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    • We have developed a protocol for the efficient differentiati

    2018-10-31

    We have developed a protocol for the efficient differentiation of functional HLCs from hPSCs that does not require the addition of recombinant growth factors and is applicable to both hESCs and hiPSCs. During the development of the protocol, it came as a surprise to discover that pluripotent microcystin lr can be differentiated to DE with a short pulse (24 hr) of CHIR99021, BIO, or Wnt3a without the inclusion of activin A. The rationale to examine GSK-3 inhibition as a route to producing DE has a basis in the observations of Lickert and colleagues (Engert et al., 2013), who demonstrated that Wnt signaling regulates SOX17 expression. A number of other publications also describe the priming activity of GSK-3 inhibitors such as CHIR99021 (Hannan et al., 2013; Kunisada et al., 2012; Tahamtani et al., 2013; Tan et al., 2013). In this study, we clearly demonstrate that GSK-3 inhibition alone is sufficient to produce DE, a finding that in itself represents an opportunity for major saving in hepatocyte-production costs. Importantly, this first stage provided productive DE, which expressed a battery of DE markers including SOX17 and FOXA2, and passed through the equivalent developmental points as growth factor DE (Figure 2A). The use of DMSO to drive DE toward a hepatic fate is well established in the field (Hay et al., 2008; Soto-Gutiérrez et al., 2007; Sullivan et al., 2010), and we show here that it is equally successful when employed following CHIR99021 treatment as with activin A and Wnt3a. We observed a rapid change in morphology, coupled with the appearance of the hepatic progenitor markers HNF4A and AFP, both at the transcriptional and protein levels. The final phase of this protocol was to take hepatic progenitors and provide the environment for hepatocyte maturation in order to generate HLCs. We chose to utilize a combination of two small molecules of the glucocorticoid family, DEX and hydrocortisone-21-hemisuccinate (HC), and assessed the literature for potential mimetics of commonly used growth factors. We identified a potent HGF mimetic called N-hexanoic-Tyr, Ile-(6) aminohexanoic amide, or dihexa, originally developed as an anti-dementia drug (McCoy et al., 2013). This molecule was identified in a screen that assessed the capacity to potentiate the biological activity of HGF. We therefore employed the small molecules DEX, HC and dihexa in phase III of the protocol. Initially, phase III was optimized using the commercially available base medium HepatoZYME that has been shown to provide supportive conditions for hepatocyte maturation (Szkolnicka et al., 2014). We demonstrated the dependency of dihexa in this system (data not shown), but it transpired that HepatoZYME is not growth factor free and contains the morphogen EGF (Garcia et al., 2001). We then translated our findings to the routinely used hepatocyte maturation medium L-15, which others have used supplemented with a combination of growth factors and small molecules (Hay et al., 2008; Sullivan et al., 2010). We supplemented L-15 with the aforementioned small molecules and efficiently generated HLCs from both hESCs and hiPSCs, which displayed typical hepatic morphology and expressed a number of hepatic markers at the transcriptional and protein levels. More importantly, the smHLCs exhibited key functional attributes including serum protein production and cytochrome P450 metabolism. It should be stated that we used the P450-Glo assay system (Promega) to initially assess function with respect to CYP activity as in line with many other publications. But, in order to gain a thorough insight into the metabolic potential of these cells, we will need to assess them using the more robust and accepted methods including mass spectrometry and high-performance liquid chromatography. It should be noted that the above method is not fully defined at this point, as the present L-15 formulation contains 8.3% serum. We are currently investigating avenues to generate a completely defined system for the production of hepatocytes, but the method described here represents a significant step toward this goal and is applicable to both hESC and hiPSC lines. Importantly, it provides a means to cut the costs associated with hepatocyte production. A combination of this small-molecule approach with the steadily advancing field of cell-culture automation may eventually allow for the production of high-quality hepatocytes from stem cells at large scale for industrial and clinical translation. In the meantime, this approach in our hands produces cells of equivalent phenotype and function to current approaches and addresses some of the cost, quality, and supply-chain concerns associated with reliance on recombinant growth factors.