Pancreas organogenesis: from lineage determination to morphogenesis.

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Annu. Rev. Cell Dev. Biol. 2013.29. Downloaded from www.annualreviews.org by Georgetown University on 08/06/13. For personal use only.

Pancreas Organogenesis: From Lineage Determination to Morphogenesis Hung Ping Shih,∗ Allen Wang,∗ and Maike Sander Departments of Pediatrics and Cellular & Molecular Medicine, Pediatric Diabetes Research Center, University of California, San Diego, La Jolla, California 92093-0695; email: [email protected]

Annu. Rev. Cell Dev. Biol. 2013. 29:19.1–19.25

Keywords

The Annual Review of Cell and Developmental Biology is online at http://cellbio.annualreviews.org

pancreas, development, progenitor, exocrine, endocrine, pluripotent stem cell

This article’s doi: 10.1146/annurev-cellbio-101512-122405 c 2013 by Annual Reviews. Copyright All rights reserved ∗

Equal contributions.

Abstract The pancreas is an essential organ for proper nutrient metabolism and has both endocrine and exocrine function. In the past two decades, knowledge of how the pancreas develops during embryogenesis has significantly increased, largely from developmental studies in model organisms. Specifically, the molecular basis of pancreatic lineage decisions and cell differentiation is well studied. Still not well understood are the mechanisms governing threedimensional morphogenesis of the organ. Strategies to derive transplantable β-cells in vitro for diabetes treatment have benefited from the accumulated knowledge of pancreas development. In this review, we provide an overview of the current understanding of pancreatic lineage determination and organogenesis, and we examine future implications of these findings for treatment of diabetes mellitus through cell replacement.

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Contents


INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.2 OVERVIEW OF PANCREAS DEVELOPMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.2 LINEAGE DECISIONS DURING PANCREAS DEVELOPMENT . . . . . . . . . . . . . . . 19.5 Pancreas Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.5 Establishing and Maintaining Pancreatic Identity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.7 Signaling During Early Pancreatic Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.8 Compartmentalizing the Organ: Patterning of Tips and Trunk . . . . . . . . . . . . . . . . . . . 19.9 Development of the Acinar Lineage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19.10 The Endocrine Versus Ductal Cell-Fate Choice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19.11 Differentiation of the Islet Cell Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19.12 PANCREAS MORPHOGENESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19.13 Intracellular Signals Governing Pancreas Morphogenesis . . . . . . . . . . . . . . . . . . . . . . . . .19.14 Coordinating Morphogenesis and Cell Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . .19.14 Morphogenetic Changes and Endocrine Cell Differentiation . . . . . . . . . . . . . . . . . . . . . .19.15 APPLICATION OF DEVELOPMENTAL PRINCIPLES TOWARD STEM CELL DIFFERENTIATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19.15 Generating Pancreatic Progenitors from Human Pluripotent Stem Cells . . . . . . . . . .19.16 Beyond Progenitors: How to Generate Functional Endocrine Cells In Vitro . . . . . . .19.16 Human Pluripotent Stem Cells as a Model for Understanding Pancreas Biology and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19.17 FUTURE PROSPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19.18

INTRODUCTION The pancreas is essential for nutrient metabolism and has both exocrine and endocrine functions. The exocrine pancreas consists of acinar and ductal cells, whose prime role is nutrient digestion. The acinar cells secrete pancreatic juice containing enzymes that catalyze the breakdown of proteins, carbohydrates, and lipids. Upon release from the acinar cells, the pancreatic juice is transported into the duodenum via the ductal system (Figure 1). Functioning largely independent from exocrine cells, the endocrine pancreas is responsible for regulating glucose homeostasis by releasing hormones into the blood stream. The endocrine pancreas comprises five endocrine cell types, which reside in small cell clusters called islets of Langerhans. The biology of the pancreas has been studied intensely, largely driven by the hope of finding better treatments for devastating pancreatic diseases, such as diabetes mellitus, pancreatitis, and pancreatic adenocarcinoma. In particular, advancements in stem cell technology have recently sparked optimism that diabetes could be cured by harnessing stem cells for therapeutic use. This has led to heightened interest in understanding embryonic development of the pancreas, specifically the events involved in cell fate decisions and endocrine cell differentiation. Recent successes in deriving pancreatic cells from human pluripotent stem cells (hPSCs) prove exemplary for the fruitful application of developmental principles to stem cell differentiation.

OVERVIEW OF PANCREAS DEVELOPMENT The invention of genetic tools, in particular transgenic mice, has been instrumental in defining the molecular events leading to pancreas organogenesis. Genetic lineage-tracing experiments revealed 19.2

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Stomach Vein

Liver

Pancreatic ducts

Artery Endocrine cells

Gall bladder

Spleen

Islet of Lagerhans


Common bile duct

Pancreas

Duodenum Acinar cells

Figure 1 Overview of adult pancreas anatomy. The pancreas lies behind the stomach in the abdomen and attaches to the duodenum. For illustration purposes, the stomach is outlined with a dashed line and the duodenum is shown as a cut view. Exocrine acinar cells secrete pancreatic juice containing enzymes for the breakdown of proteins, carbohydrates, and lipids. The pancreatic juice is transported through a ductal network that releases the juice into the duodenum. The endocrine pancreas consists of small cell clusters, called islets of Langerhans, containing five endocrine cell types, namely glucagon-producing α-cells, insulin-producing β-cells, somatostatin-producing δ-cells, and pancreatic polypeptide-producing PP cells. Note, the figure shows the islet organization characteristic for mice with centrally located β-cells. Pancreatic islets are surrounded by a dense capillary network through which the hormones are released into the blood stream. See inset for an enlarged cutaway view of the pancreas.

a common origin of pancreatic exocrine and endocrine cells from a pool of progenitor cells in the gut endoderm (Gu et al. 2002). In mice, pancreas development first becomes morphologically evident around embryonic day (e) 8.75 with the emergence of epithelial buds on opposing sides of the foregut endoderm (Figure 2a). The two pancreatic buds subsequently elongate alongside the presumptive duodenum and stomach and, as a result of gut rotation, eventually fuse into a single organ by e12.5 (Figure 2b). Simultaneously, cell proliferation leads to a rapid size increase of the pancreatic buds, which also change their shape and begin to form branched tubular structures in a process known as branching morphogenesis. During this early phase of pancreatic development, which has been referred to as the primary transition (Pictet et al. 1972), the still-undifferentiated pancreatic epithelium is tightly surrounded by mesenchymal cells. During the secondary transition from e12.5 until birth, the pancreatic epithelium continues to expand and branch into a complex yet highly ordered tubular network (Figure 2c). These morphologic changes are paralleled by the differentiation of endocrine, acinar, and ductal cells. By the end of the secondary transition, the mouse pancreas has largely acquired the topological organization of the mature organ, with clusters of acinar cells coalesced around the ends of the ductal network and endocrine cell aggregates scattered throughout the organ (Figure 2c,d ). www.annualreviews.org • Pancreas Organogenesis


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Dorsal pancreas Mesenchyme Microlumen Basement membrane Blood vessel

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Over the past two decades, significant progress has been made in deciphering key molecular mechanisms that underlie the multiple lineage decisions leading to the formation of the pancreatic cell types. In comparison, far less is known is about the regulation of pancreas morphogenesis and whether a connection exists between morphogenetic events and cell differentiation. This review covers current knowledge of these subjects and discusses how these discoveries have begun to enable the development of cell-replacement strategies for diabetes.

LINEAGE DECISIONS DURING PANCREAS DEVELOPMENT


Pancreas Induction Prior to outgrowth of the pancreatic buds, a ventral and dorsal prepancreatic region is specified in the gut endoderm around e8.5 (Slack 1995). Although ventral and dorsal pancreatic buds arise independently in distinct locations, the molecular programs governing their specification are remarkably similar. In contrast to most epithelial cells of the early gut endoderm, the expression of sonic hedgehog (Shh) is selectively excluded from the presumptive pancreatic endoderm (Hebrok et al. 1998, Kim & Melton 1998). Transgenic misexpression experiments in mice revealed that the exclusion of Shh from the prepancreatic region is necessary for the induction of pancreatic markers (Apelqvist et al. 1997). Like Shh, retinoic acid (RA) also participates in patterning the gut endoderm. Absence of RA leads to anteriorization of the endoderm, resulting in complete pancreas agenesis in zebrafish and lack of the dorsal pancreas in Xenopus and mice (Chen et al. 2004, Mart´ın et al. 2005, Molotkov et al. 2005, Stafford & Prince 2002). RA seems to induce the dorsal pancreatic program by a Shh-independent mechanism, because loss of RA is not associated with ectopic Shh expression in the prepancreatic region in mice (Mart´ın et al. 2005). The ventral foregut region gives rise to both ventral pancreas and liver, which develop in close proximity to each other (for review, see Zorn & Wells 2009). Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signaling are critical prohepatic cues, which are secreted by the cardiac mesoderm surrounding the prehepatic region (Deutsch et al. 2001, Rossi et al. 2001). The discovery of signals necessary for normal pancreas and liver induction has found practical applications in directed differentiation protocols of hPSCs, which use RA and inhibitors of Shh and BMP to induce pancreatic identity in vitro (D’Amour et al. 2006, Nostro et al. 2011). Dorsal bud emergence appears to also be regulated by extrinsic signals from endothelial cells. The dorsal and ventral pancreatic buds develop from regions of foregut endoderm that are in direct contact with the endothelium of the fusing dorsal aortae and vitelline veins, respectively ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 2 Illustrated overview of pancreatic organogenesis. (a) The dorsal and ventral pancreatic epithelium evaginates into the surrounding mesenchyme between embryonic day (e) 9.5 and e11.5 in mice. At this stage, the pancreatic epithelium is comprised of a multilayered core of unpolarized cells engulfed by a basement membrane. Scattered microlumens (light yellow) arise between epithelial cells. Blood vessels surround but have not yet penetrated the epithelial buds. (b) At e12.5, the outer tip cell layer ( green) of the pancreatic epithelium forms recognizable branch protrusions. In the trunk portion of the pancreatic epithelium, microlumens fuse to form a primitive plexus, and groups of newly polarized cells ( purple) organize into rosettes around a lumen. At the same time, blood vessels begin to intercalate into the epithelium and contact trunk cells. (c) At e15.5, the luminal plexus progressively remodels into a single-layered epithelium consisting of highly branched primitive ducts (also known as progenitor cords) and newly differentiated acinar cells. Ngn3-expressing endocrine precursors (orange) delaminate and migrate away from the progenitor cords to form endocrine clusters. Blood vessels are intercalated between nascent branches of the pancreatic ductal tree. (d ) In the mature pancreas, acinar cells cap the endings of small terminal ducts and form functional exocrine secretory units. Endocrine cells are clustered in so-called islets of Langerhans, which are penetrated by a dense network of blood vessels. See insets for enlarged views of depicted areas. www.annualreviews.org • Pancreas Organogenesis


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(Lammert et al. 2001). Tissue recombination experiments and studies of endothelium-deficient Flk1−/− mice have shown that the absence of endothelial cells prevents induction of early pancreatic genes (Lammert et al. 2001, Yoshitomi & Zaret 2004). However, contrary to these reports in mice, studies in zebrafish revealed pancreas specification in the absence of vascular endothelium (Field et al. 2003), a discrepancy that may reflect species differences.

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Pdx1 Prox1

Primary transition

Oc1

Ptf1a Foxa1/2 Tcf2 Sox9 Gata4/6 Hes1


Pancreatic progenitors Secondary transition

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Ptf1a c-Myc

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Establishing and Maintaining Pancreatic Identity Once the ventral and dorsal prepancreatic domains are specified, several transcription factors become specifically expressed in the prospective pancreatic regions. Among the earliest transcription factors that mark this region are Pdx1, Ptf1a, and Sox9 (Ahlgren et al. 1996, Guz et al. 1995, Kawaguchi et al. 2002, Krapp et al. 1998, Seymour et al. 2007). Gata4/6, Foxa1/2, Tcf2, Onecut1/2, Hes1, Prox1, and Mnx1 are also expressed in the prepancreatic domain, but their expression expands more broadly throughout the foregut endoderm (Figure 3a) (comprehensively reviewed in Gittes 2009, Pan & Wright 2011, Seymour & Sander 2011). Mice lacking any one of these factors display varying degrees of pancreas hypoplasia or agenesis; however, they still show pancreatic budding, which indicates that absence of one single factor does not entirely prevent initiation of the pancreatic program (reviewed in Gittes 2009, Mastracci & Sussel 2012, Pan & Wright 2011, Seymour & Sander 2011). Although budding of the pancreas is still observed, evidence strongly indicates that these transcription factors are important for determining pancreatic fate. For example, Ptf1a deletion leads to a reallocation of subsets of pancreas-fated progenitors to other endodermal lineages. This was shown by in vivo lineagetracing experiments, which revealed a contribution of Ptf1a-deficient cells to adjacent duodenal and common bile duct endoderm (Burlison et al. 2008, Kawaguchi et al. 2002). Further evidence that these early pancreatic transcription factors possess pancreatic fate-determining activity comes from gain-of-function experiments in Xenopus, which have shown that constitutively active forms of either Pdx1 or Ptf1a can allocate liver progenitors to the pancreatic lineage (Horb et al. 2003, Jarikji et al. 2007). Also, when ectopically expressed, Pdx1 and Ptf1a in combination are sufficient to convert duodenal precursors to the pancreatic fate (Afelik et al. 2006). Thus, in a developmental context, Pdx1 and Ptf1a can confer pancreatic identity to nonpancreatic cells. The extent to which Pdx1 and Ptf1a are also capable of activating pancreatic genes in nonpancreatic adult cells remains to be investigated. A better understanding of context-dependent effects of these early pancreatic transcription factors on gene expression could lead to novel strategies for reprogramming fibroblasts directly into pancreatic cells, as shown recently for neurons and cardiomyocytes (Qian et al. 2012, Thier et al. 2012). In addition to their role in pancreas specification, all early pancreatic transcription factors are necessary for further development of pancreatic buds (for reviews, see Gittes 2009, Pan & Wright 2011, and Seymour & Sander 2011). Mechanistically, the profound effect on the development of the organ caused by loss of only one single transcription factor may result from these early factors functioning in a transcriptional network with extensive cross-regulation between individual factors. For example, the expression of both Tcf2 and Ptf1a depends on Onecut-1 (Haumaitre et al. 2005, Maestro et al. 2003); simultaneously, Onecut-1 expression depends on Tcf2 and Ptf1a ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 3 Lineage decisions during pancreas development. (a) During the primary transition, the transcription factors Pdx1, Prox1, Onecut-1 (Oc1), Ptf1a, Foxa1/2, Tcf2, Sox9, Gata4/6, and Hes1 mediate expansion of pancreatic progenitor cells and maintain pancreatic identity. (b) At the onset of the secondary transition, pancreatic progenitors adopt either tip or trunk identity. Notch activity promotes the trunk while repressing tip identity. Cross-repression between Ptf1a and Nkx6.1 mediates tip/trunk separation. (c) Tip cells adopt an acinar phenotype, which is maintained by a positive-regulatory loop between PTF1-L and Nr5a2. Trunk cells are bipotential for the ductal and endocrine cell fate. The ductal versus endocrine fate decision is controlled by graded Notch activity. High Notch promotes the ductal fate by activating both Hes1, a repressor of Ngn3, and Sox9, an Ngn3 activator. At low Notch activity, only Sox9 is activated, leading to endocrine differentiation. (d ) Endocrine precursors further differentiate into five different cell types, of which only α- and β-cells are depicted. Cross-repression between the α-cell determinant Arx and β-cell determinants Pax4, Nkx6.1, and Pdx1 separates the two lineages. www.annualreviews.org • Pancreas Organogenesis


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(Poll et al. 2006, Thompson et al. 2012). Similar cross-regulation has been demonstrated between Sox9, Pdx1, Hes1, and Ptf1a (Ahnfelt-Rønne et al. 2012; Hale et al. 2005; Seymour et al. 2007, 2012; Thompson et al. 2012). Thus, elimination of a single factor has repercussions that ultimately affect multiple components of the pancreatic program. The advent of genome-scale tools and the availability of stem cell–based in vitro models of pancreas development (D’Amour et al. 2006, Kroon et al. 2008) now enable experiments to globally define the relevant transcriptional networks. Such knowledge should help inform reprogramming strategies for deriving pancreatic cells from other somatic cells.

Signaling During Early Pancreatic Growth


Whereas we have a relatively detailed understanding of the transcription factors regulating early pancreatic development, less is known about the signals controlling their expression. Both Hes1 and Sox9 are regulated by Notch signaling (Kageyama et al. 2007, Shih et al. 2012), which suggests that Notch signaling is important for early pancreas growth. As evidence to support this, embryos deficient for the Notch effector RBP-jκ have been shown to exhibit early arrest in pancreatic growth, similar in degree to that seen after Hes1 or Sox9 deletion (Apelqvist et al. 1999, Fujikura et al. 2006, Jensen et al. 2000, Seymour et al. 2007). Hes1 deletion is also associated with upregulation of the cell cycle inhibitor p57 in pancreatic progenitors (Georgia et al. 2006). Thus, there appears to be a direct link between Notch activity, transcription factor expression, and cell cycle regulation in the early pancreas. Known and putative roles for Notch in the developing pancreas have been expertly reviewed (Afelik & Jensen 2012). In addition to Notch signaling within the epithelium, pancreatic growth is also controlled by extrinsic signals from the surrounding mesenchyme. Seminal studies in the 1960s demonstrated the importance of the mesenchyme for early pancreatic growth (Golosow & Grobstein 1962, Rutter et al. 1964, Wessells & Cohen 1966). However, we have begun only recently to understand how mesenchymal signals control growth of the early pancreas. The best-understood pathway that relays pro-proliferative signals from the mesenchyme to the epithelium is the FGF signaling pathway. During the primary transition, FGF10 is highly expressed in pancreatic mesenchyme, whereas its receptor FGFR2 is expressed throughout the epithelium (Bhushan et al. 2001, Dichmann et al. 2003, Seymour et al. 2012). Gain- and loss-of-function experiments in mice further demonstrated that FGF10 stimulates progenitor cell proliferation and is required for early growth of the pancreatic buds (Bhushan et al. 2001). FGF10 has been shown to regulate Ptf1a and Sox9 expression in the epithelium ( Jacquemin et al. 2006, Seymour et al. 2012), which explains why FGF10 deletion results in a phenotype similar to that observed in either Ptf1a- or Sox9-deficient pancreas (Bhushan et al. 2001, Kawaguchi et al. 2002, Seymour et al. 2007). During the early growth phase of the pancreatic buds, Sox9, FGFR2, and FGF10 form a feed-forward loop, with mesenchymal FGF10 maintaining epithelial Sox9 expression and Sox9 upholding receptivity to FGF10 by cell-autonomously controlling FGFR2 expression (Seymour et al. 2012). If this loop is disrupted at any point, pancreatic epithelial cells can no longer maintain their identity, and liver genes are activated instead (Seymour et al. 2012). This demonstrates genetic coupling of early pancreatic growth and maintenance of organ identity. Additional signaling pathways critical to epithelial growth include the EGF, Wnt, and BMP pathways (Ahnfelt-Rønne et al. 2010, Miettinen et al. 2000, Murtaugh et al. 2005). Wnt and BMP signals appear not to be relayed to the epithelium; instead, they are transduced by mesenchymal cells (Ahnfelt-Rønne et al. 2010, Jonckheere et al. 2008, Landsman et al. 2011). It is unknown whether the ligand is secreted by the epithelium or the mesenchyme itself. Thus, signaling between the mesenchyme and epithelium likely involves multiple signaling loops. Recent in vitro studies 19.8

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by Sneddon et al. (2012) on pancreatic lineage intermediates derived from hPSCs supported this contention, demonstrating that coculture with mesenchyme effectively expands progenitors and that this effect is specific both to the mesenchymal cell line used and to the progenitor being amplified. They further showed that no single growth factor or combination of factors is sufficient to reproduce the magnitude of effect of coculture with mesenchyme, reinforcing the notion that multiple factors cooperate to stimulate epithelial expansion. The identification of factors orchestrating pancreatic growth should be a high priority to aid the development of scalable protocols for the in vitro production of pancreatic cells.


Compartmentalizing the Organ: Patterning of Tips and Trunk Shortly after the primary transition, the nascent pancreatic buds have not yet acquired the macroscopic structure of the mature organ, nor have they formed the differentiated cell types necessary for pancreatic function. Instead, they are almost entirely composed of multipotent progenitor cells (MPCs). The transition of noncommitted MPCs into an organ with different cell compartments is a multistep process, of which the first step is the segregation of a tip and trunk domain at the onset of the secondary transition (Figure 3b) (Zhou et al. 2007). During this time, the developing organ continues to grow rapidly. Concomitantly, the pancreatic epithelium undergoes dynamic structural changes, resulting in multiple protrusions that bud from the edges. These structures, marked by expression of Ptf1a, c-Myc, and Cpa, represent the tip domain; conversely, the inner cells representing the trunk domain are identified by Nkx6.1/6.2, Sox9, Tcf2, Onecut-1, Prox1, and Hes1 (Figure 3b) ( Jacquemin et al. 2003, Klinck et al. 2011, Kopinke et al. 2011, Kopp et al. 2011, Schaffer et al. 2010, Solar et al. 2009, Vanhorenbeeck et al. 2007, Wang et al. 2005, Zhou et al. 2007). These morphological changes mark the beginning of a complex sequence of cell rearrangements (Villasenor et al. 2010), resulting in the mature organ structure (discussed in detail in the section on Pancreas Morphogenesis). In addition to the changes in organ shape, the formerly multipotent MPCs have taken the first step toward lineage commitment. Lineage-tracing experiments revealed that the trunk predominantly gives rise to the endocrine and ductal cell lineages, whereas the tips are quickly restricted to an acinar fate (Figure 3b) (Kopinke et al. 2011, Kopp et al. 2011, Pan et al. 2013, Solar et al. 2009, Zhou et al. 2007). The transcription factors Nkx6.1/6.2 and Ptf1a act as master regulators during this process. These factors are initially coexpressed in MPCs, but their expression domains entirely segregate during tip and trunk compartmentalization: Nkx6.1/6.2 promote trunk identity and segregate to trunk cells, and Ptf1a has an equivalent role in the tip compartment (Schaffer et al. 2010). Mechanistically, this process is initiated by transcriptional cross-repression between Nkx6.1/6.2 and Ptf1a (Schaffer et al. 2010). The example of Ptf1a, which first specifies pancreatic fate and later governs acinar cell development (Kawaguchi et al. 2002, Krapp et al. 1998, Masui et al. 2010), illustrates that developmental transcription factors can regulate different processes depending on cellular context. Genetic studies have provided insight into the signaling pathways regulating tip/trunk segregation, revealing that Notch signaling plays an essential role in promoting trunk identity. Repression of Notch signaling causes excessive tip formation at the expense of the trunk population (Afelik et al. 2012, Horn et al. 2012, Magenheim et al. 2011b). Conversely, constitutively active Notch prevents tip formation and promotes expansion of the trunk domain partly by activating the trunk marker Nkx6.1 (Afelik et al. 2012, Esni et al. 2004, Murtaugh et al. 2003, Schaffer et al. 2010). Although Notch signaling clearly plays a pivotal role in coordinating tip and trunk segregation, it remains unknown how Notch is regulated during this process. www.annualreviews.org • Pancreas Organogenesis


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Development of the Acinar Lineage


In addition to their role in digestion, pancreatic acinar cells are an attractive target for β-cellreplacement therapy, because they are plentiful and retain the cellular plasticity to undergo reprogramming into β-cells (Zhou et al. 2008). Moreover, because they are the initiating cell type for precursor lesions of pancreatic cancer (Kopp et al. 2012), acinar cells are also a study focus for oncologists. As discussed, acinar cells predominantly arise from precursor cells in the tip domain (Pan et al. 2013, Zhou et al. 2007). By e15.5, most tip cells have undergone acinar cell differentiation, and subsequent expansion of the acinar domain is largely driven by cell proliferation (Pan et al. 2013, Zhou et al. 2007). Acinar cell differentiation is synergistically orchestrated by the transcription factors Ptf1a, Rbp-jl, and Nr5a2/LRH-1, which exhibit first tip cell–specific and later acinar cell–specific expression (Holmstrom et al. 2011; Krapp et al. 1996, 1998; Masui et al. 2007, 2010; Rose et al. 2001; Zhou et al. 2007). Prior to acinar cell differentiation, Ptf1a interacts with the b-HLH transcription factor Rbp-jκ (Beres et al. 2006, Masui et al. 2007, Miyatsuka et al. 2007). Together, these factors are responsible for the activation of Rbp-jl, a constitutively active, acinar-restricted paralog of Rbp-jκ (Masui et al. 2007). During the secondary transition, Rbp-jl then replaces Rbpjκ in the complex with Ptf1a. This complex, termed PTF1-L, establishes the acinar phenotype by direct activation of essential acinar genes (i.e., genes encoding digestive enzymes and proteins for exocytosis of zymogen granules), as well as Ptf1a and Rbp-jl themselves (Masui et al. 2007, 2008, 2010; Rose et al. 2001). Thus, PTF1-L induces and maintains the acinar phenotype through an autoactivation loop that sustains high expression of acinar genes (Figure 3c). Similarly, Nr5a2, a direct target gene of Ptf1a, is necessary for acinar cell differentiation (Holmstrom et al. 2011, Thompson et al. 2012). Nr5a2 binds to and regulates a set of genes similar to those regulated by the PTF1-L complex, and Nr5a2 and PTF1-L cooperate in the regulation of acinar cell–specific genes (Holmstrom et al. 2011). Together, these studies demonstrate that Ptf1a promotes acinar cell differentiation by initiating and stabilizing a gene-regulatory network that drives acinar gene expression. Although Ptf1a is an indispensable coordinator of acinar cell development, forced expression of Ptf1a alone is not sufficient to effectively induce acinar cell differentiation (Schaffer et al. 2010). Initiation of acinar differentiation may require other transcription factors functioning in parallel with Ptf1a. One candidate is Mist1, which is strongly expressed in acinar cells but does not depend on the PTF1-L complex for its expression (Masui et al. 2010, Yoshida et al. 2001). Mist1-deficient mice exhibit loss of differentiated characteristics of acinar cells, including loss of cellular polarity and defective exocytosis (Pin et al. 2001). Thus, the PTF1-L complex, Nr5a2, and Mist1 are all required to maintain the acinar phenotype. Genome-wide analysis of Mist target genes may reveal whether Mist1 occupies genomic regions similar to those of PTF1-L and Nr5a2 and could therefore regulate acinar development jointly with these factors. Less understood than acinar cell specification and differentiation are the mechanisms underlying the rapid proliferative expansion of acinar cells until birth. Unlike Mist1 and Ptf1a, which negatively regulate acinar cell expansion ( Jia et al. 2008, Rodolosse et al. 2004), Nr5a2 promotes proliferation of exocrine cell lines (Benod et al. 2011, Botrugno et al. 2004), revealing Nr5a2 as a potential candidate for driving acinar cell expansion. Additional candidates include c-Myc and β-catenin, whose inactivation in early pancreatic progenitors causes acinar cell hypoplasia (Bonal et al. 2009, Murtaugh et al. 2005, Nakhai et al. 2008, Wells et al. 2007). Temporally and spatially controlled inactivation of other acinar factors in mice may yield additional insight into molecular control of acinar cell differentiation, expansion, and maintenance.

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The Endocrine Versus Ductal Cell-Fate Choice Whereas tip cells are fated to become acinar cells, the trunk domain is bipotential and produces ductal and endocrine cells (Figure 3c) (Kopinke et al. 2011, Kopp et al. 2011, Solar et al. 2009). After the tip and trunk domains have separated at around e12.5, cells in the trunk undergo extensive morphogenetic changes to form a 3-D network of tubules lined by a single layer of polarized epithelial cells (Hick et al. 2009, Kesavan et al. 2009, Villasenor et al. 2010). This network of tubules is referred to as the primitive ducts or progenitor cords and is the epithelium that gives rise to all endocrine cells (Hick et al. 2009, Kopp et al. 2011). During the secondary transition, a subset of cells in the progenitor cords initiate expression of the transcription factor Neurogenin3 (Ngn3), which marks the onset of endocrine cell differentiation (Gradwohl et al. 2000, Gu et al. 2002, Johansson et al. 2007, Schwitzgebel et al. 2000). Trunk epithelial cells that do not activate Ngn3 eventually contribute to the ductal tree (Beucher et al. 2012, Magenheim et al. 2011b, Wang et al. 2010). Thus, precise control of Ngn3 induction is the key factor balancing the endocrine versus ductal cell-fate decision. Many of the same transcription factors (e.g., Sox9, Tcf2, Onecut-1, Hes1, Prox1, and Glis3) that segregate to the trunk domain during tip/trunk separation later become restricted to the ducts and excluded from the endocrine compartment (Figure 3c) (Delous et al. 2012, Kang et al. 2009, Maestro et al. 2003, Pierreux et al. 2006, Shih et al. 2012, Wang et al. 2005, Westmoreland et al. 2012, Zhang et al. 2009). Consistent with their duct-specific expression, their inactivation in mice causes defects in ductal cell morphology and abnormal architecture of the ductal tree. For example, pancreata deficient for Sox9, Glis3, or Onecut-1 exhibit ductal cysts and lack ductal primary cilia, a functionally important organelle of ductal epithelial cells (Kang et al. 2009, Pierreux et al. 2006, Shih et al. 2012). Whether these ductal malformations reflect a functional requirement of these factors in the maintenance of ductal epithelial properties or a role in ductal cell differentiation is unknown. Conditional inactivation of the genes encoding ductal factors in developing and mature ducts should further define their role in ductal development and function. This knowledge could be relevant to understanding the pathogenesis of duct-related illnesses, including pancreatitis and pancreatic ductal adenoma carcinoma. Unlike ductal cell development, endocrine differentiation has been studied intensely, driven by hopes that such knowledge could help produce replacement β-cells for diabetes therapy. Because activation of Ngn3 expression is essential for initiating endocrine cell differentiation, there have been substantial efforts to understand Ngn3 regulation. Interestingly, many transcription factors controlling ductal development, such as Sox9, Tcf2, Glis3, and Onecut-1, are also necessary for Ngn3 induction ( Jacquemin et al. 2000, Kang et al. 2009, Kim et al. 2012, Lynn et al. 2007, Maestro et al. 2003, Seymour et al. 2008, Zhang et al. 2009), which indicates dual roles for these factors in the development of both lineages. However, Ngn3 is expressed in only a subset of cells within the primitive ducts, which raises the question of how its expression is repressed in the majority of embryonic ductal cells. Biochemical and genetic evidence suggests that the transcription factor Hes1 plays an important role in repressing Ngn3 transcription and preventing widespread Ngn3 activation (Ahnfelt-Rønne et al. 2012, Apelqvist et al. 1999, Jensen et al. 2000, Lee et al. 2001). Recently, Hes1 was found to accelerate Ngn3 protein degradation (Qu et al. 2013), which suggests that additional posttranscriptional mechanisms contribute to Ngn3 regulation. Supporting this idea, Villasenor et al. (2008) reported that Ngn3 mRNA is detected in a much broader domain than is the protein. Generally, the detailed mechanisms controlling Ngn3 expression are still poorly understood. Progress has been slow owing to the lack of a cell system in which Ngn3 regulation can be studied ex vivo. Because Ngn3 activation follows a pattern during pancreatic differentiation of hPSCs similar to that seen in vivo

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(D’Amour et al. 2006, Xie et al. 2013), hPSCs could prove an informative model for further studies. Because ducts simultaneously express positive and negative regulators of Ngn3, their relative levels will determine whether or not a progenitor activates Ngn3 and adopts endocrine identity. Evidence suggests that the Notch pathway plays an important role in this decision. Constitutive activation of Notch signaling prevents Ngn3 activation and promotes ductal cell differentiation (Greenwood et al. 2007, Murtaugh et al. 2003). This suggests that bipotential ductal/endocrine progenitors must escape Notch signaling to initiate endocrine differentiation. Surprisingly, Notch has been found to induce both negative and positive regulators of Ngn3. High Notch signaling promotes the expression of both Hes1 and Sox9, resulting in Ngn3 repression; conversely, lower Notch activity promotes the expression of Sox9 alone, allowing for Ngn3 activation (Figure 3c) (Shih et al. 2012). Thus, the select activation of Ngn3 in small subsets of progenitors appears to be controlled by graded Notch activity in the progenitor epithelium. How individual cells within the progenitor cords acquire different levels of Notch activity remains unknown. After the secondary transition, endocrine precursors gradually stop arising from the ductal epithelium, which suggests either that postnatal ducts lose competency to form endocrine precursors or that proendocrine signals are missing (Kopinke et al. 2011, Kopp et al. 2011, Solar et al. 2009). Studies of pancreatic injury models and clonal analysis of ductal cells in vitro indicate that adult ducts retain the capacity to activate Ngn3 and thus are competent to initiate, at least to some extent, endocrine programs ( Jin et al. 2013, Kopp et al. 2011). Adult ductal cell plasticity is a field of intense study that has been reviewed comprehensively (Desgraz et al. 2011, Reichert & Rustgi 2011).


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Differentiation of the Islet Cell Types Once Ngn3 is expressed, progenitors exit the cell cycle and delaminate from the progenitor cords into the surrounding stromal tissue (Gouzi et al. 2011, Miyatsuka et al. 2011). Although individual endocrine precursors are unipotent (Desgraz & Herrera 2009), Ngn3+ cells as a whole generate five different endocrine cell types: glucagon-producing α-cells, insulin-producing βcells, somatostatin-producing δ-cells, pancreatic polypeptide–producing PP-cells, and ghrelinproducing ε-cells. Johansson et al. (2007) have shown that the competence of endocrine precursors to produce different endocrine cell types is temporally controlled. When Ngn3 expression was induced at different developmental time points from a conditional Ngn3 transgene, progenitors first differentiated into α-cells, then β- and δ-cells, and finally PP-cells. The mechanisms controlling this change in developmental potential are unknown. Studies in the developing neurons, muscle, and pituitary suggest that Notch signaling could be involved (Mizutani & Saito 2005, Mourikis et al. 2012, Zhu et al. 2006). Here, the duration of exposure to Notch is critical for determining progenitor cell competence. If the pancreas uses a similar mechanism, optimizing Notch exposure time could be key to maximizing β-cell yield in directed differentiation protocols for hPSCs. When differentiating into hormone-producing cells, Ngn3+ endocrine precursors undergo dynamic changes in gene expression, resulting in activation of both Ngn3-dependent transcription factors (Pax4, Arx, Rfx6, NeuroD1, Pax6, Isl1, and IA2) and hormones. Analysis of mouse mutants revealed that these factors control many aspects of endocrine development, including cell differentiation, cell maintenance, and islet cell–type identity (comprehensively reviewed in Mastracci & Sussel 2012, Pan & Wright 2011). Here we focus on the mechanisms governing islet cell–type identity, specifically on the cell fate choice between the β- and α-cell lineage. As with the mechanisms governing tip/trunk segregation, fate distinction between β- and αcell precursors is thought to rely on mutual repression between opposing lineage determinants. 19.12

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The transcription factors Pax4, Pdx1, and Nkx6.1 act as critical β-cell determinants (Collombat et al. 2003, Gannon et al. 2008, Henseleit et al. 2005, Holland et al. 2002, Sosa-Pineda et al. 1997), whereas Arx determines α-cell identity (Collombat et al. 2003, 2005). Forced expression of Pax4, Pdx1, or Nkx6.1 in endocrine precursors favors the β-cell fate at the expense of α-cells (Collombat et al. 2009, Schaffer et al. 2013, Yang et al. 2011). Conversely, Arx gain of function results in excess α-cells and reduced β-cells (Collombat et al. 2007). Loss-of-function experiments revealed reciprocal phenotypes, although the consequences of Pdx1 deletion in endocrine precursors are yet to be explored. Both Pax4 and Nkx6.1 are direct transcriptional repressors of Arx; conversely, Arx represses Pax4, Nkx6.1, and Pdx1 by direct or indirect mechanisms (Figure 3d ) (Collombat et al. 2005, 2007; Schaffer et al. 2013; Yang et al. 2011). One interesting but mechanistically poorly understood observation is that some transcription factors, which can change the lineage identity of endocrine precursors, lose this ability when misexpressed in differentiated endocrine cells. For example, Pdx1 or Nkx6.1 is insufficient to convert differentiated α-cells into β-cells (Schaffer et al. 2013, Yang et al. 2011). This suggests that endocrine differentiation restrains developmental plasticity. The molecular mechanisms restricting cell plasticity during cell differentiation are poorly understood. Recent studies indicate a role for DNA methylation (Dhawan et al. 2011, Papizan et al. 2011), but additional studies are needed to understand how such epigenetic mechanisms limit cell plasticity. A better understanding of these mechanisms could enable strategies for reversing epigenetic constraints in efforts to reprogram non-β endocrine cells into β-cells.

PANCREAS MORPHOGENESIS Coincident with the differentiation of endocrine and exocrine cells, the pancreas develops into a highly branched organ, wherein groups of acinar cells coalesce around a small ductal lumen to form functional exocrine secretory units, and ductal cells organize into the tubular network of the pancreatic ductal system (Pictet et al. 1972). This process, called branching morphogenesis (Figure 2), is driven by dynamic cell rearrangements and spatiotemporally controlled cell proliferation. Although pancreatic cell differentiation and its molecular control have been studied since the 1990s, knowledge of how the pancreas acquires its final shape and becomes a functional organ is still rudimentary. Two landmark studies describing the cellular changes associated with pancreatic tubulogenesis (Kesavan et al. 2009, Villasenor et al. 2010) now provide the framework for exploration of the mechanisms governing pancreatic branching morphogenesis. Prior to the initiation of epithelial branching at around e12, the pancreatic buds form a multilayered core of unpolarized cells engulfed by a single basement membrane abutting the mesenchyme (Figure 2a) (Hick et al. 2009, Kesavan et al. 2009, Villasenor et al. 2010). Pancreatic tubulogenesis begins with individual cells acquiring apicobasal polarity: Cells develop a defined apical membrane that faces a central lumen and a basal surface attached to a layer of extracellular matrix (ECM) (Kesavan et al. 2009, Villasenor et al. 2010). Groups of newly polarized cells then form rosettes, wherein adjacent cells are linked by intercellular junctional complexes around a nascent central lumen (Figure 2b, inset) (Villasenor et al. 2010). Newly formed microlumens are initially unconnected but eventually fuse into a luminal plexus (Figure 2c). Remodeling of the luminal plexus produces a tubular network lined by a polarized, monolayered epithelium attached to a basement membrane (Figure 2c, inset) (Kesavan et al. 2009, Villasenor et al. 2010). At birth, the tubes have organized into a highly branched ductal tree, connecting exocrine secretory units to the ductal system (Figure 2d, inset). Concomitant to epithelial branching, blood vessels and nerves also begin to penetrate the epithelium, intercalating between nascent branches of the pancreatic ductal tree (Figure 2b–d ) (Lindsay et al. 2006, www.annualreviews.org • Pancreas Organogenesis


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Magenheim et al. 2011a, Pierreux et al. 2010). The stereotypical organization of cells in the mature pancreas illustrates that proper pancreas morphogenesis requires highly coordinated cell rearrangements involving acinar, ductal, endocrine, endothelial, neuronal, and mesenchymal cells. Currently, little is known about the molecular cues governing these cell rearrangements; about how pancreas morphogenesis is coordinated with cell differentiation; or about how differentiation of pancreatic endocrine and exocrine cells influences surrounding nonepithelial cells, and vice versa.

Intracellular Signals Governing Pancreas Morphogenesis


In many organs, including pancreas, epithelial morphogenesis is controlled by Rho family GTPases, which regulate cell polarity, adhesion, and migration by organizing the actin cytoskeleton (Heasman & Ridley 2008). Kesavan et al. (2009) demonstrated that deletion of the Rho GTPase Cdc42 interrupts cell polarization during early morphogenesis, disrupting epithelial morphology and aborting tubulogenesis at microlumen formation. Premature arrest of tubulogenesis was also observed in mice deficient in RhoA GTPase inhibitor Strad13 (Petzold et al. 2013), whereas pancreas-specific deletion of Rho GTPase Rac1 had no obvious effect on pancreas morphogenesis (Heid et al. 2011). This implies that different Rho GTPases have distinct roles in shaping the organ. Studies in other organs show that Rho GTPases are regulated by Ephrin, EGF, and ECMintegrin signaling (Heasman & Ridley 2008, Huveneers & Danen 2009, Poliakov et al. 2004). The hypobranched pancreas seen in Ephrin and EGF receptor–mutant mice suggests that these signaling pathways could also influence Rho GTPase activity in the pancreas (Miettinen et al. 2000, Villasenor et al. 2010). How extrinsic cues coordinate epithelial morphogenesis and differentiation in a spatiotemporally controlled manner is an active area of research.

Coordinating Morphogenesis and Cell Differentiation Evidence is emerging that mesenchymal cells, blood vessels, and ECM provide locally restricted signals that influence morphogenesis and cell fate decisions in the developing pancreas. Tip/trunk segregation not only marks the first cell fate choice of pancreatic progenitors but also coincides with the formation of branches in the tip region (Figure 2b, inset) (Zhou et al. 2007). The tissue remodeling associated with branch formation results in the repositioning of mesenchymal and epithelial cells and basement membrane components (Kesavan et al. 2009, Landsman et al. 2011). Real-time imaging and static analysis of pancreas morphogenesis suggest that new branches form by tip-splitting (i.e., individual tips repeatedly split into new tips) (Puri & Hebrok 2007, Villasenor et al. 2010). Whether this is driven directly by differential proliferation of tip cells or indirectly by cell remodeling in the trunk is unknown. However, tip cells have been observed to proliferate more quickly than trunk cells (Petzold et al. 2013, Zhou et al. 2007), which implies that tip proliferation could drive branch formation. One candidate cue for regulating tip-cell proliferation is FGF, which is secreted by mesenchymal cells that surround the tips when branching is initiated (Bhushan et al. 2001). Removal of mesenchyme as an FGF source halts morphogenesis of pancreatic explants, whereas adding FGF to cultures restores branching and cell proliferation (Miralles et al. 1999). Similar to their association with mesenchymal cells, emerging tip cells are also directly apposed to the basement membrane at the beginning of epithelial branching (Kesavan et al. 2009, Villasenor et al. 2010). Explant culture studies have shown that knockdown or blocking of antibodies against the ECM component laminin-1 reduces expression of early acinar cell markers and impairs branching (Kesavan et al. 2009, Li et al. 2004). Because the branching process leads to increased cellular exposure to ECM, the process of morphogenesis as such could cause cells to adopt acinar identity. 19.14

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Whereas mesenchymal cells and ECM components favor acinar identity and promote branching, blood vessels have the opposite effect. Forced VEGFA expression in pancreatic progenitors caused increased expression of the trunk marker Hes1 and concomitant loss of the tip/acinar markers Cpa and amylase, whereas VEGF ablation favored acinar formation and caused hyperbranching (Magenheim et al. 2011a, Pierreux et al. 2010). Consistent with their trunk-promoting effect, blood vessels remain physically associated with the trunk and become separated from the tips (Magenheim et al. 2011a, Pierreux et al. 2010) (Figure 2b–c, inset). These examples illustrate that organ morphogenesis and cell differentiation are intricately linked. Further studies should focus on identifying the extrinsic and intrinsic cues mediating cross talk between endothelial, mesenchymal, and epithelial cells and on deciphering how these cues govern morphogenesis and cell differentiation. To analyze spatiotemporally dynamic intercellular interactions more precisely, it would be helpful to develop a system for studying pancreas organogenesis in vitro under defined conditions. A stem cell–based 3-D differentiation system could enable significant progress.

Morphogenetic Changes and Endocrine Cell Differentiation Morphogenetic changes are also associated with the initiation of endocrine differentiation in the progenitor cords. As cells initiate Ngn3 expression, they acquire a droplet shape and delaminate from the progenitor epithelium (Figure 2c, inset) (Gouzi et al. 2011, Rukstalis & Habener 2007). Gouzi et al. (2011) have shown that expression of Snail2, a protein that induces cell motility by triggering epithelial-to-mesenchymal transition (EMT) (Barrallo-Gimeno & Nieto 2005), is Ngn3 dependent. Ectopic Ngn3 expression also triggers EMT, which suggests a dual role for Ngn3 in inducing both cell differentiation and delamination (Gouzi et al. 2011). The extent to which these processes are interdependent is unknown. Conditional inactivation of Snail2, or of other effectors of EMT, may explain the connection between endocrine cell differentiation and delamination. Another mechanism that could enable cell delamination is asymmetric cell division, in which cells divide so that only one daughter cell inherits the apical membrane and remains in the progenitor epithelium, while the daughter cell inheriting the basal membrane escapes from the epithelial sheath and differentiates. This mechanism is associated with the asymmetric distribution of cell fate determinants to the daughter cells and is relevant for the differentiation of neurons and epidermal cells (reviewed in Knoblich 2008). It is unknown whether endocrine precursors are created by asymmetric cell divisions in progenitor cords. The knowledge may be important for devising appropriate in vitro culture conditions for production of functional endocrine cells from hPSCs. Rapidly improving real-time imaging technology, which now allows for the visualization of fluorescently labeled cells in whole organ explants at single-cell resolution, will likely prove useful for defining the cellular events associated with the emergence of endocrine precursors from the progenitor cords.

APPLICATION OF DEVELOPMENTAL PRINCIPLES TOWARD STEM CELL DIFFERENTIATION The ultimate goal of understanding pancreas development is to gain insight into pancreasassociated diseases and to develop novel therapies to treat such diseases. With the knowledge amassed by studying pancreas development, investigators are exploring novel therapeutic avenues for treating diabetes by replacing or regenerating β-cell mass. Such innovative strategies for diabetes treatment include increasing β-cell numbers by boosting their intrinsic ability to replicate; fate-converting other pancreatic cells into β-cells (reviewed in Desgraz et al. 2011); www.annualreviews.org • Pancreas Organogenesis


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and most fascinatingly, both as a therapy and as a tool for study, generating β-cells in vitro by differentiating hPSCs.

Generating Pancreatic Progenitors from Human Pluripotent Stem Cells


The enthusiasm for deriving transplantable β-cells from hPSCs is the direct result of landmark achievements in cell transplantation, in particular the successful allogenic transplantation of islets, which has allowed diabetic patients to forego insulin injections for years (Shapiro et al. 2006). Unfortunately, the amount of donor islets required for widespread treatment significantly exceeds availability, and even when donor islets are available, recipients must undergo lifelong immunosuppression to prevent rejection. Although these limitations have prevented its widespread use in diabetic patients, the insulin independence achieved with islet transplantation provides proof of the principle that cell transplantation can be a viable therapy for diabetes. Unlike allografts, which are susceptible to donor shortages, hPSCs could yield an unlimited source of transplantable β-cells. Additionally, patient-derived induced pluripotent stem cells would obviate immunosuppression. For these reasons, there is currently a major push to generate pancreatic β-cells in vitro. Protocols have been established for deriving pancreatic precursors from hPSCs. hPSCs can be differentiated with high efficiency to definitive endoderm by adding high levels of Activin A, mimicking the proendodermal role of Nodal during in vivo gastrulation (reviewed by Zorn & Wells 2009). A landmark study by D’Amour et al. (2006) established a protocol to guide hPSC-derived endoderm toward the pancreatic lineage. Similar protocols have since been published (Kroon et al. 2008, Mfopou et al. 2010, Nostro et al. 2011, Rezania et al. 2012, Xu et al. 2011). The majority of pancreatic differentiation protocols induce pancreatic fate by simultaneously treating cells with RA and inhibitors of Shh and BMP, recapitulating the signaling environment that specifies the pancreas in vivo. Such treatment generates the in vitro equivalent of early multipotent pancreatic progenitors, expressing PDX1, SOX9, PTF1a, and NKX6.1. These in vitro–generated progenitors also pass the functional test of having the potential to form mature endocrine, ductal, and acinar cells when transplanted into mice (Kelly et al. 2011, Kroon et al. 2008, Rezania et al. 2012, Schulz et al. 2012, Xie et al. 2013). The resulting endocrine cells are functional, responding appropriately to glucose and properly regulating glucose homeostasis in diabetic animal models (Kroon et al. 2008, Rezania et al. 2012, Xie et al. 2013). Further evidence for their islet-like phenotype is that the transcriptional signature and cellular composition of hPSC-derived endocrine clusters are strikingly similar to those of human islets (Xie et al. 2013). Having accomplished the in vitro differentiation of functional pancreatic progenitors, the field has attempted to optimize differentiation efficiencies across different cell lines. In addition, to allow for large-scale cell production, strategies have been developed to expand developmental lineage intermediates in vitro (Cheng et al. 2012, Schulz et al. 2012, Sneddon et al. 2012). However, because it is unknown whether transplantation of pancreatic progenitors can cure diabetes in humans, the ultimate goal is to generate functional β-cells in vitro.

Beyond Progenitors: How to Generate Functional Endocrine Cells In Vitro Currently, endocrine cells that arise in vitro are non–glucose responsive and often coexpress multiple hormones. For pancreatic differentiation of hPSCs to fulfill its full therapeutic and scientific promise, methods to generate functional pancreatic cells in vitro must be developed. As discussed in the sections on Lineage Decisions During Pancreas Development and Pancreas Morphogenesis, above, the developing pancreas receives extrinsic signals from the mesenchyme, ECM, and endothelium. However, these components are not supplied in the majority of hPSC 19.16

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differentiation protocols. Coculture with mesenchymal cell lines has been explored as a strategy to expand intermediate precursor populations, consistent with the role of mesenchyme in early pancreatic progenitor expansion (Sneddon et al. 2012). Possible instructive signals from the mesenchyme have been added to current pancreatic differentiation protocols and have resulted in increased efficiency of PDX1 induction (Guo et al. 2013). The role of mesenchyme and ECM at later steps of pancreatic development in vivo, especially during endocrine cell differentiation, is still poorly understood. Both should be considered as a possible source for signals when developing strategies to generate functional β-cells in vitro. Blood vessels may also be involved in islet development, because both native islets and in vivo– matured, transplanted hPSC-derived endocrine cells are highly vascularized with endocrine cells in direct contact with blood vessels. Although endothelial cells are clearly important for proper sensing of blood glucose levels and release of hormones, their role in pancreatic lineage determination is only beginning to be unveiled. Because blood vessels promote trunk cell formation at the expense of tip cell formation (see section on Coordinating Morphogenesis and Cell Differentiation) (Magenheim et al. 2011a, Pierreux et al. 2010), endothelial cells could provide critical missing cues for favoring the endocrine cell fate during in vitro differentiation. As discussed in the section on Pancreas Morphogenesis, the embryonic pancreas undergoes extensive morphogenetic changes in a 3-D epithelium, which may be coupled with cell-lineage decisions. Unlike in vivo development, in vitro pancreatic differentiation of hPSCs is primarily performed in 2-D culture systems. This difference between in vitro and in vivo differentiation conditions could, at least in part, explain our inability to generate functional β-cells in vitro. Protocols to generate functional cells of other endodermal lineages (e.g., the intestine) have employed 3-D culture systems. Such 3-D systems support proper morphogenesis and have been shown to produce mini organs with remarkable similarity to their in vivo counterparts (Sato et al. 2009, Spence et al. 2011). Organotypic culture systems may also facilitate the production of functional β-cells from hPSCs. An important question is whether the generation of islets or of only β-cells should be attempted. Engrafted hPSC-derived progenitors differentiate into functional islet-like clusters that contain all endocrine cell types, in a ratio similar to that in human islets (Xie et al. 2013). Although it is currently unknown whether non-β islet cells are required for β-cell function, evidence suggests that paracrine signaling between endocrine cell types may be important in humans (reviewed in Caicedo 2012). Therefore, future studies should consider the production of entire islets and not just of β-cells.

Human Pluripotent Stem Cells as a Model for Understanding Pancreas Biology and Disease Much information about pancreas development and organogenesis has been gleaned from animal models; there are, however, some limitations to these models. At a technical level, transient developmental cell populations are too sparse in embryos to isolate sufficient cells for large-scale genomic or biochemical analyses. The ability to differentiate such lineage intermediates from hPSC in virtually unlimited quantities has enabled such studies and recently has led to the identification of core transcriptional and epigenetic mechanisms of pancreatic lineage determination (Xie et al. 2013). Moreover, species-specific differences in gene regulation limit the use of animal models for studying human disease. One example is maturity onset diabetes of the young (MODY), which in humans results from single-gene mutations that are not always phenocopied in mice (Haumaitre et al. 2006, Mayer et al. 2008). It is hoped that protocols for the in vitro production of functional β-cells will facilitate studies aimed at understanding disease mechanisms of diabetes. Advances www.annualreviews.org • Pancreas Organogenesis


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in genome editing now allow us to model effects of gene mutations or single-nucleotide polymorphisms in hPSCs. Thus, the production of β-cells in vitro not only will be a significant step toward regenerative medicine but also will help advance our understanding of the pathogenesis of diabetes.

FUTURE PROSPECTS


This review has covered the tremendous progress we have made in understanding pancreatic organogenesis and has briefly discussed the progress made in recapitulating this process in vitro using hPSCs. Reflecting on this body of work, a few conclusions become strikingly clear. First, much remains to be understood about the complex process of pancreas organogenesis, specifically, how morphogenesis is coupled with cell fate specification. Although we have made significant progress in understanding many of the transcription factors that are required at various stages of pancreas development, the signals that promote and regulate each developmental decision are still poorly understood. Clearly, the current success of in vitro pancreatic differentiation of hPSCs has relied heavily on studies employing animal models. Therefore, we must continue to apply the lessons learned in vivo to achieve the critical goal of generating functional β-cells in vitro.

DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS We thank members of the Sander laboratory for constructive comments on the manuscript. We apologize to our colleagues whose references were omitted owing to space constraints. Work in the Sander laboratory is supported by grants from the National Institutes of Health (NIH), the Juvenile Diabetes Research Foundation, and the California Institute for Regenerative Medicine. H.P.S. and A.W. were supported by postdoctoral fellowships from the Juvenile Diabetes Research Foundation. A.W. was additionally supported by the NIH training program in diabetes research T32 DK7494-27. LITERATURE CITED Afelik S, Chen Y, Pieler T. 2006. Combined ectopic expression of Pdx1 and Ptf1a/p48 results in the stable conversion of posterior endoderm into endocrine and exocrine pancreatic tissue. Genes Dev. 20:1441–46 Afelik S, Jensen J. 2012. Notch signaling in the pancreas: patterning and cell fate specification. WIREs Dev. Biol. 2:531–44 Afelik S, Qu X, Hasrouni E, Bukys MA, Deering T, et al. 2012. Notch-mediated patterning and cell fate allocation of pancreatic progenitor cells. Development 139:1744–53 Ahlgren U, Jonsson J, Edlund H. 1996. The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/PDX1-deficient mice. Development 122:1409–16 Ahnfelt-Rønne J, Jørgensen MC, Klinck R, Jensen JN, Fuchtbauer E-M, et al. 2012. Ptf1a-mediated control ¨ of Dll1 reveals an alternative to the lateral inhibition mechanism. Development 139:33–45 Ahnfelt-Rønne J, Ravassard P, Pardanaud-Glavieux C, Scharfmann R, Serup P. 2010. Mesenchymal bone morphogenetic protein signaling is required for normal pancreas development. Diabetes 59:1948–56 Apelqvist A, Ahlgren U, Edlund H. 1997. Sonic hedgehog directs specialised mesoderm differentiation in the intestine and pancreas. Curr. Biol. 7:801–4 19.18

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www.annualreviews.org • Pancreas Organogenesis


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Organogenesis and morphogenesis. Preface.

From pancreas morphogenesis to β-cell regeneration.

Three-dimensional pancreas organogenesis models.

In vitro pancreas organogenesis from dispersed mouse embryonic progenitors.

Transcriptional control of mammalian pancreas organogenesis.

Endothelium-derived essential signals involved in pancreas organogenesis.

Editorial: Organogenesis: From Development to Disease.

Stochastic priming and spatial cues orchestrate heterogeneous clonal contribution to mouse pancreas organogenesis.

Lineage determination in acute leukemias.

Morphogenesis and distribution of the endocrine pancreas in adult lampreys.

Single-Cell Lineage Tracing Reveals that Oriented Cell Division Contributes to Trabecular Morphogenesis and Regional Specification.

From gradients to axes, from morphogenesis to differentiation.

Endoderm Jagged induces liver and pancreas duct lineage in zebrafish.

ECM Signaling Regulates Collective Cellular Dynamics to Control Pancreas Branching Morphogenesis.

Blood vessel crosstalk during organogenesis-focus on pancreas and endothelial cells.

Novel pancreas organogenesis markers refine the pancreatic differentiation roadmap of embryonic stem cells.

In vitro organogenesis from pluripotent stem cells.

Organogenesis from Callus Culture of Hordeum vulgare.

Morphogenesis in Selaginella. III. Meristem determination and cell differentiation.

Distribution of neurosensory progenitor pools during inner ear morphogenesis unveiled by cell lineage reconstruction.

SNF-Mediated Lineage Determination in Mesenchymal Stem Cells Confers Resistance to Osteoporosis.

Hypocotyl adventitious root organogenesis differs from lateral root development.

Morphogenesis of somatostatin- and insulin-secreting cells in the lamprey endocrine pancreas.

Mapping the route from naive pluripotency to lineage specification.