The cells used in our studies appear

The ezh2 pathway used in our studies appear reliably over time in primary hepatocyte cultures in the presence of growth factor supplemented medium and were first described as BAML cells [6]. Whether they already pre-exist as stem/progenitor cells in the liver or derive from other cell types in the presence of growth factors in culture, is currently not known. Terminal differentiation of those cells under adherent culture conditions in vitro, using standard maturation conditions (a variety of conditions from previous reports were tested beforehand) was limited, resulting in induction of hepatic gene expression and hepatocyte functions but not reaching primary hepatocyte ranges. For further improvement of hepatic terminal differentiation, we followed a strategy of ectopic sequential expression of a combination of transcription factors. First, we generated an ALDP cell line, which uniformly expressed Foxa2. Foxa transcription factors are required for normal development of endoderm derived organs such as liver and pancreas [28]. Recent studies identified that Foxa proteins act as “competence factors” and facilitate binding and transcriptional activity of other transcription factors in endoderm cells and tissues [28]. Moreover, it has been shown that Foxa2 is required for normal liver homeostasis also in the adult liver, as >43% of genes expressed in the liver were associated with Foxa2 binding [29]. Standard liver differentiation conditions were sufficient to maximally induce secretion of Aat in Foxa2 transgenic cells. In contrast, we have observed an incremental increase in albumin secretion in single, double and triple transduced cells, whereas albumin-mRNA levels already peaked in the Foxa2-only setting, indicating that maturation of specific secretory functions requires elevated levels of Hnf4α and C/ebpα, too. Ishii et al. recently demonstrated enhanced expression of Albumin, Afp, Tat and epithelial cell adhesion molecule (Epcam) genes in bone marrow derived mesenchymal stromal cells over-expressing only Foxa2. The differentiated cells also showed hepatocyte-specific functions including glycogen production and urea secretion, compared to HUH-7 or HepG2 controls, but no comparison was made with adult primary human hepatocytes [21]. In a second step, Foxa2 transgenic ALDPC were co-transduced with lentiviral vectors expressing either Hnf4α or C/ebpα. The hepatocyte nuclear factor 4α is known as a key regulator of both hepatocyte differentiation during embryonic development and maintenance of a differentiated phenotype in the adult liver [30,31]. Recent studies using hepatocyte-specific Hnf4α-knockout mice have shown that Hnf4α is essential for the generation of hepatic epithelium [31]. The CCAAT/enhancer-binding protein alpha (C/ebpα) maintains the differentiated state of hepatocytes and directs transcription of many genes expressed in the liver such as albumin or ornithine cycle enzymes involved in urea production. Conditional knockdown of C/ebpα revealed an important role in hepatic glucose, nitrogen, bile acid and iron metabolism - all of which represent highly differentiated hepatocyte functions [28]. Cultures of ALDPC transduced with Foxa2, Hnf4α and C/ebpα adopted an epithelial morphology and contained more cells with two or more nuclei, which is a hallmark of adult hepatocytes (Figs. 6 and 7) and indicative of inhibition of full cell division. The expression of liver specific genes in triple transduced ALDPCs approached in many cases levels in the range of the primary hepatocyte day 1 standard (Fig. 3). However, some genes like CYP450 enzymes and Apoc3, although maximally induced in triple transduced cells, have proven to be difficult to up-regulate to levels found in primary hepatocytes. Expression of additional transcription factors, the application of histone deacetylase inhibitors, which have been shown to improve hepatic differentiation of ESCs, and in particular three dimensional culture systems allowing the formation of organ-like epithelial structures, might be required in order to maximally induce the expression of those genes.