(HEPATOLOGY 2011) In recent years many efforts have been aimed at generating pluripotent stem cells from somatic cells by inducing high levels of expression of a combination of transcription factors including Sox2, Oct3/4 (henceforth referred to as Oct4), Nanog, Klf4, and cMyc.1-5 Initially, retroviral transduction of four reprogramming factors, Oct4, Sox2, cMyc, and Klf4, was shown to be sufficient to convert mouse fibroblasts to embryonic stem cell-like phenotype.6 Later, pluripotent stem cells were generated from adult mouse liver and stomach cells,7 human somatic cells,8 and human buy Idelalisib primary hepatocytes.9 Recently, generation of pluripotent stem cells without viral integration
by repeated transfection of expression plasmids10, 11 and by protein transfer12, 13 have overcome the risks of tumorigenicity associated with viral integration. Although these reprogramming factors are expressed in stem cells, their expression in adult somatic cells with high potential for clonal expansion such as hepatocytes has been less explored. Primary hepatocytes show limited ability to proliferate in culture except under the influence of chemically defined HGM medium containing the primary Copanlisib clinical trial mitogens hepatocyte growth factor (HGF), and epidermal growth factor (EGF) (henceforth referred to as growth factors [GF]).14
Under these conditions, hepatocytes undergo multiple proliferative cycles, express altered levels of hepatocyte-associated transcription factors, and lose characteristic gene expression markers such as albumin. In the presence of specific matrix components such as Matrigel, they redifferentiate and revert to a mature
hepatocyte phenotype.15 In the present study we examined whether primary hepatocytes express any of the iPS-reprogramming factors in culture and if their expression changes as a result of growth factor-induced proliferation. Transcription factor REST (RE-1 silencing transcription factor; also called NRSF) has been shown to regulate the expression of these self-renewal and reprogramming factors in mouse embryonic stem cells.16 Thus, we looked at REST expression to see if it was see more expressed in our model and if it regulates the expression of any of the reprogramming factors. Primary rat hepatocytes were plated on collagen-coated plates and incubated in the presence of HGM medium with (+GF) or without growth factors (−GF) over a period of 10 days. Plates were harvested at day 0 (2-hour plated), 2, 4, 6, 8, and 10 after plating for analysis of message and protein by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blot analysis, respectively. EGF, epidermal growth factor; GF, growth factor; HGF, hepatocyte growth factor; MESC, mouse embryonic stem cells; MTG, Matrigel; PHX, 70% partial hepatectomy; REST, RE-1 silencing transcription factor.