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    • To address the question of whether these HLCs

    2018-11-12

    To address the question of whether these HLCs could serve in cellular therapies, a variety of markers of hepatocyte differentiation were considered. For example, production of albumin, a classic marker of hepatocyte differentiation, was observed in HLCs (Fig. 2). However, production of albumin has been shown to be non-predictive of response to cell-based therapy (Dunn et al., 1991; Koike et al., 1996; Nahmias et al., 2007). Alternatively, mitochondria play a crucial role in many cellular functions relevant to cell based therapy including energy production, differentiation, apoptosis, and ammonia detoxification. Mitochondria are the site of several enzymes of the urea cycle and thus essential to ammonia detoxification and prevention of hepatic encephalopathy. Our data indicate significant maturation of mitochondria in HLCs compared to their hiPSCs precursors. However the mitochondria of HLCs remain noticeable less mature than the gold standard - primary hepatocytes. Our experience with hiPSCs and HLCs is analogous to the maturation of ESCs. Reviewed evidence suggests that respiratory activity in mitochondria of ESCs is low to minimize generation of reactive oxygen species which may damage and mutate DNA during early development (Parker et al., 2009). As stem Obeticholic Acid of the early embryo commit to differentiation, their mitochondria undergo dramatic functional maturation. Successful differentiation of mammalian ESCs involves transcription and replication of the mtDNA genome, replication of mitochondria, and up-regulation of enzymes required for aerobic metabolism (Chen et al., 2008; Cho et al., 2006; Chung et al., 2007; Schieke et al., 2008; Suhr et al., 2010). These changes are needed to fulfill the elevated ATP requirements of fully differentiated cells. In the case of primary hepatocytes, they are rich in mitochondria to fulfill their many vital roles including metabolism, synthesis of blood proteins and biotransformation. Our results suggest that HLCs derived from hiPSCs are capable of many of these functions, but they do not appear to detoxify ammonia to urea under ex vivo conditions. Our results also demonstrate that cultured HLCs express higher levels of AFP and lower levels of albumin than human hepatocytes produced by in vivo expansion in genetically engineered mice (Azuma et al., 2007). Our observations are similar to other reports of significant but lower than normal production of albumin by HLCs (Cai et al., 2007; Sancho-Bru et al., 2011; Si-Tayeb et al., 2010; Song et al., 2009). The high levels of AFP production in HLCs are unexplained, but suggest that HLCs exhibit an inability to turn off early stage gene(s) as the mechanism of persistent immature phenotype. As mentioned above, the liver is a major site of detoxification, and ammonia is arguably the most important endogenous toxin which requires clearance by the liver (Denis et al., 1983). Ammonia accumulation causes cerebral edema, the most feared complication of acute liver failure (Haussinger et al., 1992). A functioning urea cycle must be present for ammonia to be effectively detoxified and eliminated from the body (Ytrebo et al., 2009). Therefore, ammonia removal and urea production are important considerations for ex vivo cell therapies. Our data indicate that HLCs are inferior to primary hepatocytes at both ammonia detoxification and ureagenesis. The CYP enzymes, found in high levels in the human liver, are also important to detoxification of endogenous and exogenous wastes. Our HLCs expressed high levels of mRNA from CYP3A4 and CYP2C19 and detectable CYP3A4 and CYP2C19 enzyme activity. However, the CYP activities of HLCs were significantly below that of primary human hepatocytes. Mitochondria play a vital role in the balance between pluripotency, cellular differentiation, and replication (Armstrong et al., 2010). TFAM, POLG and POLG2 are the key genes in regulating mtDNA replication. All three genes are nuclear-encoded, but produce transcription factors which are translocated to the mitochondria. We observed that the expression of POLG and POLG2 increased progressively in hiPSCs, HLCs, and primary hepatocytes; however, expression of TFAM declined with maturity. Our data is consistent with reports that an increase in the number of TFAM molecules above the optimal mtDNA:TFAM stoichiometry has inhibitory effects on mtDNA transcription and replication (Garstka et al., 2003; Webb and Smith, 1977). Therefore, a reduction in levels of TFAM protein may explain, in part, the increased transcription of mtDNA through the 4 stages of differentiation (Kanki et al., 2004).