These data together with our previous studies indicate that C18 4 cells possess

After washes, cells were incubated by FITC coupled secondary antibody, and analyses were performed using Accuri C6 flow cytometer and Cflow software. The SSCs derived cells without primary antibody but were incubated by FITC coupled Amuvatinib c-kit 阻害剤 secondary antibody served as a negative control. Western blots Cells were lysed with RIPA buffer for 30 min on ice. After 30 min lysis on ice, cell lysates were cleared by centrifugation at 12,000 g, and the concentration of protein was measured by BCA kit. Ten micrograms of cell lysate from each sample were used for SDS PAGE, and Western blots were performed according to the protocol we described previously. The chosen anti body included CK8, phos ERK12, phos Smad2, Smad23, cyclin A, cyclin B, cyclin D1, cyclin E, and ACTB.<br><br> After extensive washes in PBS, the blots were detected by chemiluminescence. Albumin synthesis of hepatocyte like cells derived from SSCs by ELISA Primary mouse hepatocytes were isolated Afatinib ic50 from liver tissues using 0. 03% collagenase IV and 0. 025 EDTA and cultured in Williams E 100 nM insulin 15% FBS according to procedure as described previously. Culture medium from SSCs and the differentiated cells was collected over 2 days from equivalent numbers of cells. Albumin production in the medium from the differentiated cells and primary mouse hepatocytes was determined by mouse Albumin ELISA Quantitation Kit according to the manufacturers instructions. Albumin secretion was normalized to per 105 cells.<br><br> Urea assays of hepatocyte like cells derived from SSCs After exposure of the cells to 2 mM ammonium AG-490 Tyrphostin AG490 chloride for 24 h, urea productions in the culture media of SSCs, mature hepatocyte like cells derived from SSCs, and primary mouse hepatocytes were measured using Urea Assay Kit. Fresh culture medium supplemented with 2 mM am monium chloride was used as a negative control. Urea production was expressed as mM urea nitrogen per 105 cells within 24 h. Uptake and release of indocyanine green of hepatocyte like cells derived from SSCs Indocyanine green was suspended in DMSO for a stock at 100 mgml and freshly diluted in HCM to a working concentration of 1 mgml. Hepatocyte like cells derived from SSCs and primary mouse hepatocytes were incubated with the diluted ICG for 30 min at 37 C. After extensive washes, positive foci were counted and photographed under the microscope, and the cells were returned to HCM and incubated for 20 h.<br><br> Release of cellular ICG stain was examined, and undifferentiated C18 4 cells were used as a negative control while primary mouse hepatocytes served as a positive control. Statistical analysis All experiments were performed independently at least 3 times. All the values were presented as mean SEM, and statistically significant differences between SSCs and differentiated cells were determined using the analysis of variance and a 2 tailed t test. Results Direct transdifferentiation of SSCs to hepatic stem like cells We first verified the identity of the C18 4 cells using various markers for germ cells and SSCs. Immunocyto chemistry revealed that C18 4 cells expressed VASA, UCHL1, GFRA1, and RET, suggesting that the C18 4 cells are phenotypically SSCs.