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Attenuation of oxidative stress in U937 cells by polyphenolic-rich bark fractions of Burkea africana and Syzygium cordatum

BACKGROUND: Oxidative stress has been implicated in the progression of various diseases, which may result in the depletion of endogenous antioxidants. Exogenous supplementation with antioxidants could result in increased protection against oxidative stress. As concerns have been raised regarding synthetic antioxidant usage, the identification of alternative treatments is justified. The aim of the present study was to determine the antioxidant efficacy of Burkea africana and Syzygium cordatum bark extracts in an in vitro oxidative stress model. METHODS: Cytotoxicity of crude aqueous and methanolic extracts, as well as polyphenolic-rich fractions, was determined in C2C12 myoblasts, 3T3-L1 pre-adipocytes, normal human dermal fibroblasts and U937 macrophage-like cells using the neutral red uptake assay. Polyphenolic content was determined using the Folin-Ciocalteau and aluminium trichloride assays, and antioxidant activity using the Trolox Equivalence Antioxidant Capacity and DPPH assays. The extracts efficacy against oxidative stress in AAPH-exposed U937 cells was assessed with regards to reactive oxygen species generation, cytotoxicity, apoptosis, lipid peroxidation and reduced glutathione depletion. RESULTS: B. africana and S. cordatum showed enrichment of polyphenols from the aqueous extract, to methanolic extract, to polyphenolic-rich fractions. Antioxidant activity followed the same trend, which correlated well with the increased concentration of polyphenols, and was between two- to three-fold stronger than the Trolox antioxidant control. Both plants had superior activity compared to ascorbic acid in the DPPH assay. Polyphenolic-rich fractions were most toxic to the 3T3-L1 (IC(50)’s between 13 and 21 μg/ml) and C2C12 (IC(50)’s approximately 25 μg/ml) cell lines, but were not cytotoxic in the U937 and normal human dermal fibroblasts cultures. Free radical-induced generation of reactive oxygen species (up to 80%), cytotoxicity (up to 20%), lipid peroxidation (up to 200%) and apoptosis (up to 60%) was successfully reduced by crude extracts of B. africana and the polyphenolic-rich fractions of both plants. The crude extracts of S. cordatum were not as effective in reducing cytotoxic parameters. CONCLUSION: Although oxidative stress was attenuated in U937 cells, cytotoxicity was observed in the 3T3-L1 and C2C12 cell lines. Further isolation and purification of polyphenolic-fractions could increase the potential use of these extracts as supplements by decreasing cytotoxicity and maintaining antioxidant quality