Embryonic development of the endocrine pancreas

C H A P T E R

12 Embryonic development of the endocrine pancreas Spencer R. Andrei*, Maureen Gannon*,†,‡,§,¶ *

Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, United States, †Department of Veterans Affairs, Tennessee Valley Health Authority, Nashville, TN, United States, ‡Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, United States, §Program in Developmental Biology, Vanderbilt University, Nashville, TN, United States, ¶Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, United States O U T L I N E Introduction to pancreas development

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Pancreas development Foregut endoderm compartmentalization Pancreatic buds and branching morphogenesis Exocrine versus endocrine development during secondary transition Endocrine development during late gestation

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Introduction to pancreas development Pancreas development refers to the complex series of events occurring from conception through adulthood that orchestrate the formation of a fully functional, ­ hormone-producing pancreas in mammals. Understanding the underlying developmental mechanisms directing pancreas organogenesis is essential to fully comprehend physiological and pathophysiological processes in the mature adult organ. Limited supply of tissue, taken together with the well-known ethical restrictions regarding human specimens, has thwarted the scientific community from elucidating the precise developmental mechanisms directing human pancreas maturation; however, the ability to genetically manipulate mouse strains has allowed researchers to determine the extent to which a myriad of instructive or permissive factors interact to coordinate proper growth of the Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, Volume 2 https://doi.org/10.1016/B978-0-12-814831-0.00012-9

Postnatal islet development and function Maturation of postnatal islets Communication between endocrine cells in postnatal islets

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Conclusions

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Acknowledgments

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References

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­ ancreas. In fact, the majority of our current knowledge p on human pancreas development has been deduced from data acquired from other species such as rodents.1–4 In this chapter, we compare and contrast developmental similarities in both human and rodent pancreata. The adult pancreas is composed of exocrine and endocrine tissues which uniquely differ in their respective functionalities. The exocrine compartment (nearly 98% of the adult pancreas) is composed of acinar and ductal cells, which secrete and transport digestive enzymes into the duodenum. The endocrine portion of the pancreas consists of the islets of Langerhans which contain five hormone-producing cell types including the α-cells (glucagon), β-cells (insulin), δ-cells (somatostatin), PP cells (pancreatic polypeptide), and ε-cells (ghrelin) the primary function of which is to regulate blood glucose levels in a dynamic manner. Understanding the developmental processes leading to the formation of these

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tissue compartments is fundamental when investigating pancreatic structure and/or function. In fact, numerous investigations have utilized the information obtained from developmental biology (with regard to the spatiotemporal expression patterns of genes and transcription factors) to serve as a model by which to direct the differentiation of stem cells into functional, hormone-­ producing pancreatic endocrine cells in vitro.5–10 Each species reaches developmental milestones in a temporally unique manner that is predicated upon their respective gestational periods. However, progression through the different stages of pancreas formation across mammalian species is highly comparable. Here we highlight similarities and differences between human pancreas development and islet differentiation and that of the mouse, the most commonly used model system for studying pancreas development (Fig.  1 and Table  1). Around embryonic day (e) 5.5–6.5 in mice and human gestational day (hd) 14–16, the events of gastrulation result in the formation of three germ layers (ectoderm, endoderm, and mesoderm). The primitive gut tube from which the pancreatic buds arise derives from the definitive endoderm with an overlying mesodermal layer.11 Hormone-positive cells can be detected within the pancreatic epithelium shortly after bud formation in mice around e10.5 and human gestational week (hw) 8–10 which is coincident with the first phase of expression of neurogenin 3 (Neurog3; a transcription factor expressed in endocrine progenitor cells).12 Following delamination of endocrine cells from the pancreatic epithelium, ­hormone-producing cells form “clusters” that eventually coalesce into islets by e18 in mice and hw20. This is a notable temporal disparity considering islets are formed in mice nearly 85% of the way through their gestational period whereas humans are only ~50% through gestation. Additional differences between mouse and human islets include the timing and pattern of innervation and microcirculation,13 the inherent transcriptional landscape,14 and the well-documented architectural differences.15 Therefore, one should always consider the potential drawbacks of rodent-to-human comparisons when analyzing developmental mechanisms across species. This chapter briefly covers the developmental processes of the pancreas as a whole while harboring an in-depth focus at the precise mechanisms leading to development of the endocrine compartment in both humans and mice.

Pancreas development Foregut endoderm compartmentalization Specific digestive organs arise from precise regions along the anterior-posterior axis of the embryonic gut

tube that are compartmentalized based on the spatiotemporal expression of certain transcription factors (Fig. 2). The anterior-most portion of the embryonic gut tube is the foregut which is demarcated by Sox2 expression and gives rise to the esophagus, forestomach, lungs, and trachea. Immediately posterior to the Sox2-expressing anterior foregut is the posterior foregut which gives rise to the antral stomach, duodenum, liver, gall bladder, and pancreas. The portion of the posterior foregut marked solely by Pdx1 expression gives rise to the antral stomach and common bile duct, whereas regions in which Pdx1, Ptf1a, and Mnx1/Hb9 are co-expressed develop into the dorsal and ventral pancreatic buds. The Cdx2+ midgut gives rise to the small intestine with anterior Pdx1 co-­ expression marking the region that will give rise to the duodenum. Lastly, the posterior-most portion of the gut tube is the hindgut which is Cdx1/Cdx2 ­dual-positive and gives rise to the colon.

Pancreatic buds and branching morphogenesis An intricate system of secreted signaling molecules derived from the notochord and dorsal aorta initiate highly orchestrated spatial rearrangements that direct the formation of the pancreatic buds. At e8.0 in the mouse, the notochord is positioned immediately dorsal to the prepancreatic endoderm. Notochord-derived signals such as Activin-βB and FGF2 inhibit sonic hedgehog (Shh) expression in the dorsal posterior foregut endoderm, allowing for the initiation of pancreatogenesis. Failure to inhibit Shh expression in the prepancreatic endoderm results in aberrant pancreas development and overlapping intestinal phenotypes.16 Shortly thereafter, the dorsal aortae fuse together, resulting in the displacement of the notochord and removing the contact point between the notochord and dorsal endoderm. Signals from the dorsal aorta induce Pdx1 and Ptf1a expression within the dorsal posterior foregut endoderm. Pdx1 is essential for pancreatic outgrowth in both mice and humans (Fig. 3), and also regulates differentiation of specific cell types within the rostral duodenum.17–19 Ptf1a is also required for the outgrowth of the pancreatic epithelium from the duodenum in mice and humans and in its absence, cells destined for the pancreatic fate instead of differentiate into intestinal epithelial cells20, 21 (Fig.  3). Endodermal epithelial cells that co-express Pdx1 and Ptf1a bud outward as a plexus into the overlying mesenchyme to form dorsal (e9.5, hd26) and ventral (e10, hd32) pancreatic buds (Fig. 1). Initially, two ventral buds emerge from the ventral endodermal epithelium but only one survives to fuse with the dorsal bud later in gestation. This period of time following bud evagination and prior to definitive differentiation is referred to as the “primary transition.” Endodermally derived cells within the plexus are

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2° transition

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1° transition

FIG. 1  Timeline of mouse and human pancreas development.

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TABLE 1  Gestational timeline comparison of mouse and human development Mouse embryonic Pancreatogenic day (e) features

Human gestational day (hd)

9.0–9.5

Pdx1 expression in prepancreatic endoderm

29–31

9.5–10.0

Pancreatic bud formation

30–33

10.0–14.0

Organ outgrowth

33–45

MPC proliferation Tip-vs-trunk remodeling initiates 14.0–14.5

Trunk-and-tip cell separation observed

45–47

14.5–15.5

Endocrine lineage 47–52 demarcated by Neurog3 expression

15.5–16.5

Dorsal and ventral bud fusion completes

52–58

Adapted from Jennings RE, Berry AA, Strutt JP, Gerrard DT, Hanley NA. Human pancreas development. Development. 2015;142:3126–3137, and the UNSW Human Embryo Resource.

c­onsidered primary multipotent pancreatic progenitor cells (MPCs), with the ability to generate all endocrine and exocrine pancreatic cell lineages.22 Through an intricate process involving gut tube rotation and elongation of the dorsal and ventral pancreatic buds, fusion occurs beginning at e12.0 in mice and hd37 in humans to yield a single organ (Fig. 1). Branching morphogenesis begins around e12.5 in mice and, through distinct alterations in the organization of the epithelial

plexus, establishes the pancreatic ductal network.23 At this time, FGF10 becomes highly expressed in the pancreatic mesenchyme and induces proliferation within the endoderm as well as branching morphogenesis.24 Branching morphogenesis occurs through MPC-driven remodeling via the “tip and trunk” model where extending branch tips emerging from a trunk domain resembling a tree-like structure1, 25 (Fig.  1). Spatiotemporally expressed transcription factors regulate pancreatic cell fates within the trunk and tip domains. Initially, cells located within the tip domain embody undifferentiated secondary MPCs with the ability to give rise to any of the three pancreatic cell types (duct, exocrine, endocrine) and are demarcated by Sox9, Ptf1a, and Cpa1, as well as low levels of Pdx1 and Oc122 (Fig. 3). On the other hand, trunk cells are demarcated by high levels of Oc1, Sox9, Hnf1β, and Nkx6.1 (Fig. 3). The cells that line the cylindrical tube-like luminal “trunk” domain are bipotent in nature and give rise to either endocrine or ductal cells. Preendocrine cells activate Neurog3 expression (Fig.  3), with committed endocrine progenitors showing elevated Neurog3 and eventually delaminating from the trunk domain during a period known as the “secondary transition” (described further below). Clusters of delaminated endocrine cells give rise to hormone-producing islets through cell migration and sorting mechanisms mediated by semaphorin-to-neuropilin (Sema3a-Nrp2) signaling,26 as well as Oc1, connective tissue growth factor (CTGF), Wnt, and other signaling cascades.3 Neurog3lo cells remain proliferative and contribute to mature duct cell formation if they fail to upregulate Neurog3 and delaminate.27 These mechanisms operate in concert to ensure a proper, homeostatic population of endocrine, duct, and acinar cells that compose the mature adult pancreas.

FIG. 2  Transcription factor regionalization along the anterior-posterior axis of the gut tube. Reprinted with permission from Guney MA, Gannon M. Pancreas cell fate. Birth Defects Res C Embryo Today Rev. 2009;87:232–248.

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FIG.  3  Transcription factor expression patterns throughout the stages of endocrine cell differentiation. Figures reprinted with permission from Offield MF, Jetton TL, Labosky PA, et al. Pdx-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development. 1996;122:983– 995, Kawaguchi Y, Cooper B, Gannon M, Ray M, MacDonald RJ, Wright CV. The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors. Nat Genet. 2002;32:128–134, Gradwohl G, Dierich A, LeMeur M, Guillemot F. Neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc Natl Acad Sci U S A. 2000;97:1607–1611. Copyright (2000) National Academy of Sciences, United States.

Exocrine versus endocrine development during secondary transition The secondary transition (occurring at approximately e13.5 in mice and hd39 in humans; Fig. 1) is the period of pancreatic development during which the exocrine and endocrine compartments become functionally distinct from the secondary MPCs located within the tip domain and bipotential trunk cells, respectively. The ability of secondary MPCs to differentiate into any pancreatic cell type is conserved until acinar gene expression begins within cells located at the tips at approximately e14.0, suggesting that exocrine/endocrine differentiation is controlled spatially as well as temporally. The spatiotemporal manner by which exocrine and endocrine differentiation occurs is a crucial component to a thorough understanding of cell lineage allocations in the pancreas. Cell lineage determinations in the developing pancreas are most often described by their unique transcriptional programs. Below we cover the different transcription factors and the downstream targets that are involved in pancreas cell fate allocations. Exocrine cell fate allocation Acinar and ductal cells make up the exocrine portion of the pancreas. Acinar cells are considered the functional unit of the exocrine pancreas where they synthesize, store, and secrete digestive enzymes into the

ductal epithelial network, ultimately leading to the duodenum. Acinar cells are derived from tip-localized secondary MPCs that activate the transcription factor Mist1 and maintain low levels of Oc1 and Pdx1 expression (Oc1lo, Pdx1lo) and high levels of Ptf1a (Pdx1hi) (Fig. 3). Bipotent trunk progenitors differentiate into ductal cells upon Foxa2, Hnf1β, Sox9, and Oc1hi expression (Fig. 3). Although the exocrine compartment of the pancreas holds significant physiological and pathophysiological relevance, this chapter focuses on the developmental processes of the endocrine portion of the pancreas. Endocrine cell fate allocation Endocrine lineage allocation is primarily regulated by the pro-endocrine transcription factor Neurog3, which is induced by combined activity of the Oc1 and Pdx1 transcription factors. Neurog3 expression is initially observed in scattered cells within the prepancreatic epithelium at e9.5. Cells exhibiting sustained activation of Neurog3hi give rise to all five types of hormone-­ producing endocrine cells including glucagon-producing α-cells, ­insulin-producing β-cells, somatostatin-secreting δcells, ghrelin-producing ε-cells, and PP cells that secrete PP. Differentiation of each of these cell types requires Neurog3 expression28 (Fig.  3). Transgenic overexpression of Neurog3 throughout the mouse pancreatic epithelium results in an increase in endocrine specification, but generates only increased numbers of α-cells

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(the first h ­ormone-expressing cells to differentiate).29 Furthermore, it has been demonstrated that the temporal nature of Neurog3 expression is the primary determinant in progenitor-to-endocrine cell differentiation. The earliest Neurog3-expressing progenitors favor α-cell commitment whereas β- and δ-cells arise from progenitors demonstrating Neurog3 expression at later timepoints.30 After cells expressing high levels of Neurog3 pass through the commitment state to the endocrine cell fate, the pro-endocrine precursors ultimately delaminate from the trunk epithelium. Through an asymmetric cell division, Neurog+ cells generate an apical daughter cell that maintains a connection with the epithelial layer and a basal daughter cell that does not; apical cells conserve their progenitor-like capabilities while basal cells differentiate into endocrine cells. Delamination results from Snail2 activation (within Neurog3+ cells) leading to inhibition of E-cadherin expression, thus allowing endocrine progenitors to migrate radially into the parenchyma from the epithelial trunk domain in an epithelial-to-­ mesenchymal transition.31 In late gestation in the mouse (~e18.5), delaminated endocrine cells organize into clusters that serve as the beginning stages to islet formation (Fig. 1). Endocrine cell subtype specification: α- and β-cells Although endocrine progenitors expressing Neurog3 exhibit reduced Pdx1 expression, cells destined for the β-cell lineage upregulate Pdx1. Many studies have demonstrated a clear, essential role for Pdx1 in β-cell specification and differentiation. Pdx1 activity simultaneously promotes β-cell differentiation while repressing the α-cell fate.32 Indeed, loss of Pdx1 from embryonic ­insulin-expressing cells results in loss of the β-cell phenotype and maturity onset diabetes33 and inactivation of Pdx1 in adult β-cells induces the loss of β-cell identity and function and activation of the α-cell program.32 Pdx1 binds to and activates many genes essential for normal β-cell function including insulin, SLC2A2, and glucokinase. Transgenic overexpression of Pdx1 in glucagon-­expressing cells postnatally results in α-to-β cell conversion, while hypomorphic Pdx1 alleles result in increased numbers of α-cells and decreases in β-cells, leading to impaired glucose tolerance.34 Heterozygosity for inactivating mutations in Pdx1 in mice and humans leads to an increased susceptibility for type 2 diabetes.18, 19, 35 An in-depth review of the role of Pdx1 and transcriptional regulation of pancreatogenesis has been published.36 Pax4 and Arx are two transcription factors that have significant roles in the determination of endocrine cells toward the α-, β-, or δ-cell fates. Pax4 is first detected at e9.5 in the pancreatic epithelium37 but is maintained throughout adulthood only in β- and δ-cells.38 Pax4 is necessary and sufficient for commitment to the β-cell lineage: embryonic overexpression or adult islet-restricted

overexpression promotes the β-cell fate38, 39 (although these findings are controversial40), whereas Pax4 deletion in mice results in an increase in ε cells at the expense of β-cells.41 Furthermore, overexpression of Pax4 reduces glucagon expression in differentiating human embryonic stem cells (hESCs).42 Similarly, Arx is first observed at e9.5 in the pancreatic anlagen and at e14.5 within glucagon+ and PP cells.41 Arx activation has been demonstrated to favor the α-cell fate. Expression of Arx in the pancreatic epithelium during development and in adult β-cells increases the α-to-β/δ-cell ratio.43 Moreover, mice in which the Arx gene has been inactivated exhibit reduced α-cell differentiation and their conversion into functional β-like cells.44 Taken together, these studies suggest opposing roles of Pax4 and Arx in pancreatic endocrine cell fate specification with Pax4 favoring the β-cell fate and Arx promoting α-cell differentiation. In fact, Pax4 and Arx have been shown to directly inhibit the expression of the other through direct promoter binding, suggesting they both directly and indirectly reciprocally repress their respective endocrine cell fates.41 The MafA and MafB transcription factors also demonstrate a unique spatiotemporal expression program in the developing pancreas. In contrast to other broadly expressed transcription factors, MafA and MafB are only expressed in hormone-positive cells. The MafA and MafB expression patterns in mouse insulin+ cells are dynamic in nature with MafB being expressed in immature insulin there should be no spaces between the word before the + sign and the + sign...check the whole document+ cells and MafA expression becoming elevated as β-cell terminal differentiation occurs and MafB is silenced. MafB is first detected at e10.5 in glucagon+ or insulin+ cells in the pancreatic epithelium, whereas MafA expression begins at e13.5 and only in insulin+ cells.45–47 In mice, MafB expression in insulin+ cells is silenced shortly after birth and becomes restricted to α-cells in adults.48 MafB is required for differentiation of both α- and β-cells, while MafA is essential for proper functionality of β-cells in adult islets. The expression patterns of MafA and MafB differ between rodents and humans. In mice, there is a developmental transition from MafB to MafA expression in β-cells. In contrast, in humans MafB expression is not silenced in β­ -cells but remains elevated throughout adulthood concomitant with MafA. Moreover, initiation of MafA expression occurs significantly earlier in mice (secondary transition) whereas onset of MafA expression in humans is roughly around age 11.49 An in-depth review of the roles of MafA and MafB in pancreatogenesis has been published.49 Endocrine cell subtype specification: δ, PP, and ε cells Much less is known about the transcription factors involved in δ-, PP-, and ε-cell lineage determination. Somatostatin-secreting δ-cells play physiologically important roles in islet function, inhibiting both insulin and

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Postnatal islet development and function

glucagon secretion, but somatostatin itself does not appear to have any role in normal pancreas development.50 A recent study conducted by Zhang et al. (2014) identified Hhex as a crucial transcriptional regulator required for δ-cell differentiation and islet function.51 Other studies have demonstrated that β-cell specific Pdx1 deletion results in an increase in α- and δ-cell numbers as well as β-cell dysfunction.52 Sosa-Pineda et al.37 showed that Pax4 inactivation leads to a loss of β- and δ-cells, whereas others have demonstrated that Arx null mice exhibit increases in δ cells while α-cells are decreased.53 β-cell specific Pdx1 deletion results in an increase in α- and δ-cell numbers due to noncell autonomous effects.52 Inactivation of the Mnx1 gene switches insulin-­ producing β-cells to a δ-like cell fate, suggesting that Mnx1 normally represses the δ-cell lineage.54 The precise transcriptional program required for PPsecreting PP cell development has yet to be fully elucidated. Early investigations yielded contradictory results with regard to when PP cells are first observed during development55, 56; however, Johansson et  al.30 utilized Neurog3 mutant mice to demonstrate that pancreatic progenitors have the ability to become PP cells around e10.5 until nearly e14.5 in mice. Ablation of embryonic PP cells results in decreased numbers of insulin- and ­somatostatin-expressing cells suggesting common lineage differentiation pathways or noncell autonomous regulation of β- and δ-cell differentiation by PP cells.57, 58 The hunger hormone ghrelin is expressed in the pancreatic ε cells that were first identified in 2002.59 Ghrelin+ cells are relatively scarce in the adult pancreas when compared to the other hormone-producing cells but are also derived from Neurog3+ progenitors in the embryonic pancreas and are most prominent in embryonic endocrine clusters and in neonatal islets.59 Mice lacking the Nkx2.2 transcription factor exhibit a dramatic increase in ghrelin+ cells while nearly all other pancreatic endocrine cell lineages are lost or reduced.60 Similarly, embryos deficient for Pax6 demonstrate an increase in ghrelin+ cells.61 Although several studies have demonstrated that ghrelin is not required for normal pancreas development or endocrine differentiation,62 further studies will be required in order to precisely determine the role of ghrelin, as well as the factors preluding ε-cell determination, in the pancreas.

Endocrine development during late gestation There are several factors involved in pancreas development that have their main effect on endocrine differentiation during late gestation. For example, Foxa2 is present during the very early stages of pancreatogenesis but does not appear to affect development until late gestation, when it represses terminal differentiation of α-cells. Furthermore, glucagon+ cells in Foxa2

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knockout mice lose their capacity to differentiate and the islets show a disrupted architecture.63 Foxa2 mutant mice show early postnatal lethality due to hyperinsulinemia and hypoglycemia caused by irregular insulin secretion.64 In contrast, Foxa1 appears to play a more important role in early pancreatogenesis since Foxa1/2 double knockouts demonstrate markedly attenuated early pancreas development.65 These studies suggest a dynamic interplay between Foxa1 and Foxa2 in a manner similar to MafA and MafB where Foxa1 regulates early developmental processes of the pancreas and Foxa2 is predominantly involved in later differentiation stages. Although expressed in embryonic insulin+ cells, MafA is necessary only in later stages to promote β-cell maturation66, 67 and maintain β-cell differentiation through activation of genes regulating functionality such as Pdx1, insulin, G6pc2, and GLUT2.68–71 MafA null mutant mice become glucose-intolerant postnatally, although endocrine cell development does not appear to be affected.72

Postnatal islet development and function Maturation of postnatal islets Endocrine terminal differentiation and islet morphogenesis continue postnatally (Fig.  1). Glucose responsiveness, a characteristic trait of postnatal islet maturation, is regulated by an intricate network of genes that are expressed after birth. Immature β-cells secrete insulin at low glucose with a poor response to elevated glucose, while mature β-cells have low-basal insulin secretion and demonstrate strong glucose-stimulated insulin secretion (GSIS). Historically, mouse islets were considered structurally and functionally mature upon expression of MafA and Glut2 concomitant with a loss in MafB. However, recent studies have demonstrated that urocortin 373, 74 and synaptotagmin 4 are also molecular markers of β-cell maturation.75 The expression of the glucose transporter, GLUT2, and glucokinase regulate the ability of the pancreas to respond to changes in plasma glucose levels and therefore insulin secretion, while prohormone convertase (PC1/3) is required for insulin protein processing. Although GSIS is attained by the second week after birth in the mouse,75 full islet maturation is not attained until 3 weeks of age, at which time pancreas innervation is complete. Species-to-species distinctions in islet composition and morphogenesis exist in mammals15, 76, 77 though the precise underlying mechanisms governing these differences have not been fully characterized. Pancreatic endocrine cells initially assemble into a mantle-core islet morphology during the early postnatal period. In adults, mouse, rat, and pig islets have

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a β cell-rich core surrounded by an α cell-rich mantle. Juvenile human islet architecture resembles these other species; however, with age, human islets show more of a lobular phenotype where α-cells can be found within the islet core78 (Fig.  1). Additionally, the ratio of ­endocrine-to-endocrine cell types differ in human islets when compared to rodents, and is more similar to islets from nonhuman primates.15 The β-to-α/δ-cell ratio is considerably lower in humans than rodents and it is speculated that this contributes to the lower threshold for GSIS observed in human islets.77 Other contributors could be species differences in islet gene expression. For example, rodents express Glut2 while human islets predominantly express the h ­ igher-affinity Glut1. In addition, human β-cells express both MafA and MafB, and MafA is known to regulate β-cell function in the mouse. Further investigations will need to be conducted in order to clarify the precise mechanisms and cellular processes underlying differential gene expression and islet architectural composition in rodent and human pancreatic islets.

investigations have demonstrated a link between the Cx36 connexin gene and susceptibility to type 2 diabetes. 86 The autocrine effects of glucagon on α-cells are well established; glucagon induces its own secretion in the face of hypoglycemia-mediated induction. However, the extent to which insulin acts in an autocrine manner on ­β-cells remains to be fully elucidated. Early investigations suggested that insulin acted via a negative feedback loop to inhibit further insulin release87; however, more recent studies have demonstrated that insulin plays a homeostatic role in β-cells where it regulates gene transcription and calcium cycling, as well as proliferation and survival.88 Communication and signaling mechanisms occurring between α-to-β-cells is also of great interest due to the reciprocal regulatory nature of glucagon and insulin by changes in glucose concentrations. Several studies have demonstrated inhibitory roles of β-cell secreted factors on neighboring α-cells. Mice lacking α-cell insulin receptors demonstrated glucose intolerance and hyperglucagonemia indicating that insulin normally acts in a paracrine manner to inhibit glucagon secretion.89 Insulin Communication between endocrine cells in also reduces ATP-induced KATP activity simultaneously postnatal islets with a GABA-induced α-cell hyperpolarization resulting α- and β-cell interactions in closure of voltage-dependent calcium channels and 90, 91 Zn2+, Many studies have confirmed direct and indi- subsequent inhibition of glucagon secretion. rect interactions between the different hormone-­ co-secreted with insulin, reduces electrical activity of producing cell types within the mature pancreatic KATP channels thereby reducing glucagon release and 92 islet. Cell-to-cell communication between endocrine GSIS from α-cells. cells is required for efficient regulation of glucose homeostasis. Perhaps the most frequently discussed Interactions with other hormone-expressing cells cell-to-cell interaction is that which occurs between Somatostatin release from δ-cells inhibits both glucatwo adjacent β-cells. Cell adhesion between two gon and insulin secretion from adjacent α- and β-cells, β-cells is mediated by several molecules, includ- respectively, through G-inhibitory protein-mediated ing cadherins. E-cadherin is a calcium-dependent signaling mechanisms including inhibition of adenyadhesion molecule that forms homodimers with lyl cyclase/cAMP-mediated secretion as well as K+ cadherins on neighboring cells, thus facilitating a ­channel-induced hyperpolarization.93 Somatostatin is direct cell-to-cell connection. The importance of generally considered to inhibit α- and β-function under E-cadherins in pancreatogenesis is substantial where stimulatory conditions, while basal secretion of glucait has been demonstrated to regulate β-cell aggrega- gon and insulin are not altered in somatostatin-deficient tion, 79–81 insulin secretion, 82 and gap junction com- mice.94 Interestingly, β-cells co-secrete urocortin 3 simulmunication, 83 among others. N-cadherin is involved taneously with insulin which inhibits somatostatin sein endocrine cell sorting to generate the proper islet cretion from δ-cells.95 architecture, as well as insulin and glucagon secreThere is comparatively very little information tion. 84 Normal β-cell functionality is also partially available describing PP- or ε-cell interactions with regulated by gap junctions through which adjacent neighboring endocrine cells of the pancreas. Ghrelin+ β-cells share cytoplasmic materials that allow for ε-cells are scarce in the adult islet where they are biosynthesis and synchronous release of insulin. thought to be local regulators of insulin release. When specific subunits of gap junctions, known as ­ε-cells have been demonstrated to inhibit insulin reconnexins, are mutated or deleted in mice, islets lose lease in humans and rodents in a G inhibitory protein-­ their normal synchronization of calcium oscillations regulated manner in β-cells.96 Further studies will that occur in a characteristic pulsatile manner, and need to be conducted in order to fully elucidate the thus demonstrate a reduced capacity to secrete in- extent to which these other cell types interact within sulin in response to glucose. 85 Additionally, clinical the pancreatic islets. B. Bioengineering and regeneration of the endocrine pancreas



Conclusions

Conclusions Principles of developmental biology and organogenesis have provided the foundational knowledge from which several directed differentiation protocols for generating in  vitro replacement β-cells from stem cell populations are based. We have made significant progress in the area of hESC and induced pluripotent stem cell (iPSC)-based in vitro differentiation into functional ­β-cells. Furthermore, transdifferentiation of other pancreatic endocrine cell types, or closely related endodermally derived cell types, into insulin-producing, β-like cells is becoming more feasible. Both of these strategies from which to devise β-cells are based on basic fundamental principles in developmental biology with regard to the spatiotemporal manner in which key transcription factors are expressed. The step-wise transitions between transcription factor expression patterns are tightly regulated in normal pancreatogenesis. The generation of future therapeutic agents designed to combat diabetes relies on these principles. Current insulin replacement regimens for patients with diabetes merely alleviate the

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symptoms related to the disease rather than restore endogenous insulin production. However, several recent investigations have yielded promising for β-cell regenerative medicine. The process of stem cell differentiation into definitive endoderm, foregut endoderm, pancreatic endoderm, and finally endocrine cells is illustrated in Fig. 4 (based on D’Amour (2006)97 and Kroon (2008)98). These investigations were among the first to demonstrate the distinctions in endocrine cell functionality when cells are harvested from different stages of the differentiation process. As such, current clinical trials are being conducted to test whether pancreatic endoderm stage cells encapsulated into a subdermally implanted, biocompatible, and semipermeable cell containment device are capable of differentiating into insulin-producing β-cells (Fig. 5). The encapsulation device is designed to protect the progenitor cells (and subsequently differentiated insulin-producing cells) from contact with infiltrating immune cells, while the porosity of the device would allow for efficient gas and nutrient exchange and insulin release into the surrounding tissue. If successful, this

FIG. 4  In vitro differentiation of human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) into endocrine cells.

FIG. 5  Schematic representation of encapsulation device for cell-based β-cell replacement therapies. B. Bioengineering and regeneration of the endocrine pancreas

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strategy would be a monumental advancement for the care of patients with diabetes; however, development of a high-throughput method from which to derive mature, glucose-responsive insulin-producing β-cells from hESCs or iPSCs sources is essential.

Acknowledgments S.R.A. was supported by the Vanderbilt Integrated Biological Systems Training in Oncology Training Grant (NIH 1T32CA119925-01A2). M.G. was supported by grants from the American Diabetes Association ­(1-16-IBS-100), NIH/NIDDK (R01DK105689 and R24DK090964), and the Department of Veterans Affairs (1 I01 BX003744-01).

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