The activation of Wnt/��-catenin during human MSC differentiation into hepatocytes is associated with abnormal proliferation, expression of CSC markers, spheroid formation and the generation of liver cells with tumoral characteristics, in contrast to hepatocytes differentiated without Wnt/��-catenin activation. Exploration Gemcitabine HCl of the differences between cancer stem cells from normal stem cells is crucial not only for the understanding of tumor biology but also for the prevention of potential complications derived from future liver therapies with human MSC. Materials and Methods Ethics Statement This study was approved by the Reina Sofia University Hospital Review Board. The procedures followed were in accordance with the ethical standards of the ethic committee from Hospital Reina Sof��a and with the Declaration of Helsinki.
All samples were collected after written informed consent. Human mesenchymal stem cells (MSCs) isolation Human bone marrow (BM) was aspirated from the iliac crest of healthy donors. Fresh BM was cultured in flasks (FalconTM, BD Pharmigen, Franklin Lakes, NJ) seeding 10 ��l BM cells/cm2 with alpha-minimum essential medium (��-MEM) supplemented with 2 mM L-glutamine, 15% fetal bovine serum (FBS) (BioWhittaker, Switzerland), 100 U/ml Penicillin, 0.1 mg/ml Streptomycin and 1 ng/ml of fibroblast growth factor-basic (FGF-b, Peprotech EC, London, UK) [58]. Cells were allowed to adhere for 48 h and non-adherent cells were washed out with phosphate-buffer saline (PBS) 100 mM pH 7,4 (Sigma-Aldrich, St Louis, MO). After 48 h, ��-MEM supplemented with 10% FBS and 1 ng/ml FGF-b was added twice weekly.
All cultures were maintained at 37��C in a humidified atmosphere containing 5% CO2. When adherent cells reached 90% confluence they were detached with 0.25% trypsin-EDTA (BioWhittaker, Switzerland), washed twice with PBS, centrifuged at 1800 rpm, 5 minutes and 4��C and replated in 6-well plates (SPL life sciences, Korea) at 103 cells/cm2 and cultured under the same conditions. In vitro hepatic differentiation Two different differentiation protocols were applied to confluent human MSCs for their differentiation into hepatocytes. An explicative diagram is included in Supporting Information (Figure S2). In the first protocol (conditioned medium 1, CM1), cells were cultured in ��-MEM containing 10% FBS, 20 ng/ml hepatocyte growth factor (HGF) and 10 ng/ml fibroblast growth factor-7 for 21 days.
The second protocol (conditioned medium 2, CM2) AV-951 is based in the article by Kuan der Lee [17]. Briefly, human MSCs were previously treated with epidermal growth factor (EGF) 20 ng/ml and FGF-b 10 ng/ml for 48 h. Then, 20 ng/ml HGF, 10 ng/ml FGF-b and 0.61 g/L nicotinamide were added for one week. Finally, cells were treated for fourteen days with 1 ��M dexamethasone, 20 ng/ml oncostatin and 10 ��l/ml ITS.
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