Small-Molecule-Driven Hepatocyte Differentiation of Human Pluripotent Stem Cells

“…well documented that BIO and CHIR99021 are potent pharmacological GSK3-specific inhibitors that result in activation of the Wnt signaling pathway (Sato et al., 2004; Sineva and Pospelov, 2010), so we next assessed the ability of the protein Wnt3a alone to drive differentiation toward DE. Figure…”

“Meanwhile, cell culture and production technologies are rapidly improving, and the first instances of pluripotent-cellderived therapies have entered clinical trials (Schwartz et al., 2012).”

“…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).”

“…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).”

“…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…”

“We monitored developmentally relevant markers of primitive streak (T, MIXL1, GSC, and FOXA2), mesendoderm (T and FOXA2), and definitive endoderm (HHEX, CER1, SOX17, and FOXA2).”

“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 a…”

“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).”

“…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).”

“…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).”

“We finally settled on a modified formulation of Leibovitz L-15 medium (L-15), which has been described in the literature as a standard method to generate mature hepatocytes (Hay et al., 2008; Sullivan et al., 2010).”

“There are numerous reports of the utility of the small molecule DMSO in stem cell differentiation and specifically in the generation of hepatic progenitors (Hay et al., 2008; Rambhatla et al., 2003; Soto-Gutierrez et al., 2006; Sullivan et al., 2010).”

“We finally settled on a modified formulation of Leibovitz L-15 medium (L-15), which has been described in the literature as a standard method to generate mature hepatocytes (Hay et al., 2008; Sullivan et al., 2010).”

“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).”

“…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).”

“…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…”

“4 j 939–952 j May 12, 2015 j a2015 The Autho there was an increase in expression of PROX1 and TBX3, which are believed to interact in vivo to promote themigration and proliferation of hepatoblasts from the primary liver bud (Sosa-Pineda et al., 2000; Lüdtke et al., 2009).”

“We monitored developmentally relevant markers of primitive streak (T, MIXL1, GSC, and FOXA2), mesendoderm (T and FOXA2), and definitive endoderm (HHEX, CER1, SOX17, and FOXA2).”

“…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).”

“…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).”

“We observed a rapid upregulation of NODAL within 4 hr of exposure to CHIR99021, which is indicative of a transition toward a PS population (Figure 3) (Lu et al., 2001).”

“Phase I of differentiation consists of a 24-hr treatment with RPMI-B27 ± insulin (RPMI 1640 GlutaMAX + B27 supplement, both from Life Technologies) plus 3–4 mM CHIR99021 (Stemgent), followed by a 24-hr treatment with RPMI-B27 alone.”

“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.”

“…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…”

“…of PROX1 and TBX3, which are believed to interact in vivo to promote themigration and proliferation of hepatoblasts from the primary liver bud (Sosa-Pineda et al., 2000; Lüdtke et al., 2009). rs The human embryonic stem cell line H1 was differentiated in RPMI-B27 supplemented with 3 mM CHIR99021.”

“Initially, phase III was optimized using the commercially available base mediumHepatoZYME that has been shown to provide supportive conditions for hepatocyte maturation (Szkolnicka et al., 2014).”

“To corroborate that the source of the HLCs is from DE rather than from yolk sac, we demonstrated that the DE origin marker CYP7A1 was expressed (Asahina et al., 2004) (Figure 6F).”

“The rationale to examine GSK-3 rs 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.”

“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).”

“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).”

“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 Cell Reports j Vol. 4 j 939–952 j May 12, 2015 j ª2015 The Authors 939 (A) The normal…”

“…BIO and CHIR99021 are potent pharmacological GSK3-specific inhibitors that result in activation of the Wnt signaling pathway (Sato et al., 2004; Sineva and Pospelov, 2010), so we next assessed the ability of the protein Wnt3a alone to drive differentiation toward DE. Figure 2C demonstrates…”

“There are numerous reports of the utility of the small molecule DMSO in stem cell differentiation and specifically in the generation of hepatic progenitors (Hay et al., 2008; Rambhatla et al., 2003; Soto-Gutierrez et al., 2006; Sullivan et al., 2010).”

“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).”

“H1 hESCs (WiCell) and 207 hESCs (Ström et al., 2010) and Detroit 551 (ATCC CCL-110) hiPSCs were maintained at 37 C/5% CO2 in feeder-free conditions using growth-factor-reduced Matrigel (Sigma-Aldrich) and E8 Medium (Life Technologies), with routine passaging performed at a 1:3 ratio using 0.5 mM…”

“See also Figure S2. progenitors, we observed maintenance of FOXA2 levels and an induction of GATA4, both of which are known to be pioneer factors critical to promote the hepatic gene expression program (Kaestner, 2005).”

“Following studies of the utility of GSK-3 inhibition in priming pluripotent cells for endodermal differentiation (Tahamtani et al., 2013) and reports that Wnt/b-catenin signaling regulates SOX17 expression and is essential for endoderm formation (Engert et al., 2013), we set out to establish…”

“However, HepatoZYME is a proprietary reagent and contains the growth factor EGF and thus was unsuitable for this application (Garcia et al., 2001).”

“Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) offer a potentially limitless source of cells for industrial and clinical translation, and the everadvancing field of cellular reprogramming has redefined the limits of cell plasticity (Taylor et al., 2010).”

“Other studies have used small molecules to replace growth factors to drive down the cost of the method (Hay et al., 2007, 2008; Siller et al., 2015); however, those publishedmethods both still rely upon the use of undefined components, highlighting the need to develop defined systems.”