Nodal and Wnt Signaling in Endoderm Formation

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The Role of Activin/Nodal and Wnt Signaling in Endoderm Formation Catherine Payne,* Jason King,† and David Hay* Contents 208 209 210 212 214 214 214

I. Introduction II. TGFb Signaling III. WNT/b-Catenin Signaling IV. Applied Biology V. Conclusions Acknowledgments References

Abstract Human embryonic stem cells (hESCs) are located within the inner cell mass of the preimplantation blastocysts. hESCs exhibit two important properties, the ability to generate exact copies of themselves, termed self-renewal, and pluripotency, the ability of stem cells to differentiate into every cell type of the embryo. This means that in theory it may be possible to generate an inexhaustible supply of primary human somatic cells in vitro which are suitable for application in regenerative medicine. Maintaining stem cell self-renewal and eliciting differentiation are dependent on the coordination of a number of signaling pathways which include members of the transforming growth factor beta (TGFb) and Wnt families. The work in our laboratory has focused on the efficient generation of hepatocyte-like cells (HLCs) from hESCs and induced pluripotent stem cells (iPSCs). In order to mimic signaling during primitive streak and endoderm development, we have utilized TGFb and Wnt signaling pathways in vitro. This has resulted in the generation of homogeneous populations of HLCs exhibiting liver specific function. This chapter will focus on TGFb and Wnt signaling pathways and their role in primitive streak, endoderm, and HLC development. ß 2011 Elsevier Inc.

* MRC Centre for Regenerative Medicine, The University of Edinburgh, Edinburgh, United Kingdom Roslin Cellab, Roslin Biocentre, Roslin, Midlothian, Scotland, United Kingdom

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Vitamins and Hormones, Volume 85 ISSN 0083-6729, DOI: 10.1016/B978-0-12-385961-7.00010-X

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2011 Elsevier Inc. All rights reserved.

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I. Introduction Human development is a complex process involving the integration of multiple signaling pathways regulating gene transcription. These pathways are essential for patterning the embryo and eliciting differentiation to all the three germ layers: endoderm, mesoderm, and ectoderm (Tam and Loebel, 2007). In particular, we will focus on the generation of definitive endoderm (DE) as a precursor to HLCs. DE differentiation is preceded by the ingression of the epiblast (primitive ectoderm) to form the primitive steak (PS) from which both endoderm and mesoderm arise (Ginsburg et al., 1990; Tam and Loebel, 2007; Tam et al., 2004). The two major factors involved in PS formation, Activin/Nodal and Wnt, also play important roles in endoderm and mesoderm specification (Ding et al., 1998; Gadue et al., 2006; Liu et al., 1999; Yamamoto et al., 2001). Following commitment, DE lines the ventral region of the developing embryo forming the foregut, midgut, and hindgut (Kaestner, 2005; Zhao and Duncan, 2005). The foregut endoderm develops into the lung, thyroid, liver, and the ventral rudiment of the pancreas and is patterned by factors secreted from the adjacent mesenchymal structures (Dessimoz and Grapin-Botton, 2006; Lemaigre and Zaret, 2004; Zaret, 2001) in a concentration-dependent manner (Serls et al., 2005). The region of the foregut which becomes the liver bud is adjacent to the cardiac mesoderm (CM) and the septum transversum (ST). Both structures secrete factors which pattern the foregut endoderm (the CM produces FGFs while the ST produces BMPs) (Douarin, 1975; Rossi et al., 2001). In vitro, hESC-derived DE can be differentiated to hepatic endoderm using low levels of FGF2 while intermediate and high concentrations induces a pancreatic cell fate and pulmonary fate, respectively (Ameri et al., 2010). Following specification, the emerging liver bud invades the ST and the resident liver stem cell, the hepatoblast, differentiates into two of the major cell types found in the liver, hepatocytes and cholangiocytes (for a review, see Dancygier, 2010). We have studied human development in vitro and in vivo and applied this knowledge to pluripotent stem cells. In doing so, we have generated a system for deriving large numbers of high fidelity HLCs in vitro. These HLCs exhibit many of the attributes of primary human hepatocytes and will play important roles in developing cell-based assays which model human drug toxicity and disease. Additionally an inexhaustible supply of HLCs has an important role to play in developing cell-based therapies for human liver disease (for a review, see Dalgetty et al., 2009).

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II. TGFb Signaling TGFb signaling plays an important role in embryonic development and involves the interplay of a large number of ligands, regulators, and receptors (Heldin et al., 2009; Kitisin et al., 2007; Fig. 10.1). The TGFb superfamily are soluble ligands, including nodal, Activin A, BMPs, and

Cerberus

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cofactor FoxHI FoxA2 MIX/BIX

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P300/CBP

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Figure 10.1 Smad-mediated pathway of Activin/Nodal and BMP signaling. Activin binding (1) to the Activin receptor (ActR) type II dimer induces coassociation of a type I Activin receptor dimer (in the case of Activin, this is ALK4) which leads to the phosphorylation and activation of ALK4 by the type II receptor’s kinase (3). The Smad 2/3 complex is recruited to ALK4 (4) via interaction with SARA. Phosphorylation of Smad 2/3 (5) results in a conformational change that enables it to bind Smad 4 (7). This complex enters the nucleus (8) and associates with transcriptional cofactors (9) and P300/CBP to drive expression (10). Prior to Nodal binding, a member of the EGFCFC coreceptor family, such as Cripto, must first bind to ALK4 to stabilize the tetrameric ALK4-ActRII receptor complex. Subsequent signaling is via the same Smad 2/3 pathway. BMP signaling also requires assembly of a tetrameric receptor complex; however, these can be more varied involving an additional type II receptor (BMPRII) and three different type I receptors (ALK2, ALK3, and ALK6). Instead of using Smad 2/3þ4, BMP signaling can use Smads 1/5/8þ4. Nodal signaling can be inhibited extracellularly by Lefty and Cerberus. Inhibitory Smads 6 and 7 are strongly induced by Activin, TGF-b, and BMP and act as intracellular antagonists of Smad-mediated signaling.

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TGFb (Kitisin et al., 2007), which bind to type II and type I receptors. Nodal/Activin A bind to the extracellular portion of the Activin receptor type 2 (ActRIIA or ActRIIB) dimer, causing a conformational change and phosphorylation of the glycine serine rich domain (GS) on the Activin receptor type 1B (ALK4). BMPs also bind to several type II receptors (ActRIIA, ActRIIB, and BMPR2) activating the type I receptor (ALK2, ALK3, or ALK6; Heldin et al., 1997; Kitisin et al., 2007; Moustakas, 2002; Fig. 10.1). Following activation, the type I receptor recruits the receptorregulated Smads to the receptor complex through the SMAD anchor for receptor activation (SARA). The activated type I receptors then phosphorylate the intracellular messenger Smads (Heldin et al., 1997). There are at least eight Smad proteins which can be split into three classes: (i) receptoractivated Smads (R-Smads) include Smad 1, Smad 2, Smad 3, Smad 5, and Smad 8/9; (ii) the comediator or common Smad (co-Smad), Smad 4; and (iii) inhibitory Smads (I-Smads) include Smad 6 and Smad 20–22. Following Activin/Nodal signaling, Smads 2 and 3 dissociate from SARA and bind Smad 4. The Smad 2/3/4 complex then translocates to the nucleus and activates gene expression (Fig. 10.1). BMP signaling induces the activation of Smad 1, 5, or 8 which bind to Smad 4 and translocate to the nucleus and regulate gene transcription (Feng and Derynck, 2005; Heldin et al., 1997; Itoh and ten Dijke, 2007; Kitisin et al., 2007; Massague et al., 2005; Moustakas, 2002; Fig. 10.1). Activin/Nodal signaling plays a central role in patterning of vertebrate embryos and in vitro Activin A binds the same receptor as Nodal and likely mimics Nodal signaling (Gadue et al., 2006). The role of Nodal signaling in the mouse during development has been studied using insertional mutagenesis and receptor complex nulls. Both types of mutants failed to form an elongated primitive streak, displaying excessive ectoderm commitment and deficiency in endoderm and mesoderm. This was in part rescued by hypomorphic expression of Nodal revealing a degree of DE differentiation (Lowe et al., 2001). The requirement of high concentrations of Activin/ Nodal signaling in the formation of the primitive streak and subsequently DE was further supported by studies in hESCs (D’Amour et al., 2005) demonstrating a pivotal role of TGFb signaling in human endoderm development.

III. WNT/b-Catenin Signaling Wnt signaling also plays an important role in embryonic development. The Wnt signaling pathway is a complex network involving a large number of ligands, regulators, and receptors (Logan and Nusse, 2004; Moon et al., 1997; Fig. 10.2). There are at least 19 Wnt genes and 10 frizzled receptors

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Figure 10.2 Wnt/b-Catenin signaling pathway. In the absence of Wnt, LRP is bound by Dickkopf (DKK) and prevented from interacting with Frizzled. b-Catenin is bound by CK1a, Axin, GSK3b, and APC and phosphorylated to establish the “Destruction ¨ b-Catenin to the proteasome for degradation. When Wnt Complex” which targets binds Frizzled, LRP and Disheveled (DVL) associate with Frizzled and promote the dissociation of the destruction complex, to release APC and enable the remaining proteins to form a complex with LRP/Frizzled/DVL. b-Catenin is then free to translocate to the nucleus where it displaces the GRO/HDAC/CTBP repression complex from the TCF (T cell factor)-promoting expression.

which are part of the G protein-coupled receptor family which can be broadly divided into canonical and noncanonical signaling pathways. The canonical Wnt pathway involves the binding of Wnt to its cell-surface receptor, Frizzled, and its coreceptor low density lipoprotein receptor protein (LRP5/6). This causes the activation of the Dishevelled (DSH) family proteins, which in turn recruits axin to the plasma membrane and inhibits the assembly of the destruction complex (Axin, GSK3b APC, and CKI) which promotes b-Catenin stability and subsequent nuclear translocation (Logan and Nusse, 2004; Moon et al., 1997). In the nucleus, b-Catenin interacts with the T cell factor/lymphocyte enhancing binding factor (TCF/LEF) family of transcription factors displacing the transcriptional repressor Groucho and HDAC (Fig. 10.2; Arce et al., 2006; Clevers, 2006; Moon, 2005; van Noort and Clevers, 2002; Wu and Nusse, 2002). In the absence of Wnt signaling, b-Catenin is tagged for degradation by the

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“destruction complex” and does not lead to transcriptional activation by b-Catenin (Clevers, 2006; Kimelman and Xu, 2006). There are other Wnt pathways which do not involve the frizzled receptor/b-Catenin pathway and are referred to as noncanonical. This signaling pathway either elicits an increase in intracellular calcium through interactions with protein kinase C or activates small Rho GTPases, such as c-Jun N terminus kinase ( Jnk) (Kuhl et al., 2000; Nateri et al., 2005; Topol et al., 2003; Westfall et al., 2003). For the simplicity, we will concentrate on canonical signaling and its role in PS formation. Canonical Wnt signaling has been shown to play important role in PS and endoderm development through knockout studies. The effects of knocking out the intracellular messenger, b-Catenin, have been studied in mice. While b-Catenin null embryos formed blastocysts that implanted and developed to egg cylinder stage embryos, they did not form the primitive streak, mesoderm, or endoderm (Haegel et al., 1995). Further work in the field demonstrated that Wnt3 was essential for PS formation demonstrating the importance of Wnt3 in primary axis formation in the mammals (Liu et al., 1999). More recently, canonical Wnt signaling was shown to be essential in the proper development of the liver bud in zebra fish (Ober et al., 2006). In support of this, canonical Wnt signaling has been shown important in human liver development and hESC differentiation to both PS and HLCs (Hay et al., 2008).

IV. Applied Biology The coordinated expression and function of both Activin/Nodal and Wnt are essential for proper PS and derivative cell-type formation in both mouse and human development (Gadue et al., 2006; Hay et al., 2008). When coupled to pluripotent stem cells, it provides the biologist with an enormous potential to generate efficient levels of endoderm or mesoderm derivatives for in vitro and in vivo experimentation. In this section, we will discuss the importance of this technology with a focus on the generation of HLCs from pluripotent stem cell populations via the PS. The real impact of deriving hepatocyte-like cells (HLCs) from pluripotent stem cell is the provision of a resource from the desired genetic background. The current gold standard are primary human hepatocytes which are human in origin and possess broad range function upon isolation. Unfortunately, high-level liver function is lost within 24–48 h of isolation. Other limitations include limited supply and expense. Alternative approaches have employed primary rat, porcine, and murine hepatocytes which are useful, but often not indicative of human liver function and also possess limited clinical application. Transformed human hepatocytes have

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also been used to model human liver function and while the lines can be propagated in vitro, they exhibit poor differentiated function which limits their use (Sharma et al., 2010). Therefore the provision of high fidelity HLCs from a renewable and scalable source, such as hESC or iPSC, would benefit a number of downstream regenerative medical applications playing a role in society’s health and wealth. The accurate prediction of human drug toxicity is one example. The costs of successful drug development are heavily influenced by compound attrition rate. For every new drug that reaches the market, 5000–10,000 compounds have been tested in preclinical trials. Therefore, any strategies which streamline and standardize the process of predictive toxicology testing would impact drug attrition levels and therefore cost. Stem cell-derived liver technology could also be applied to the creation of models which allow us to model “disease in a dish.” The recent advances in cellular reprogramming of human somatic cells have allowed us to generate iPSCs from human somatic cells (for a review, see Dalgetty et al., 2009). This in essence has given us the ability to create human libraries of stem cells which display the genetic background of interest. We have shown that it is possible to translate the know-how developed in hESCs and apply that to iPSCs to efficiently deriving hepatocytes (Sullivan et al., 2010) which hold great potential in developing a better understanding of human liver toxicity and disease. Presently, end stage liver disease is on the increase in the Western world. Although highly successful, orthotopic liver transplantation is not the answer to this problem due to limited availability of organs. This is compounded by the dire prediction that these incidences to liver disease will rise sharply in the next 10 years. As such, there is an urgent need to develop alternative treatment strategies. One example of this is the bioartificial liver (BAL) device, which is viewed as a pragmatic approach to this problem (for a review, see Carpentier et al., 2009). While a number of groups have developed promising technologies, the ability to translate those to a cost-effective good manufacturing practice (GMP) BAL is proving difficult. This is mainly due to the numerous biological issues associated with primary hepatocytes and cell lines and may benefit from large-scale production of hESC- or iPSC-derived HLCs. Another approach to treating liver disease, and probably the most challenging, is the provision of functional mass by cell transplantation. Encouraging experiments have shown previously that primary adult hepatocytes successfully transplant into diseased organs resulting in a modest benefit (Bilir et al., 2000; Strom et al., 1997). However, the issues of primary human hepatocyte scarcity and heterogeneity mean that this approach is not feasible in the clinic. While hESC HLCs offer great potential here, there are numerous cell culture challenges that face this strategy, such as their defined, cost-effective, and GMP scalable production (Hannoun et al., 2010). Probably, the greatest challenge that faces cell therapy in the liver and other tissues is the safe transplantation of the derivative cells without tumor formation in vivo (Basma et al., 2009).

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V. Conclusions Applying the knowledge acquired from a number of developmental studies has proved vital in developing high fidelity somatic cell models from pluripotent stem cells. In particular, we have studied Activin/Nodal and Wnt pathways which play an important role in primitive streak, DE, and HLC formation from both hESC and iPSCs. As such, pluripotent stem cellderived HLCs are likely to revolutionize the way in which we model human liver development and disease, measure human drug toxicity, and provide novel cell therapies for treating human disease.

ACKNOWLEDGMENTS Drs. Payne and King were funded by a grant from the UK Stem Cell Foundation and Scottish Enterprise. Dr. Hay was funded by a RCUK Fellowship.

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