Mammalian mitochondria are composed of approximately differe

Mammalian mitochondria are composed of approximately 1000 different proteins, but only 13 proteins are encoded by the mitochondrial genome. We selected Atp5g, Atp8 and Ucp2 as protein markers of the mitochondrial genome. We observed progressively higher expression of Ucp2, Atp5g and Atp8 in hiPSCs, HLCs, and primary hepatocytes. It has hitherto been believed that the ratio of mtDNA to nuclear DNA reflects the tissue concentration of mtDNA per cell. Control of mtDNA copy number is crucial for successful differentiation of ESCs (Facucho-Oliveira et al., 2007), and presumably also of hiPSCs. With the hepatic differentiation of hiPSCs, we observed an increased mtDNA copy number. MitoTracker staining also rose to reflect the expanded number of mitochondria in the Cycloheximide manufacturer during hepatic differentiation. Our hiPSCs were derived from human cardiac fibroblasts. These parental fibroblasts possessed a large supply of mitochondria. However, reduction and dedifferentiation of mitochondria was essential for proper energy production and for prevention of DNA damage by oxidative stress in their conversion to hiPSCs. In turn, re-differentiation of hiPSCs to HLCs involved resupplying mitochondrial mass to meet the elevated ATP requirements of differentiated hepatocytes (Brown, 1992). It is possible that the difference in maturity profile and functionality between our HLCs and primary hepatocytes can be explained by our original source of somatic cells, the cardiac fibroblasts. Prior work with hiPSCs derived from fibroblasts suggests that mitochondrial numbers returned to their pre-reprogrammed state or lower levels after re-differentiation (Armstrong et al., 2010; Suhr et al., 2010). Whether the lower mitochondrial functionality of our HLCs was due to their cell of origin is unknown. Alternatively, the mitochondrial complement of HLCs may require more time and more replicative cycles to realize its full potential (Suhr et al., 2010). Further work is needed to clarify the role of original somatic cell type and other factors in re-programming iPSCs to new mature cell types.
Conclusion In conclusion, our results confirm that functional HLCs can be generated from hiPSCs. However, our studies suggest that HLCs produced in vitro are not sufficiently mature to serve as ex vivo cell therapies, such as an extracorporeal BAL. Our results support those of Takayama et al. (2012), and suggest that a more native milieu is needed to complete hepatic differentiation of HLCs. Other reports suggest that in vivo transplantation of HLCs derived from hiPSCs can rescue rodents from lethal drug-induced acute liver failure (Chen et al., 2012), reduce liver fibrosis in a mouse model (Asgari et al., in press), enhance liver regeneration in mice (Espejel et al., 2010), and stabilize chronic liver disease (Choi et al., 2011). These in vivo studies suggest that novel techniques are still needed to complete hepatic differentiation of HLCs and expand them in numbers sufficient for human therapy. One possibility for expansion and maturation of HLCs is a genetically engineered large animal, similar but larger than the source of human hepatocytes in our study (Azuma et al., 2007), to serve as an in vivo hepatocyte incubator (Hickey et al., 2011). The future success of ex vivo cell therapies depends on novel techniques to provide an abundant, high quality supply of functionally normal hepatocytes.
Acknowledgements This work was supported by grant(s) from the (NIH P30DK084567), Marriott Foundation, Wallace H. Coulter Foundation, Nature and Science Foundation of Jiangsu Province (10KJB320006 to Y. Yu), the National Natural Science Foundation of China (81070361 to Y. Yu) and Jiangsu Province\'s Outstanding Medical Academic key program (RC2011067 to Y. Yu).
Introduction Maintenance of human embryonic stem cells (hESCs) in an undifferentiated state is realised by a multitude of extrinsic factors whose effects are mediated by overlapping intracellular signal transduction pathways. Those implicated to date have included pathways stimulated by ligands such as basic Fibroblast Growth Factor (bFGF), Wnt and members of the Transforming Growth Factor Beta (TGFβ) superfamily (Okita and Yamanaka, 2006). Binding of bFGF to its cognate receptor normally results in activation of receptor tyrosine kinase activity and subsequent stimulation of the Ras-activated mitogen-associated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K)/Akt/Protein kinase B (PKB) and Phospholipase C-gamma (PLC-γ) pathways, the former two of which have been shown to be active in hESCs (Dvorak et al., 2005; Kang et al., 2005) In contrast, binding of Wnt proteins to cell surface receptors of the Frizzled (FZD) family activates Dishevelled family proteins which in turn inhibit a complex of proteins (axin/GSK3/APC) responsible for the proteolytic degradation of the β-catenin intracellular signalling molecule and transcription co-factor. Stabilising the cytoplasmic pool of β-catenin by pharmacological inhibition of GSK3 promotes embryonic stem cell renewal (Sato et al., 2004). In addition, TGFβ and Nodal, expressed by undifferentiated hESCs, and Activin-A, normally secreted by fibroblast feeders which are supportive of hESC renewal, all work to oppose induction of trophoblast/primitive endoderm differentiation by Bone Morphogenic Protein (BMP)-4, another member of the TGFβ superfamily (Beattie et al., 2005; Sato et al., 2003). This is mediated by the activation of different cell surface receptors (Type II vs I) and downstream transcription factors (SMAD2/3 vs SMAD1/5/8) (James et al., 2005; Valdimarsdottir and Mummery, 2005).