The differentiation of neuronal cells from mouse embryonic stem cells

With new technologies emerging today, the importance of stem cells in the cell therapy of nervous system diseases is supported by recent studies. Therefore, the development of neuronal cell differentiation protocols from stem cells is of great importance. In our study, the differentiation of neuronal and neuroglial cells from mouse embryonic stem (ES) cell line and their analysis with neuronal cell markers are aimed. Mouse ES cells were differentiated to neurogenic series cells by adding N2 and bFGF to the culture medium on coated Fibronectin dishes. For the identification of differentiated cells, they were evaluated by light microscopy using immunhistochemistry techniques and by electron microscopy. Indirect immunohistochemical staining method was performed with SSEA-1 (mouse embriyonic stem cells marker), Nestin (neural precursor cells marker), βIII-Tubulin (neuronal cells marker), MAP-2 (neuronal cells marker), GFAP (astrocyte marker), and O4 (oligodendrocyte marker). After 1 week of differentiation of cells, immunoreactivities of SSEA-1 and Nestin were detected to be negative and moderate, respectively. After 2 weeks culture time, the differentiation was still continuing and especially positive immunoreactivities of β-III Tubulin and MAP-2 and weak immunoreactivities of O4 and GFAP were supported neuronal differentiation. In conclusion, our results suggest that neuronal cell derived from mouse ES cells were differentiated particularly to neuron using N2+bFGF+fibronectin culture condition. Therefore, these differentiated cells may be used as a treatment method in degenerative diseases of the nervous system

In vitro antiproliferative/cytotoxic activity on cancer cell lines of a cardanol and a cardol enriched from Thai Apis mellifera propolis

<p>Abstract</p> <p>Background</p> <p>Propolis is a complex resinous honeybee product. It is reported to display diverse bioactivities, such as antimicrobial, anti-inflammatory and anti-tumor properties, which are mainly due to phenolic compounds, and especially flavonoids. The diversity of bioactive compounds depends on the geography and climate, since these factors affect the floral diversity. Here, <it>Apis mellifera </it>propolis from Nan province, Thailand, was evaluated for potential anti-cancer activity.</p> <p>Methods</p> <p>Propolis was sequentially extracted with methanol, dichloromethane and hexane and the cytotoxic activity of each crude extract was assayed for antiproliferative/cytotoxic activity <it>in vitro </it>against five human cell lines derived from duet carcinoma (BT474), undifferentiated lung (Chaco), liver hepatoblastoma (Hep-G₂), gastric carcinoma (KATO-III) and colon adenocarcinoma (SW620) cancers. The human foreskin fibroblast cell line (Hs27) was used as a non-transformed control. Those crude extracts that displayed antiproliferative/cytotoxic activity were then further fractionated by column chromatography using TLC-pattern and MTT-cytotoxicity bioassay guided selection of the fractions. The chemical structure of each enriched bioactive compound was analyzed by nuclear magnetic resonance and mass spectroscopy.</p> <p>Results</p> <p>The crude hexane and dichloromethane extracts of propolis displayed antiproliferative/cytotoxic activities with IC₅₀values across the five cancer cell lines ranging from 41.3 to 52.4 μg/ml and from 43.8 to 53.5 μg/ml, respectively. Two main bioactive components were isolated, one cardanol and one cardol, with broadly similar <it>in vitro </it>antiproliferation/cytotoxicity IC₅₀values across the five cancer cell lines and the control Hs27 cell line, ranging from 10.8 to 29.3 μg/ml for the cardanol and < 3.13 to 5.97 μg/ml (6.82 - 13.0 μM) for the cardol. Moreover, both compounds induced cytotoxicity and cell death without DNA fragmentation in the cancer cells, but only an antiproliferation response in the control Hs27 cells However, these two compounds did not account for the net antiproliferation/cytotoxic activity of the crude extracts suggesting the existence of other potent compounds or synergistic interactions in the propolis extracts_.</p> <p>Conclusion</p> <p>This is the first report that Thai <it>A. mellifera </it>propolis contains at least two potentially new compounds (a cardanol and a cardol) with potential anti-cancer bioactivity. Both could be alternative antiproliferative agents for future development as anti-cancer drugs.</p