After the development of 6-8 wk, the teratomas and tumor-like bumps were fixed and examined with HE staining
After the development of 6-8 wk, the teratomas and tumor-like bumps were fixed and examined with HE staining. the functional identification of the differentiated cells. Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells. RESULTS: Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The differentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers, hepatocyte functions and capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells towards hepatic lineages. CONCLUSION: Hepatic cells of different developmental stages from early progenitors to matured hepatocytes can be acquired in the appropriate order based on sequential induction with VPA and cytokines. research model for the better investigation and understanding of the entire developmental process of hepatocytes, from ES cells to hepatic progenitors, and then to mature hepatocytes. Furthermore, as VPA is an epigenetic modulator, so our results may also be of benefit to the research of mechanisms of epigenetic modifications during liver development. MATERIALS AND METHODS Mouse monoclonal to Calcyclin Reagents VPA was purchased from Sigma (St Louis, MO); fetal bovine serum (FBS) was purchased from Hyclone (Rockville, MD); murine leukemia inhibitory factor (LIF) was purchased from Chemicon (Temecula, CA); mouse hepatocyte growth factor (mHGF), mouse epidermal growth factor (mEGF), oncostatin M (OSM), Insulin-Transferrin-Selenium (ITS), and collagen?I?were all from R&D systems (Minneapolis, MN); Matrigel was purchased from BD Biosciences (Palo Alto, CA); sheep anti-ALB antibodies were purchased from Biodesign (Saco, ME); rabbit anti-AFP, mouse anti-CK19 were from Dako (Copenhagen, Denmark); rat anti-OCT-4 was from R&D; mouse anti-SSEA-1 was from Developmental Hybridoma Bank of Iowa University; goat anti-mouse DLK was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rat anti-A6 was presented by Dr. Valentina Factor of NIH; FITC-conjugated bovine anti-sheep IgG, FITC-conjugated rabbit anti-goat IgG, FITC-conjugated goat anti-rabbit IgG, TRITC-conjugated goat anti-mouse IgG, TRITC-conjugated goat anti-rat IgG were all purchased from Dako; FITC-conjugated goat anti-mouse IgM was from Jackson Immunoresearch Laboratories Inc. All other reagents were from Sigma (St.Louis, Hydroxyphenylacetylglycine MO). Culture of mouse ES cells Mouse ES D3 cells, provided by the Cell Biology Institute of the Chinese Academy of Sciences, were cultured on mitomycin C inactivated MEF feeder layers in high-glucose DMEM supplemented with 15% FBS, 2 mmol/L L-Glu, 0.1 mmol/L N-ME, 1% NEAA and 10 ng/mL murine LIF as described previously. Briefly, MEF feeder cells were isolated from ICR mice at embryo day 13.5 and cultured at 37C and 5% CO2. At approximately 80% confluence, the feeder cells were incubated with 10 g/mL mitomycin C for 4 h and washed three times with PBS. Then the cells were replated at 8 104 cells/cm2 on to tissue culture flasks. After allowed for attachment overnight, the ES cells were seeded. Differentiation of hepatic cells from mouse ES cells A protocol was designed to obtain the hepatic progenitor cells and then mature hepatocytes from mouse ES cells in a sequential manner (Figure ?(Figure1).1). For differentiation of hepatic progenitor cells, ES cells were cultured in DME medium as described above with the exception of the withdrawal of feeder layer and LIF, and treated with 1 mmol/L VPA for 4-6 d, then the VPA was removed and recombinant mouse HGF 10 ng/mL was added for another 6-12 d until the hepatic progenitor cells became confluent. For differentiation of hepatocytes from hepatic progenitor cells, Hydroxyphenylacetylglycine the progenitor cells were cultured in a Williams E medium supplemented with 20 ng/mL mEGF, 10 ng/mL mHGF, 10-6 mol/L insulin, 10-7 mol/L Dex, 0.5 mmol/L ascorbic acid diphosphate, 10 mmol/L nicotinamide and 10% FBS (maturation medium?I) for 6 d, and then replaced with another Williams E medium containing 1% ITS, 10 ng/mL OSM Hydroxyphenylacetylglycine and 10-6.