Partial hepatectomy Cyclopamine order was performed as described.19 Paraffin-embedded liver sections

(4 μm thick) were used for immunohistochemical staining. Antigen retrieval was achieved by heating the slides in a microwave at high power in 1× citrate buffer for 10 minutes. The tissue sections were blocked in blue blocker for 20 minutes followed by incubation with primary antibody overnight at 4°C. The primary antibody was then linked to biotinylated secondary antibody followed by routine avidin-biotin complex method. Diaminobenzidine was used as the chromogen, which resulted in a brown reaction product. Primary antibodies used were as follows: NRSF (Millipore 07-579; 1:1,000), Oct4 (ab27985; 1:250), KLF4 (SC-20691; 1:500), Nanog (ab80892; find more 1:700), and Myc (ab32072; 1:100). Data are expressed as means ± standard error (SE). Statistical differences were determined by one-way

analysis of variance (ANOVA) followed by Tukey’s honest significant difference test to determine which means were significantly different from each other or from controls using the JMP software (SAS Institute, Cary, NC). The criterion for significance was P < 0.05. We report that primary hepatocytes do express REST, Oct4, cMyc, Klf4, and Nanog in culture (Figs. 1, 2). Their expression is up-regulated with time in culture. However, this up-regulation is further enhanced in cultures incubated with growth factors (Figs. 1, 2). REST and Klf4 message showed a steady increase with time in both the groups peaking at day 10 in culture (Fig. 1A,D). Oct4, cMyc, and Nanog message showed earlier peaks at 4, 2, and 8 days, respectively (Figs. 1B,C,E, 2). Previous studies have shown maximum proliferation of dedifferentiated hepatocytes when plated in culture Casein kinase 1 with GF between days 4 and 10 by bromodeoxyuridine labeling and tritiated thymidine incorporation.15 Expression of REST, Oct4, Nanog,

Myc, and Klf4 protein was maximally induced when there was maximum proliferation in culture (days 4 to 10), suggesting their role in GF-mediated proliferation of hepatocytes in culture. To test if expression of REST and reprogramming factors is dependent on the differentiation status of cells, Matrigel was used to induce hepatocyte differentiation as described. Hepatocytes were incubated as follows: with Matrigel with GF (+MT G+GF), with Matrigel without GF (+MTG−GF), without Matrigel with GF (−MTG+GF), or without Matrigel without GF (−MTG−GF). Matrigel-induced differentiation down-regulated the growth factor induced expression of REST, Klf4, cMyc, and Oct4 message (Fig. 3A-D). The group without Matrigel and with growth factors (−MTG+GF) showed maximum expression of REST, Klf4, and cMyc, whereas the group with Matrigel and without growth factors (+MTG−GF) showed the least expression. The other two groups lay in between (Fig. 3). Albumin (Fig.