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© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 752-760 doi:10.1242/dev.097386

ABSTRACTWith the high prevalence of gastrointestinal disorders, there is greatinterest in establishing in vitro models of human intestinal diseaseand in developing drug-screening platforms that more accuratelyrepresent the complex physiology of the intestine. We will review howrecent advances in developmental and stem cell biology have madeit possible to generate complex, three-dimensional, human intestinaltissues in vitro through directed differentiation of human pluripotentstem cells. These are currently being used to study humandevelopment, genetic forms of disease, intestinal pathogens,metabolic disease and cancer.

KEY WORDS: Directed differentiation, Embryonic stem cells,Gastrointestinal disease, Gut tube, Intestinal morphogenesis,Pluripotent stem cells

IntroductionThe intestinal tract is one of the most architecturally andfunctionally complex organs of the body. The human intestine is 8 min length (Hounnou et al., 2002) and is subdivided into fivefunctional domains along the proximal-to-distal axis: the duodenum,jejunum and ileum are segments of the small intestine; and thececum and colon make up the large intestine. The intestinecomprises specialized cell and tissue types from all three germlayers. Intestinal tissues include endoderm-derived epithelium,which houses specialized intestinal stem cells (ISCs) (Sato andClevers, 2013), mesoderm-derived smooth muscle, vasculature,lymphatics and immune cells, and the ectoderm-derived entericnervous system (ENS). The contributions from all of the germ layersare required to help coordinate a myriad of complex intestinalfunctions.

The intestine is best known for its digestive function to orchestratethe breakdown of macronutrients, regulate absorption and secretewaste. Less appreciated is the central role of the intestinal endocrinesystem in coordinating systemic nutrient levels and feeding behaviorwith other organ systems via endocrine hormones. In addition, theepithelium of the intestine acts as a selective barrier by restrictingmicrobes to the gut lumen (Turner, 2009). These epithelial functionsare largely carried out by four cell types: absorptive enterocytes andthe three secretory lineages. Secretory cells involved in barrierfunction include goblet and Paneth cells, which secrete mucin andantimicrobial factors. The enteroendocrine cells are a rare butdiverse population of cell types that secrete hormones that regulatesatiety, motility, absorption, β-cell proliferation, secretion of other

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1Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center,3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA. 2Division ofEndocrinology, Cincinnati Children’s Hospital Medical Center, 3333 BurnetAvenue, Cincinnati, OH 45229-3039, USA. 3Department of Internal Medicine,University of Michigan Medical School, Ann Arbor, MI 48109, USA. 4Departmentof Cell and Developmental Biology, University of Michigan Medical School, AnnArbor, MI 48109, USA. 5Center for Organogenesis, University of Michigan MedicalSchool, Ann Arbor, MI 48109, USA.

*Author for correspondence (james.wells@cchmc.org)

hormones and digestive enzymes, among other things (Engelstoft etal., 2013). The epithelium of the intestine turns over every 5 days inmice, and this regenerative capacity is driven by a residentpopulation of ISCs (Creamer et al., 1961; Sato et al., 2009). Themechanical functions of the intestine are controlled by complexinteractions between the epithelium, smooth muscle and ENS, whichregulate peristalsis to ensure unidirectional movement of luminalcontents.

Given the cellular and functional complexity of the intestine, it isno wonder that there are a staggering number of people impacted byintestinal dysfunction. Common intestinal disorders includeinfections, irritable bowel syndrome (IBS), malabsorption and fecalincontinence. Other debilitating diseases include inflammatorybowel disease (IBD), colon cancer and diseases that, in some cases,have a genetic basis, such as Hirschsprung’s Disease. Additionally,since most oral drugs are absorbed in the intestine, the mostcommon drug side effects are intestinal. Given the plethora offunctions carried out by the intestine, there is significant interest inpreventing or reversing intestinal disease by manipulation ofintestinal cell biology; for example, by stimulating ISC growth as ameans to protect or re-establish epithelial integrity and barrierfunction following injury (Zhou et al., 2013). However,gastrointestinal (GI) disease research has largely relied on in vivoanimal studies, which are intrinsically low throughput andsometimes do not adequately mimic human physiology. Therefore,in vitro-derived human intestinal tissues represent a powerful toolfor functional modeling of congenital defects in human intestinaldevelopment, preclinical screening for drug efficacy and toxicitytesting, and for establishing models to study the mechanistic basisof diseases including IBD and cancer.

In this Primer, we discuss the current understanding of intestinedevelopment and how this information has been used to direct thedifferentiation of human pluripotent stem cells (PSCs) into intestinaltissue in vitro. This approach requires the temporal manipulation ofsignaling pathways in a step-wise manner that recapitulates earlyintestinal development (Fig. 1). These main developmental stepsinclude the formation of definitive endoderm, posterior endodermpatterning, gut tube formation, and intestinal growth andmorphogenesis (Fig. 2). Success in this area is largely due to a shiftfrom two- to three-dimensional growth conditions and the presenceof mesenchymal cell types that result in a level of tissue complexitythat more closely resembles the developing intestine in vivo. We willalso evaluate how these tissues can be used to model humanintestinal development and disease.

The first step: endoderm formationGenerating intestinal cells from PSCs requires a step to mimic theprocess of gastrulation and formation of definitive endoderm (DE)(Fig. 1). Studies of gastrulation using frog, fish, chick and mouseembryos have been essential in identifying a conserved molecularpathway that directs gastrulating cells into the endoderm andmesoderm lineages (reviewed in Zorn and Wells, 2009; Zorn andWells, 2007). Central to these processes is the Nodal family of

How to make an intestineJames M. Wells1,2,* and Jason R. Spence3,4,5

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TGFβ signaling proteins, which in frog embryos act both to initiategastrulation and to guide the specification of the mesoderm andendoderm lineages in a dose-dependent manner (Green and Smith,1990). Nodal acts through its cognate Type I (Alk4/7) and type II(ActRIIA or B) receptors, and also requires the Crypto co-receptorfor robust activation of signaling in vivo (Gritsman et al., 1999). Theserine-threonine kinase activity of the Nodal-receptor complexmediates phosphorylation and nuclear localization of a Smad2/3/4complex. Once in the nucleus, Smads interact with transcriptionalco-factors such as FoxH1/FAST1 or Mix-like homeodomainproteins to activate a highly conserved endoderm gene regulatorynetwork (Hoodless et al., 2001; Tremblay et al., 2000). The core ofthis transcriptional network in vertebrates contains the transcriptionfactors Sox17, Foxa2, Mix, Gata4/6 and Eomes. Although the exactroles of these factors might vary slightly between vertebrates, theyact to coordinate the induction the endoderm lineage (Sinner et al.,2006).

Establishing posterior identity from the DEAt the end of gastrulation there are four major signaling pathwaysthat promote the posterior patterning of the vertebrate embryo:Wnt, Fgf, retinoic acid (RA) and Bmp. In Xenopus embryos, highlevels of β-catenin dependent Wnt (Wnt/β-catenin) signalingpromotes posterior gene expression in endoderm while at the sametime inhibiting anterior gene expression in the posterior (McLin etal., 2007; Zorn and Wells, 2009). Conversely, repression of Wntby the Sfrp family of secreted Wnt antagonists is required to helpestablish the stomach-duodenum boundary (Kim et al., 2005).Bmp ligands are expressed in the posterior mesoderm and act in aparacrine manner to promote posterior fate of endoderm in frog,fish and chick embryos (Kumar et al., 2003; Rankin et al., 2011;Roberts et al., 1995; Tiso et al., 2002). Similarly, Fgf ligands that

are expressed in posterior mesoderm act on adjacent ectoderm andendoderm to promote a posterior fate (Dessimoz et al., 2006; Wellsand Melton, 2000). Finally, RA is also known to promote posteriorpatterning of endoderm (Bayha et al., 2009; Huang et al., 1998;Niederreither et al., 2003; Stafford and Prince, 2002; Wang et al.,2006b).

One of the primary means by which these pathways promote aposterior fate is through direct regulation of Caudal homeobox (Cdx)genes, first identified as master regulators of posterior fate inDrosophila (Macdonald and Struhl, 1986; Mlodzik et al., 1985).One family member, Cdx2, is expressed in a temporally andspatially dynamic manner, first being expressed in all three germlayers in early gestation, then becoming restricted to the intestinalepithelium by midgestation. Initial insights into the critical role forCdx2 during gut/intestine development came through tetraploidcomplementation assays (Chawengsaksophak et al., 2004). Theseexperiments showed that Cdx2-null mesoderm and neurectodermwere properly specified, whereas the hindgut failed to expressendodermal Shh and had delayed formation of the hindgut.Subsequent studies showed that conditional deletion of Cdx2 inendoderm using Foxa3-Cre resulted in mutant mice that formed agut tube that was truncated at the cecum, completely lacked thecolon and terminated in a blind-ended sac. Mutant intestines failedto undergo villus morphogenesis and expressed foregut-specificgenes and an esophageal program, with the epitheliummorphologically resembling the squamous epithelium of theesophagus. Collectively, these data suggested that in the absence ofCdx2, a foregut program is ectopically activated in the region of theintestine (Gao et al., 2009).

Studies in vitro and in vivo suggest that Wnt signaling mediatesposterior fate by regulating Cdx2 expression. For example,electroporation of a constitutively active form of β-catenin in the

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Fig. 1. Intestinal differentiation and morphogenesis in a dish. (A) Directed differentiation of human PSCs into human intestinal organoids (HIOs).Pluripotent stem cells (PSCs) are first differentiated into definitive endoderm (DE) (yellow) and, during differentiation, a small population of cells differentiateinto mesoderm (red). Upon the activation of WNT and FGF signaling, endoderm begins to express gut-specific transcription factors (CDX2, red nucleus), whichpersists in the epithelium throughout intestinal development. In addition, the mesenchymal cells proliferate and coalesce with the endoderm to form three-dimensional (3D) ‘spheroids’, consisting of a mesenchymal layer and a polarized epithelial layer with a lumen. Spheroids are then grown under 3D conditions in vitro and form HIOs. HIOs contain most epithelial cell types of the developing intestine, including goblet cells, Paneth cells, enteroendocrine cells andenterocytes. Other cell types have not been explicitly identified. The mesenchyme (light red) also differentiates into smooth muscle and fibroblastic cell types.(B) Directed differentiation of human PSCs (far left image shows one colony) is induced by the nodal mimetic activin A, resulting in the formation ofSOX17+/FOXA2+ DE. Human DE is then differentiated into CDX2+ gut/intestinal tissue by inducing high levels of FGF and WNT signaling (for example, byusing recombinant FGF4+WNT3A). During this differentiation process, endoderm and mesoderm undergo morphogenesis to form CDX2+ tube-like structures(middle panel) and 3D spheres that delaminate from the tissue culture dish (fourth image from the left shows a delaminated spheroid). Three-dimensionalspheroids contain CDX2+ endoderm and mesoderm, and are grown in a 3D matrix in the presence of EGF, noggin (NOG) and R-spondin (RSPO1). Over thecourse of 1 month, spheroids expand and differentiate into HIOs, which contain multiple differentiated cell types. HIOs can be repeatedly passaged every 10-14 days for more than 1 year.

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mouse foregut endoderm, genetic activation of β-catenin orstabilization of β-catenin using a Gsk3b inhibitor at E8.25 resultedin ectopic induction of Cdx2 and repression of Sox2 in the foregut(Sherwood et al., 2011). Fgf, RA and BMP signaling pathwayssimilarly regulate posterior endoderm specification by regulatingexpression of the Cdx genes (Bayha et al., 2009; Dale et al., 1992;Dessimoz et al., 2006; Keenan et al., 2006; Kinkel et al., 2008;Kumar et al., 2003; Lickert and Kemler, 2002; Northrop andKimelman, 1994). In many cases, these pathways directly regulateCdx expression through Wnt, RA and Fgf responsive elements(Haremaki et al., 2003; Rankin et al., 2011; Tiso et al., 2002).Finally, Cdx factors feed back on these posteriorizing pathways byregulating expression of key signaling components such as Wnt3,Fgf8 and the RA synthesizing enzyme Cyp26a1. These data suggestthat there is a posterior regulatory network involving the synergisticactivities of Fgf, Wnt and RA, which act to regulate expression ofthe Cdx family of transcription factors.

Using embryonic patterning and inductive cues to generateintestinal tissue in vitroActivation of Nodal signaling has been the primary approach togenerate DE cells from mouse and human embryonic stem cells(ESCs) (D’Amour et al., 2005; Kubo et al., 2004). This requiresculturing ESCs in high levels of activin A (100 ng/ml) that, over thecourse of 3 or 4 days, result in monolayer cultures of DE cellsexpressing endoderm markers such as Sox17 and Foxa2. The reasonmost protocols use the nodal mimetic activin is because it does notrequire the crypto co-receptor and is many-fold more active thannodal in promoting endoderm formation from human PSCs or inXenopus animal caps (Tada et al., 2005). Although the DE derivedfrom Nodal verses activin treatment is remarkably similar, recentstudies indicate that there are some differences in gene expressionand the in vivo differentiation potential of activin versus nodal-generated DE (Chen et al., 2013). In part, this could be due to thesignificant differences in the activity of activin versus nodal as thelevels of nodal signaling can impact the anterior-posterior (A-P)nature of the endoderm. For example, high levels of nodal/activin

signaling in the anterior primitive streak of mice promote anteriorendoderm fate (Chen et al., 2013; Spence et al., 2011b). Consistentwith this, longer activin treatment directs ESCs into an anteriordefinitive endoderm fate. Further differences between nodal andactivin-generated DE could be due their differential ability tosynergize with Wnt signaling to promote anterior endoderm fate, ashas been shown in fish and frogs (Ho et al., 1999; Zorn et al., 1999),and in ESCs (Sumi et al., 2008).

Manipulation of the posterior regulatory network has been thefocus of efforts to direct the intestinal differentiation of mouse andhuman PSC-derived DE (Ameri et al., 2009; Cao et al., 2010;McCracken et al., 2011; Ogaki et al., 2013; Sherwood et al., 2011;Spence et al., 2011b; Ueda et al., 2010). Activation of the Wntpathway in DE cultures derived from mouse ESCs directs aposterior fate, as demonstrated by Cdx2 expression. However, Caoand colleagues reported that Cdx2 expression required the presenceof fibroblast-conditioned medium (Cao et al., 2010), suggesting thatother signaling factors secreted by fibroblasts were acting with Wntto promote posterior specification. Indeed, WNT and FGF pathwayswere shown to act synergistically to induce a posterior fate in humanPSC cultures, as marked by broad CDX2 expression (Fig. 1B)(McCracken et al., 2011; Spence et al., 2011b). Moreover, thesestudies demonstrated there was a temporal signaling requirementsuch that prolonged activation of the FGF and WNT signalingpathways together was required to irreversibly specify a posteriorfate. For example, extended treatment of human DE cultures withFGF4 and WNT3A proteins for 4 days was required for stable andirreversible CDX2 expression, and was crucial for intestinalspecification. By contrast, shorter treatment for 2 days resulted intransient, reversible induction of CDX2 and was not sufficient forintestinal specification (Spence et al., 2011b). As discussed above,the WNT and FGF pathways may have multiple nodes ofintersection with RA and BMP signaling during establishment of theposterior axis in embryos. Thus, it seems reasonable to expect acombinatorial role for these pathways in activating the posteriorregulatory network in PSC cultures; however, this has not yet beendemonstrated.

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Fig. 2. Gut tube formation in thedeveloping mouse embryo. Three primarygerm layers, mesoderm (Mes), ectoderm (Ec)and endoderm (En), are specified duringgastrulation. The gut tube is specified fromthe endoderm, which has a flat andsquamous-like morphology (left, E7.5). Thedeveloping gut tube epithelium forms alongthe anterior-posterior axis (middle, E8.5),stained here by whole-mount in situhybridization for NM_029639 (Moore-Scott etal., 2007). At this point in time, the anteriorendoderm has formed the foregut tube, theposterior endoderm has formed hindgut tubeand the middle region is forming the midgut.Stained sections of the mid/hindgut regionshow that the epithelium has acquired acuboidal-like morphology. Gut tube formationis completed by E9.5 in the mouse embryo(right), with the pseudo-stratified epitheliumfully encased in mesoderm-derivedmesenchyme. Staining for desmoplakinhighlights the pseudo-stratified morphology ofthe gut tube. The boxed area and linesindicate the regions or sections enlarged inthe panels underneath.

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Contribution of other germ layers to the developingintestineThe intestines are assembled from progenitors arising from thethree germ layers. Although most studies focus on the endoderm-derived epithelium, the mesoderm and ectoderm-derived tissuesplay central roles in intestinal development and function. Themesoderm that gives rise to the intestinal mesenchyme is generatedduring gastrulation and is distributed laterally along the trunk ofthe vertebrate embryo. This lateral plate mesoderm separates intosomatic and splanchnic mesoderm, the latter of which is a distinctpopulation of cells adjacent to the developing intestinal epitheliumprior to gut tube closure in the avian embryo (Thomason et al.,2012). As the gut tube closes and the epithelium matures, thesplanchnic mesoderm forms a mesenchyme that encases theprimitive gut tube. This mesenchyme forms multiple layers withdistinct cell types and functions, including subepithelialmyofibroblasts and fibrobasts, as well as circular and longitudinalsmooth muscle. In addition, the mesoderm-derived endothelialcells and pericytes form a complex vascular plexus that requires aclose proximity to epithelial cells for nutrient and hormonetransport (Powell et al., 2011; Zorn and Wells, 2009; Zorn andWells, 2007).

As well as giving rise to multiple cell types, the mesenchyme isalso a crucial player in villus formation (Mathan et al., 1976;Karlsson et al., 2000) (see Fig. 3). Aggregates of mesenchymeadjacent to the intestinal epithelium are first visible at E14.5, andexpress platelet-derived growth factor A (Pdgfra) (Karlsson et al.,2000). At later stages, as the epithelium remodels to give rise to thevilli projecting into the lumen, the mesenchymal clusters remainassociated with the tip of the villus epithelium and express severalsignaling ligands, including Bmp2 and Bmp4. In addition, both theepithelium adjacent to the mesenchymal clusters and the clusteritself appear to be non-proliferative, suggesting that clusters may actas a signaling center to coordinate villus emergence and withdrawfrom the cell cycle (Karlsson et al., 2000). Consistent with this,inhibition of Bmp signaling leads to excessive proliferation andectopic formation of crypt-like structures on the villus epithelium,similar to what is seen in human juvenile polyposis (Haramis, 2004;Batts et al., 2006).

More recent work has demonstrated that mesenchymal clustersrespond to, and are dependent on, epithelial hedgehog (Hh)signaling (Walton et al., 2012). Hh signaling has been previouslyshown to be crucial for villus morphogenesis (Madison et al., 2005;Mao et al., 2010). The study by Walton and colleagues described amechanism by which Hh signaling controls the size and formationof Pdgfra+ mesenchymal clusters, thus allowing villusmorphogenesis to proceed. In the absence of Hh signaling, both themesenchymal clusters and the villi fail to form (Walton et al., 2012).In addition to cluster formation, new work has also revealed theimportance of the mesenchyme-derived intestinal smooth musclelayers, which generate compressive forces necessary for intestinalfolding seen in a variety of vertebrates, and which leads to anincreased intestinal surface area (Shyer et al., 2013).

Ectoderm-derived tissue is also crucial for intestine developmentand function. Within the intestinal mesoderm are two neuralplexuses that control intestinal motility. These neurons, collectivelycalled the enteric nervous system (ENS), coordinate the muscularcontractions of peristalsis, which control the mixing of food withdigestive enzymes and the movement of luminal contents throughthe GI tract. The peripheral nervous system is derived from a subsetof trunk neural crest cells (NCCs) referred to as vagal neural crestcells, which originate just caudal to the hindbrain in the regionbetween somites 1 and 7 (Burns et al., 2002; Durbec et al., 1996).Vagal NCCs migrate ventrally and invade the mesenchymesurrounding the primitive foregut tube in early somite stage embryos(reviewed by Kuo and Erickson, 2010). NCCs migrate caudally tocolonize the developing small and large intestine, and migration isregulated by several pathways including netrin, Bmp and endothelinsignaling (Goldstein et al., 2005; Jiang et al., 2003; Nataf et al.,1996). Last, NCCs differentiate into neuronal and glial lineages, andthis involves the repression of several signaling pathways, includingBmp and Notch (Chalazonitis et al., 2004; Goldstein et al., 2005).

Harnessing embryonic morphogenesis and tissue-tissueinteractions to generate more functional intestinal tissuesfrom PSCsMultilineage contributions and tissue-tissue interactions are criticalfor the development a functional intestine. There is an emerging

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Fig. 3. Milestones in murine and HIO intestinal development. The intestinal epithelium is present as a pseudostratified epithelium at ~E12.5 through E13.5in the mouse, and within 14 days of HIO specification. After this point, the appearance of mesenchymal clusters (orange), which respond to hedgehog (Hh) andplatelet-derived growth factor A (Pdgfa) secreted from the epithelium, is coincident with the onset of villus morphogenesis. In the mouse, villus morphogenesisproceeds between E14.5 and E16.5, whereas in HIOs this stage takes considerably longer. As the epithelium remodels, the columnar epithelium forms withstereotypical villus (green) and proliferative intervillus (yellow) domains. Mesenchymal clusters remain associated with the villus tip and act as a postmitoticsignaling center, signaling to the epithelium via secreted growth factors (including Bmp ligands, among others), which prevent epithelial proliferation. The fetalintervillus domain gives rise to the adult crypt by postnatal day 14 in the mouse, and after approximately 8 weeks in HIOs. The mature adult crypt houses theproliferative intestinal stem cells (yellow) and secretory Paneth cells (red), as well as several additional cell types, the most prevalent of which are enterocytes(green), goblet cells (blue) and enteroendocrine cells (purple). D

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concept that one reason for the lack of functionality of many PSC-derived cells and tissues is due the absence of organ morphogenesisin PSC monolayer cultures. For example, monolayer culture-derivedpancreatic endocrine cells lack the ability to secrete insulin inresponse to glucose (D’Amour et al., 2006) and liver hepatocyteshave an enzyme expression profile that is fetal in nature(DeLaForest et al., 2011). These non-physiological, two-dimensional (2D) monolayer cultures do not recapitulate the manymorphogenetic processes that occur during endoderm organdevelopment. Efforts to use more three-dimensional (3D)approaches for differentiation of PSCs have largely usedspontaneous aggregation into embryoid bodies. However thestochastic nature of this approach makes it hard to efficiently directdifferentiation into specific cell and tissue types. Moreover, there islittle evidence that normal morphogenesis occurs in embryoidbodies. Here, we will discuss morphogenetic processes that areimportant for intestinal development and how they have been use todirect the morphogenesis of PSCs into complex, 3D humanintestinal ‘organoids’ (HIOs; see Box 1).

Transition from 2D to 3D: formation of the primitive gut tube in theembryoShortly after gastrulation, all three germ layers undergomorphogenesis that will result in the formation of complex 3Dstructures such as the neural tube, somites and the gut tube. Gut tubemorphogenesis in birds and mammals initiates at the anterior andposterior ends of the embryo with the formation of the foregut andhindgut, and is largely completed within 36 hours. During thisprocess, the 2D DE undergoes dramatic cell shape changes, starting

as flat squamous cells, but then transitioning into a cuboidalepithelium during the morphogenesis of the foregut and hindgut.The entire process of gut tube formation is completed within 2 daysafter the end of gastrulation in mice, at which time the gut tubeepithelium has a pseudostratified morphology (Fig. 2). In addition,cell lineage tracing experiments and embryo imaging experimentsdemonstrate that endoderm and mesoderm cells are activelymigrating laterally and along the anterior-posterior axis between thegastrula and gut tube stages of development (Kimura et al., 2006;Lawson et al., 1991; Lawson et al., 1986; Lawson and Pedersen,1987).

The signaling pathways that control posterior fate, such as FGFand WNT, also regulate endoderm and mesoderm cell behaviors thatare involved in morphogenesis of the gut tube. Non-canonical Wntsignaling is involved in hindgut morphogenesis and elongation.Mutations in genes encoding either Wnt5a or the downstreammediators of non-canonical Wnt signaling, such as the disheveled-interacting protein Dapper1 (Dact1), perturb both hindgut formationand subsequent intestinal elongation (Cervantes et al., 2009; Marlowet al., 2004; Rauch et al., 1997; Tai et al., 2009; Yamaguchi et al.,1999). Fgf signaling may also play a role in morphogenesis byregulating migration of presumptive hindgut mesoderm. In chick,Fgf8 and Fgf4 act as a chemoattractant and repellant, respectively,to regulate mesoderm migration laterally and posteriorly as theembryo and hindgut extend caudally (Yang et al., 2002).

Triggering gut tube morphogenesis in PSC culturesAlthough the Wnt and Fgf pathways play distinct roles duringembryonic hindgut morphogenesis, studies using human PSC

Box 1. PSC-derived versus primary intestinal epithelial cultures: what is the difference?

One of the major advances in gastrointestinal biology includes long-term growth and expansion of individual adult ISCs in three-dimensional ‘enteroid’cultures (Ootani et al., 2009; Sato et al., 2009; Sato and Clevers, 2013; Sato et al., 2011). Enteroids are also commonly referred to as ‘mini-guts’, as wellas ‘organoids’ (Sato and Clevers, 2013). Human intestinal organoids (HIOs), however, are derived from pluripotent stem cells and are significantly differentfrom enteroids, as recently discussed at length (Finkbeiner and Spence, 2013). Briefly, enteroids are derived from fully committed adult intestinal crypts(or single ISCs) that are isolated from human or mouse intestine (Gracz et al., 2013; Wang et al., 2013). Enteroids consist of epithelium only and are biasedtowards an undifferentiated stem-cell fate due to the culture conditions (Miyoshi et al., 2012). Enteroids have proven to be an unparalleled model in whichto study molecular mechanisms by which adult intestinal stem cells are regulated and maintained, and are well suited to adult disease studies (de Lau etal., 2011; Dekkers et al., 2013; Zhou et al., 2013). HIOs, however, are generated by recapitulation of a developmental program and are therefore anunparalleled model in which to study human gastrointestinal development (Du et al., 2012). Both HIOs and adult enteroids have great potential as new invitro models for drug screening, infectious disease and cancer.

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cultures suggest that FGF and WNT synergistically act both topromote expression of CDX2 and to initiate a morphogeneticprocess that is similar to gut tube morphogenesis in vivo (Figs 1, 2)(Spence et al., 2011b). For example, in vivo, embryonic DE formsas a flat sheet of cells with a squamous-like morphology. However,as gut tube morphogenesis commences, DE cells transition to thecuboidal type of epithelium that lines the developing mid- andhindgut (Fig. 2). Similar transitions occur with PSC-derived DE,which starts as a flat sheet of cells, much like a squamousepithelium. However, following 4 days of treatment with both FGF4and WNT3a, DE cells condense into the cuboidal epithelium andform gut tube-like structures.

In addition to the epithelial morphogenesis observed in PSCcultures, the mesoderm undergoes transitions that are similar tothose observed during gut tube morphogenesis in vivo: FGF ligandsmediate mesoderm specification, proliferation and migration (Yanget al., 2002). In PSC cultures, FGF4 mediated an expansion ofmesoderm that formed the mesenchyme surrounding the epitheliumof these gut tube-like structures. It is likely that these gut tubestructures were fully committed to the intestinal lineage, as growthin 3D pro-intestinal culture conditions (Sato et al., 2009) resulted inthe formation intestinal tissue, and not other gut tube derivatives.

In addition to intestinal epithelium, PSC-derived HIOs have asignificant level of mesenchymal complexity. The mesenchyme inHIOs comes from mesoderm, which populates the early DE culturesand expands in response to FGF4 to form a layer of primitivemesenchyme surrounding the developing organoid (Spence et al.,2011b) (see Fig. 1). This primitive mesenchyme differentiates inparallel with the epithelium and forms several differentiated celltypes, such as smooth muscle, subepithelial myofibroblasts andfibroblasts, consistent with in vivo development (Spence et al.,2011b).

Milestones during midgestational intestine development are mirroredin PSC-derived intestineIn the mouse embryo, immediately after gut tube formation (aroundE9.0), the hindgut is a simple cuboidal epithelium that undergoes aseries of changes over the next 6-7 days that culminates in theemergence of the stereotypical villus structures of the postnatalintestine (Fig. 3) (Spence et al., 2011a). Between E9.5 and E12.5,the intestinal epithelium proliferates and expands in cell number,girth and luminal size, with the intestinal epithelium and lumenshaped like an oval (Sbarbati, 1982). Recent studies using geneticlineage tracing and 3D confocal microscopy demonstrate that theepithelium becomes pseudostratified around E12.5 (Grosse et al.,2011). Villus morphogenesis begins at E14.5, and gives rise to acolumnar epithelium and the stereotypical villus structures thatproject into the lumen. The intestinal mesenchyme is also involvedand acts as a signaling center to drive villus morphogenesis(Karlsson et al., 2000; Madison et al., 2005; Walton et al., 2012).

Shortly after the villi have formed, around E16.5 in mouse,proliferative intervillus progenitor cells give rise to newlydifferentiated cells on the villi, which include enterocytes, gobletand enteroendocrine cells. Of note, Paneth cells do not differentiateuntil around postnatal day 14 (P14) in mice and their differentiationoccurs simultaneously with tissue remodeling events that areresponsible for crypt emergence (Calvert and Pothier, 1990; Kim etal., 2012). In humans, crypts and Paneth cells are present by 22weeks of gestation (Trier and Moxey, 1979).

Many of the epithelial milestones that occur at these stages ofintestinal development also take place during PSC-derived intestine‘development’ in vitro, i.e. during the formation of HIOs (Fig. 3)

(McCracken et al., 2011; Spence et al., 2011b). The 3D cuboidalepithelium of the early intestinal organoids gives rise to apseudostratified epithelium, which undergoes a process mimickingvillus morphogenesis, with a few exceptions. As organoids continueto grow in culture, the pseudostratified epithelium transitions tocolumnar epithelium; however, true villi do not form. Instead, villus-like structures form, which are made up of epithelial protrusionsextending into the lumen. These structures are not true villi due theirlack of the lamina propria, which is the underlying mesenchymaltissue made up of nerve, vascular, immune and support cells normallyfound in the core of the villus. Although true villi do not seem todevelop in HIOs, the villus-like structures still behave similarly totheir in vivo counterpart and proliferation is restricted to the base ofthe villus-like structure, equivalent to the intervillus progenitordomain, and preferentially expresses molecular markers of theintervillus domain, including Sox9. Furthermore, HIOs generate cellsthat express markers for the major intestinal cell types: enterocytes,goblet, enterendocrine and Paneth cells. The co-expression ofGata4/Gata6 and presence of Paneth cells in the HIOs suggest thatthey are most similar to the small intestine. In addition to the majorcell types mentioned above, the small intestine in vivo possesses othercell types, including tuft, cup and M-cells; however, it is unclear atthis point whether HIOs possess these cell types. HIOs also appear toundergo a process similar to crypt emergence. In HIOs, the intestinalstem cell marker Lgr5 is not expressed robustly during early stages oforganoid ‘development’, but is expressed in discreet epithelialdomains after prolonged culture. It is important to point out that todate, functional validation of all cell types in organoids has not beencarried out, and many of the conclusions we have drawn are based onmarker analysis. Therefore, as this system becomes more widely used,it will be interesting to see additional levels of functional validationfor individual cell types.

Future directionsBuilding a better intestine in vitroPSC-derived HIOs exhibit several basic functional properties; forexample, they secrete mucus into the lumen and they are competentto absorb dipeptides, indicating a functional dipeptide transportsystem. Despite these observations, it is still unclear whether HIOsrepresent a ‘mature’ or ‘immature’ tissue. Recent studies havesuggested that human PSC-derived intestinal tissues are moresimilar to fetal human intestine than to adult human intestine(Fordham et al., 2013). Importantly, however, different methodsused to generate HIOs and maintain their growth in vitro confoundcomparisons from one study to another. In general, the generationof functional, mature cells or tissues from human PSC cultures hasproven challenging across many tissue types (Dolnikov et al., 2005;Si-Tayeb et al., 2010; D’Amour et al., 2006; Kroon et al., 2008).Recently, the transcription factor Blimp1 (also know an Prdm1) wasidentified as a key regulator of the transition from fetal to matureintestinal epithelium, including the formation of crypts (Harper etal., 2011; Muncan et al., 2011). Therefore, identification of signalingpathways that repress Blimp1 may be one approach to promotematuration of HIOs.

One potential reason for lack of maturation and/or functionalityis because in vitro-derived tissues lack important cell types that arecrucial for organ function. For example, even though HIOs containimportant mesoderm-derived cell types that are crucial forintestinal function, such as smooth muscle myocytes, fibroblastsand subepithelial myofibroblasts (Powell et al., 2011), they aremissing a vascular plexus and the enteric nervous system (ENS)that coordinates the muscular contractions of peristalsis. The lack D

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of the vascular and neural plexus limits the physiological relevanceof drug absorption studies and makes it impossible to studyintestinal motility disorders. Given that protocols exist forgenerating vascular endothelial cells and NCCs from human PSCs(Bajpai et al., 2010; Lee et al., 2007; Levenberg et al., 2002), itshould be possible to incorporate vascular and ENS progenitorsinto developing HIOs in an attempt to generate vascular and neuralnetworks. An example of this approach was recently demonstratedwhereby vascular progenitors were added during the differentiationof PSCs into hepatocyte progenitors (Takebe et al., 2013). Theaddition of vascular cells resulted in the formation of 3D liver budswith a well-formed vascular plexus and greatly improvedfunctionality in vivo.

Development and disease researchAlthough generating transplantable segments of intestine in vitro isstill years (or decades) away, in vitro-derived human intestinal tissueallows for unprecedented studies of human development,homeostasis and disease. Agonists and antagonists can be used todissect the role of signaling pathways during intestinalmorphogenesis, patterning and cell fate specification. Using standardgenetic gain- and loss-of-function approaches, it is straightforwardto study gene function and identify gene regulatory networksinvolved in lineage development and morphogenesis. Alternatively,iPSC technology can be used to identify the molecular basis ofhuman congenital disorders affecting the intestines such as cysticfibrosis and genetic forms of malabsorption (Wang et al., 2006a).Because of the ease of manipulation and imaging, human organoid-based approaches are a powerful tool to study wide variety of otherdisease processes. Introduction of viral and bacterial pathogens intothe lumen of organoids can allow for new studies of infectiousdisease and facilitate identification of pathways mediating host-pathogen interactions (Finkbeiner et al., 2012). Organoids derivedfrom PSCs or adult tissues may also be useful to study theenvironmental and genetic causes of cancer initiation andprogression (Ootani et al., 2009). Last, many commonly prescribeddrugs have intestinal side effects, making it a possibility that HIOscould be used as a rapid screening tool for drugs with goodabsorption and fewer intestinal complications.

As for using in vitro-derived intestinal tissues in transplantation-based therapies, current efforts have focused on seeding damagedintestinal epithelia with adult ISCs (Yui et al., 2012). This approachhas resulted in engraftment and expansion of transplanted cells;however, it is technically challenging and might be a difficultapproach to use broadly, particularly in more proximal regions ofthe intestinal tract. Moreover, this approach is not helpful forindividuals suffering from short gut syndrome. Although in vitroderivation of transplantable intestinal segments is not likely to occurin the immediate future, recent advances in tissue engineering havemade it possible to generate an artificial trachea for transplantation(Jungebluth et al., 2011). Similar approaches using endogenous orbiosynthetic scaffolds should be possible for intestine.

Human organoids are fueling a renaissance of in vitro studies ofhuman intestinal development and disease. Organoid-based researchis especially powerful when combined with new technologies suchas gene targeting, real-time high content imaging, and fluidics-basedplatforms to grow multiple tissue-type organoids on chips to mimicsystemic organ interactions.

AcknowledgementsWe thank our colleagues for many helpful discussions and apologize for notincluding additional references due to space limitations.

Competing interestsThe authors declare no competing financial interests.

FundingJ.R.S. is supported by the University of Michigan Center for Organogenesis, theUniversity of Michigan Biological Sciences Scholar Program, The National Instituteof Diabetes and Digestive and Kidney Diseases and a March of Dimes BasilO’Connor new investigator award. J.M.W. is supported by the National Institutes ofHealth. Deposited in PMC for release after 12 months.

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