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  • Early protocols for the generation of hepatocytes from

    2018-10-24

    Early protocols for the generation of hepatocytes from pluripotent cells relied on the use of embryoid body formation (Baharvand et al., 2006; Basma et al., 2009; Imamura et al., 2004). This method involves the creation of cell p38 pathway and the spontaneous differentiation of the pluripotent population to a mixed population of cells representing the three germ layers (Itskovitz-Eldor et al., 2000). Notable improvements in efficiency and functionality have since been achieved by various groups, which based their protocols on developmental signaling and utilized adherent culture conditions. However, all directed differentiation protocols for hepatocyte-like cells (HLCs) published to date have relied on the use of recombinant growth factors such as activin A, Wnt3a, hepatocyte growth factor (HGF), oncostatin M (OSM), fibroblast growth factor 4 (FGF4), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and bone morphogenetic protein 4 (BMP4) (Agarwal et al., 2008; Brolén et al., 2010; Cai et al., 2007; Chen et al., 2012; Hay et al., 2008; Liu et al., 2010; Si-Tayeb et al., 2010b; Song et al., 2009; Sullivan et al., 2010; Touboul et al., 2010). Some recent progress has been made in replacing growth factors for the differentiation of mesoderm and ectoderm (Chambers et al., 2012; Lian et al., 2012), and efforts have been undertaken to find suitable candidates for the production of definitive endoderm (DE), exemplified by the Melton group, who identified IDE1 and 2 (Borowiak et al., 2009). Recent studies have also demonstrated the utility of other small molecules in priming pluripotent cells for differentiation toward endoderm (Tahamtani et al., 2013) and mesoderm (Tan et al., 2013). To date, however, further endodermal differentiation has only been performed in combination with other recombinant growth factors (for a review, see Han et al., 2012). A number of publications have demonstrated that Wnt signaling is required to specify DE (Engert et al., 2013; Nakanishi et al., 2009), and we have found that a pulse of GSK-3 inhibition is sufficient to produce populations of DE at a high efficiency. We are then able to direct the small-molecule-generated population of DE to functional HLCs with a combination of small molecules, namely DMSO, dexamethasone (DEX), and an HGF receptor agonist N-hexanoic-Tyr, Ile-(6) aminohexanoic amide (dihexa) (McCoy et al., 2013). This stepwise approach is able to generate HLCs at high efficiency, without the inclusion of growth factors (Figure 1). The differentiated cells demonstrate similar levels of function to those derived via published growth-factor-based approaches.
    Results
    Discussion
    Experimental Procedures
    Author Contributions
    Acknowledgments