Derivation of insulin-producing cells from human embryonic stem cells

Stem Cell Research (2009) 3, 73–87

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REVIEW

Derivation of insulin-producing cells from human embryonic stem cells Dennis Van Hoof a , Kevin A. D'Amour b , Michael S. German a,⁎ a b

Diabetes Center, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA Novocell Inc., 3550 General Atomics Court, San Diego, CA 92121, USA

Received 5 June 2009; received in revised form 1 August 2009; accepted 18 August 2009 Abstract The potential of pluripotent human cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells, to differentiate into any adult cell type makes them ideally suited for the generation of various somatic cells and tissues in vitro. This remarkable differentiation capacity permits analyzing aspects of human embryonic development in the laboratory, as well as generating specialized adult human cells for screening drugs, and for replacing tissues damaged by injury or degenerative diseases, such as diabetes. Understanding and controlling the fundamental processes that drive the differentiation of specialized cells are the keys to the eventual application of this technology to patients. In this review, we discuss the different protocols developed that are aimed at deriving β-cells from hESCs. Despite many differences, successful strategies share a general adherence to the normal differentiation pathway through definitive endoderm. Mimicking normal pancreagenesis offers the best strategy for producing glucose-responsive insulin-producing cells in vitro for people with diabetes. © 2009 Elsevier B.V. All rights reserved.

Contents Diabetes mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pancreas development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lessons from the mouse: Formation of endoderm . . . . . . . . . . . . . . . . . . . . . Lessons from the mouse: Development of the pancreas from endoderm . . . . . . . . . The human pancreas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differentiation of human embryonic stem cells into pancreatic endocrine cells . . . . . . Derivation, culture, and differentiation of human embryonic stem cells . . . . . . . . Lessons from mouse embryonic stem cells: Differentiation into insulin-producing cells Formation of definitive endoderm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exogenous expression of pancreas-associated transcription factors. . . . . . . . . . . . Directed differentiation into insulin-producing cells . . . . . . . . . . . . . . . . . . . . Glucose-responsive insulin-producing cells that ameliorate hyperglycemia . . . . . . .

⁎ Corresponding author. Fax +1 415 731 3612. E-mail address: [email protected] (M.S. German). 1873-5061/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scr.2009.08.003

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D. Van Hoof et al. Conclusions and prospectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Diabetes mellitus

Pancreas development

Diabetes mellitus is a metabolic disorder characterized by elevated blood glucose levels. It can lead acutely to ketoacidosis or nonketotic hyperosmolar coma, and longterm to chronic complications, including cardiovascular disease, stroke, amputation, and microvascular damage to retina, kidney, and nerves. Cellular uptake of glucose from the blood is regulated principally by the hormone insulin. The pancreatic β-cells produce insulin as a pro-hormone that is cleaved into mature hormone plus C-peptide inside storage granules prior to secretion, which is regulated by extracellular stimuli, especially glucose. Together with other endocrine cells, including glucagon-producing α-cells, somatostatin-producing δ-cells, and pancreatic polypeptideproducing PP-cells, β-cells form the scattered islets of Langerhans, which compose 1–2% of the pancreas. Of the two major forms of diabetes mellitus, type 1 results from a complete loss of insulin due to T-cell-mediated autoimmune destruction of the β-cells, and type 2 results from relative insulin deficiency due to inadequate insulin secretion in the setting of reduced insulin sensitivity. Some patients with type 2 diabetes can achieve normal blood glucose levels by combining lifestyle modifications with oral insulin secretagogues and sensitizers. In contrast, many people with type 2 diabetes and all with type 1 diabetes require insulin replacement, usually via injection. The impossibility of perfectly matching insulin replacement to the rapidly fluctuating demands causes the complications of diabetes. Currently, transplantation of whole pancreas or purified islets from cadaveric donors offers the only cure for patients that require insulin therapy (White et al., 2009). However, preventing rejection of the allograft requires permanent treatment with immunosuppressive drugs with multiple adverse side-effects. Furthermore, at best, cadaveric tissue can provide sufficient β-cell mass for only a minute fraction of the patients that could benefit from β-cell replacement. The ability of embryonic stem cells (ESCs) to divide indefinitely, combined with their potential to differentiate into any of the N 200 different types of cells of adult mammals (Keller, 2005), suggests a possible solution to the supply problem. Only induced pluripotent stem (iPS) cells (Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Yu et al., 2007) rival the expansion and differentiation capacity of ESCs. Their genetic modification makes iPS cells unsuitable for clinical applications, although this problem can be solved by modifying the methods for their derivation (Kaji et al., 2009; Yu et al., 2009; Zhou et al., 2009; Kim et al., 2009). In this review, we will focus specifically on reports describing the generation of insulin-producing cells from human ESCs (hESCs) that might eventually provide cell-based therapies to restore the β-cell mass in people with diabetes.

Lessons from the mouse: Formation of endoderm In order to derive β-cells from hESCs in vitro, we must first understand the fundamental processes that underlie development of the pancreas in vivo, starting at the stage from which ESCs are derived (Fig. 1). Most of our knowledge of pancreatic development comes from studies in model organisms: mouse, chicken, zebrafish, and Xenopus. Despite obvious differences in scale, development of the pancreas is highly conserved among these vertebrates. Within ∼ 4 days after fertilization, the mouse egg develops into a blastocyst, comprising an outer shell of ∼ 100 trophoblast cells encasing a compact group of ∼ 25 inner cell mass (ICM) cells clustered on one side of the cavity (Theiler, 1989). From these ICM cells, all of the various tissues of the adult animal will eventually develop. ICM cells express several pluripotency-associated transcription factors, including the three key factors Oct4 (Niwa et al., 2000), Sox2 (Yuan et al., 1995; Avilion et al., 2003), and Nanog (Mitsui et al., 2003; Chambers et al., 2003), which act cooperatively (Boyer et al., 2005). Embryonic stem cells are derived from the ICM cells, and retain both their self-renewal capacity and their pluripotency. Around embryonic day 4.5 (e4.5), the blastocyst implants into the uterine epithelium and enlarges considerably. At the same time, the ICM develops into the epiblast and a layer of primitive endoderm that covers the blastocoelic side of this epiblast (for review, see (Rossant, 2004)). Eventually, the primitive endoderm will produce extraembryonic lineages, whereas the embryonic ectoderm will give rise to all definitive tissues of the embryo. At ∼ e5.5, the ectoplacental cone sprouts from the polar trophectoderm to invade the maternal tissue. Internally, cells from the trophectoderm and embryonic ectoderm divide rapidly and produce the proamniotic cavity, thus forming the egg cylinder. The outside of this egg cylinder and the inside of the trophectoderm are covered by a layer of visceral endoderm and parietal endoderm, respectively, both of which are derived from the primitive endoderm (Theiler, 1989). At the end of this stage, the embryonic ectoderm and extraembryonic ectoderm are morphologically distinct, and a longitudinal axis that establishes left-right symmetry becomes evident from the appearance of the primitive streak at the posterior end of the embryo (for review, see (Rossant and Tam, 2004)). Formation of this primitive streak is preceded by local activity of Brachyury T and Wnt3 (Rivera-Pérez and Magnuson, 2005). Gastrulation, which starts at ∼e6.5, divides the embryonic ectoderm of the epiblast into the three primary germ layers: ectoderm, endoderm, and mesoderm. The latter two layers originate from a common cluster of mesendoderm cells that aggregate at the anterior primitive streak region

Derivation of insulin-producing cells from human embryonic stem cells

75

Figure 1 (A) Schematic overview of pancreas development, and the transcription factors involved in this process during mouse embryogenesis. Developmental stages are indicated as embryonic day for mouse (black) and week of development for human (gray); note that the human developmental stage is an approximation. (B) Derivation of endocrine cells from a common precursor. The intermediate developmental stages are marked by the timely expression of pancreas-associated transcription factors.

near the node. Mesendoderm cells emigrate through the streak and then move laterally and anteriorly to develop into mesoderm and endoderm, respectively, thus displacing the

visceral endoderm to extraembryonic sites (Tam et al., 2007) (for reviews, see (Tam et al., 1993; Wells and Melton, 1999). Canonical Wnt signaling contributes to the establishment of

76 the primary axis as well as the differentiation of the mesendoderm cells (Liu et al., 1999). Nodal secretion from the node governs the decision to become mesoderm versus endoderm: low levels of Nodal induce differentiation into mesoderm, and high levels drive differentiation into endoderm (Conlon et al., 1994; Brennan et al., 2001). Exogenous Activin A can mimic the endoderm fate decision by binding to the same receptors as Nodal (De Caestecker, 2001). Definitive endoderm is marked by a transient upregulation of Brachyury T (Kubo et al., 2004), and expression of Gsc (Blum et al., 1992), Foxa2 (Sasaki and Hogan, 1993; Monaghan et al., 1993), Gata5 (Weber et al., 2000; Reiter et al., 2001), and Sox17 (Kanai-Azuma et al., 2002) as well as the primitive streak marker Mixl1 (Pearce and Evans, 1999; Hart et al., 2002). Visceral endoderm also expresses Brachyury T, Gsc, Foxa2, and Sox17 (Wilkinson et al., 1990), but it can be distinguished from definitive endoderm by its expression of Sox7 (Kanai-Azuma et al., 2002) and Mox1 and 2 (Candia et al., 1996). Recently, it has been shown that the chemokine Cxcl12b and its receptor Cxcr4a restrict the anterior migration of the endoderm in zebrafish (Nair and Schilling, 2008).

Lessons from the mouse: Development of the pancreas from endoderm After initial anterior–posterior patterning has occurred, and gastrulation has completed at ∼e7.5, the definitive endoderm begins to roll up to form the gut tube, while regionally distinct gene expression patterns emerge along the gut endoderm (for review, see (Wells and Melton, 1999)) in a process involving BMPs (Winnier et al., 1995; Solloway and Robertson, 1999), Gata4 (Narita et al., 1997), Furin (Roebroek et al., 1998), and matrix metallopeptidase 2 (Linask et al., 2005). Cells within the endoderm in the region that will become the foregut/midgut junction are specified to become pancreas as early as e8.0, and the expression of patterning genes such as the transcription factors Pdx1, Ptf1a, Hnf1b, Hhex, Foxa2, Mnx1 (Hlxb9), and Onecut1 (Hnf6a) initiate around this time (for reviews, see (Jensen, 2004; Murtaugh, 2007; Jørgensen et al., 2007)). Retinoic acid-mediated signaling plays a critical role in this patterning of the foregut and prospective pancreatic domain (Kumar et al., 2003), and acts upstream of key transcription factors such as Pdx1, which is crucial for pancreas development (Jonsson et al., 1994; Offield et al., 1996). Notably, while these transcription factors are critical for the patterning and eventual development and function of the pancreas, none are absolutely restricted to cells of the pancreatic lineage, or even endoderm. At embryonic day 9.5, the first morphological evidence of the pancreas appears as a thickening on the dorsal side of the gut that grows into the dorsal pancreatic bud, followed by the two ventral/lateral buds a day later, one of which dominates and eventually fuses with the dorsal bud. Distinct signaling events guide dorsal and ventral bud development, although suppression of hedgehog signaling plays an important role in both. Initially, Shh expression extends throughout the anterior–posterior axis of the developing gut tube endoderm, but is repressed specifically in the dorsal and ventral pancreatic buds by notochord signaling via FGF2

D. Van Hoof et al. (basic FGF) and Activin (Hebrok et al., 1998; Kim and Melton, 1998). In contrast, FGF2 signaling from the cardiac mesoderm induces liver-specific differentiation of the ventral bud, which has a pancreatic fate when FGF2 is absent (Deutsch et al., 2001). As the buds appear, the expanding pancreatic epithelium initiates the expression of the transcription factors Sox9, Nkx2.2, and Nkx6.1 (Lynn et al., 2007; Seymour et al., 2007), in addition to the gut endoderm patterning genes Pdx1, Ptf1a, Hnf1b, Hhex, Foxa2, Mnx1, and Onecut1 noted above. Interestingly, Mnx1 is required for the expansion of the dorsal bud (Harrison et al., 1999; Li et al., 1999), and Hhex is required for the ventral bud (Bort et al., 2004), while FGF10 from the overlying dorsal mesenchyme regulates the growth of the pancreatic epithelium in both the dorsal and the ventral pancreatic buds (Bhushan et al., 2001). Expansion of the dorsal and ventral pancreatic buds continues, during which the gut rotates and brings the two pancreatic rudiments in closer proximity around e12.5. While the connection of the dorsal part to the duodenum diminishes, that of the ventral part will form the main duct. Differentiation of the endocrine cells initiates slightly earlier in the dorsal bud than in the ventral bud, with glucagon-positive cells appearing first at ∼ e9.5. Differentiation of the endocrine cells requires the transient expression of the transcription factor Ngn3, which commits individual cells in the pancreatic epithelium to the endocrine lineage (Apelqvist et al., 1999; Schwitzgebel et al., 2000; Gu et al., 2002) (for review, see (Miyatsuka et al., 2008)). The broadly expressed pancreatic epithelial transcription factors Sox9, Hnf1b, Foxa2, and Onecut1 collaborate in activating Ngn3 expression (Lynn et al., 2007), while Notch-Delta signaling activates the inhibitory transcription factor Hes1, thereby inhibiting Ngn3 expression and endocrine differentiation in the majority of the pancreatic epithelial cells (for review, see (Oliver-Krasinski and Stoffers, 2008)). By e10.5, insulin-positive cells can be detected in the primitive buds, but neither these nor the early glucagonexpressing cells noted above have characteristics of mature β- and α-cells, respectively, nor are they precursors of mature islet cells (Herrera, 2000; Wilson et al., 2002). At ∼ e13, a secondary transition that peaks around e14.5 initiates, during which a second wave of Ngn3 expression occurs (Villasenor et al., 2008). Ngn3 activates the expression of a number of endocrine transcription factors including Neurod1 (Huang et al., 2000), Pax4 (Smith et al., 2003), and Nkx2.2 (Watada et al., 2003), the latter two of which govern the further differentiation into β-cells. Eventually, the combinatorial expression of multiple factors defines the identity of the specific endocrine cells present in islets (Fig. 1) (for review, see (Spence and Wells, 2007)). Basically, αcells are characterized by Pax6, Nkx2.2, Irx1 and 2, Brn4, and Arx; β-cells by Pax4, Pax6, Nkx2.2, Nkx6.1, Mnx1, MafA, and Pdx1; δ-cells by Pax4 and Pax6; PP-cells by Nkx2.2; the factors essential for development of ɛ-cells are yet unknown (for reviews, see (Oliver-Krasinski and Stoffers, 2008; Spence and Wells, 2007; Martin et al., 2007)).

The human pancreas Due to obvious ethical constraints, knowledge about human pancreas development is more limited and mostly descriptive

Derivation of insulin-producing cells from human embryonic stem cells (Bouwens et al., 1997; Polak et al., 2000). Yet, recent studies show that, in addition to the obvious difference in duration of pancreagenesis, some dissimilarities at the developmental, morphological, and gene expression level (Jeon et al., 2009) exist between the mouse and the human pancreas (for review, see (Gittes, 2009)). Around the fifth week of human development (5WD), the ventral pancreatic bud migrates toward the dorsal bud and they fuse a week later. Between 7WD and 8WD, the pancreas consists of pyramidal cells that are in contact with loose and dense peripancreatic mesenchyme (Polak et al., 2000). At this stage, the first endocrine cells start to appear; many of which are positive for insulin, glucagon, and somatostatin, simultaneously, whereas pancreatic polypeptide-containing cells can be detected from 9WD onward (Polak et al., 2000). By 11WD, the percentage of polyhormonal cells has decreased drastically, while the endocrine cell density has enlarged considerably. As indicated by Ki-67 expression, a marker for dividing cells, the hormone-positive cells in human islets cease to proliferate after ∼ 20WD (Bouwens et al., 1997), even though the highest increase in endocrine tissue occurs between 21WD and 40WD (Stefan et al., 1983). Additionally, differentiated human islets have a very limited proliferative capacity after 12WD, suggesting that a hormone-negative precursor cell population is the foremost source for islet neogenesis (Bouwens et al., 1997). The overall morphology of the mature pancreas differs between humans and mice: the human pancreas forms a single, smooth organ with a fibrous stroma, whereas the mouse pancreas has an irregular, lobular structure of more loosely connected tissue. In contrast to the organized mouse islets, which are composed of a large core of β-cells enveloped by a layer of α-cells and other endocrine cells, primate islets contain endocrine cells that are scattered in a more random pattern (Wieczorek et al., 1998). The human pancreas contains approximately one million islets consisting of a thousand endocrine cells each, ∼75% of which are βcells. In addition, a significant fraction of the β-cells in the adult human pancreas are found outside of organized islets (Butler et al., 2003). Diabetic mice require 2000 to 3000 human islets to establish normoglycemia (Gaber et al., 2004; Kroon et al., 2008). However, transplantations of donor-derived islets (White et al., 2009) have shown that the number of functional β-cells that can be extracted from a single cadaveric pancreas is often not enough to restore euglycemia in a human diabetic individual. This, again, illustrates the need for alternative sources of β-cells to treat the vast and rising number of diabetic patients.

Differentiation of human embryonic stem cells into pancreatic endocrine cells Derivation, culture, and differentiation of human embryonic stem cells As noted above, ESCs are derived from the ICM of blastocyststage embryos, and can self-renew indefinitely when grown in serum-containing medium on mitotically inactive mouse feeder cells, while retaining the potential to differentiate into any adult cell type (Thomson et al., 1998; Reubinoff et

77

al., 2000; Cowan et al., 2004). Recently, procedures have been developed to derive and maintain hESCs on human feeder cells as well as feeder-free matrices in various growth factor-supplemented basal media (Rao and Zandstra, 2005; Klimanskaya et al., 2005; Ludwig et al., 2005). In addition, hESCs can be transferred from feeders to feeder-free conditions and vice versa without affecting their pluripotency, although such transfers do affect their protein expression profile (Van Hoof et al., 2008). Irrespectively, advancements like these allow the generation of clinicalgrade cell lines by omitting nonhuman components. Spontaneous differentiation of ESCs can be induced by growth in suspension to form aggregates (i.e., embryoid bodies) in the absence of factors that support self-renewal. However, individual lines show vast differences in their propensity to differentiate into particular lineages (Osafune et al., 2008). Numerous groups have reported the derivation of endocrine hormone-expressing cells from spontaneously differentiating cultures (Lavon et al., 2006; Xu et al., 2006; Segev et al., 2004; Baharvand et al., 2006; Otonkoski et al., 1993; Treutelaar et al., 2003; Humphrey et al., 2003; Delacour et al., 2004; Brolén et al., 2005), but this approach is unlikely to form a viable platform for generating sufficient numbers of pure populations of cells for clinical use. Lineage-specific differentiation of ESCs proved most successful when these cells were exposed to signals mimicking those that normally drive differentiation during embryonic development.

Lessons from mouse embryonic stem cells: Differentiation into insulin-producing cells The derivation of mouse ESCs (mESCs) (Evans and Kaufman, 1981; Martin, 1981) preceded that of the first hESC lines by ∼ 17 years (Thomson et al., 1998). Hence, the first protocols designed to promote differentiation into insulin-secreting cells specifically were developed for mESCs. These protocols relied on either transfection with an expression construct harboring an antibiotic resistance gene under control of the insulin promoter (Soria et al., 2000), or embryoid body formation (Shiroi et al., 2002). Insulin-producing cells were also successfully derived from hESCs (Assady et al., 2001), implying for the first time that hESCs can differentiate into cells with β-cell characteristics. More specific multistage procedures developed for the differentiation of mESCs into neural tissues also generated cells containing islet hormones (Lumelsky et al., 2001); (Hori et al., 2002). However, the absence of C-peptide as well as insulin mRNA revealed that intracellular insulin did not originate from de novo synthesis in these cells, but from uptake from the culture media (Rajagopal et al., 2003; Sipione et al., 2003; Hansson et al., 2004; Paek et al., 2005a,b). In addition, most of the islet markers used, such as the transcription factors described above, cannot uniquely identify cells of pancreatic lineage, because they also mark cells of other lineages, especially neural ectoderm. Recently, the protocol developed originally for the differentiation of mESCs (Lumelsky et al., 2001) was modified and applied to hESCs (Mao et al., 2009), but the characterization of the cells generated in this study is fairly limited. Although C-peptide-positive cells were reported to

78 be present at the final stage, transplantation of these cells did not fully protect streptozotocin-treated mice from developing diabetes; yet, their fasting blood glucose levels approximated that of healthy animals. These findings suggest that, even though the transplanted cells are unable to respond adequately to hyperglycemia, they produce sufficient insulin to decrease elevated blood glucose levels over time.

Formation of definitive endoderm Initially, most studies focused primarily on analyzing the gene products expressed at the end of the differentiation procedures. However, the intervening steps, especially the generation of definitive endoderm, an essential step for pancreagenesis in vivo, were not addressed. Deriving endoderm from hESCs proved challenging, until incubation with Activin A (De Caestecker, 2001) under low serum conditions resulted in cultures comprising ∼ 80% definitive endoderm cells (D'Amour et al., 2005). Suppression of PI3K activity and reduction of insulin/insulin-like growth factor signaling proved critical for efficient definitive endoderm formation, as it induces the cells to go through a mesendoderm stage in response to Activin/Nodal signals (McLean et al., 2007). The efficiency of definitive endoderm generation improved further with exposure of hESCs to a combination of Activin A and Wnt3a in the complete absence of serum on the first day, followed by two days in medium supplemented with Activin A and 0.2% serum (D'Amour et al., 2006). These differentiation methods (D'Amour et al., 2005; McLean et al., 2007) result in transient upregulation of the mesendoderm markers Brachyury T (Kubo et al., 2004), Foxc1, and Mixl1 (Pearce and Evans, 1999; Hart et al., 2002), and sustained expression of the definitive endoderm markers Cer1, Cxcr4 (Nair and Schilling, 2008), Foxa2 (Sasaki and Hogan, 1993; Monaghan et al., 1993), Gata4 (Narita et al., 1997) and 6, Gsc (Blum et al., 1992), Hhex (Bort et al., 2004), and Sox17 (Kanai-Azuma et al., 2002) from day 3 after the onset of differentiation (Liu et al., 2007). Recently, this Activin A-based differentiation strategy has also been applied to hESCs grown on human feeder cells (Zhou et al., 2008), and with a lower efficiency under feederfree conditions in the absence of any xeno factors (Zhou et al., 2008; King et al., 2008). Furthermore, wortmannin improves the yield of definitive endoderm cells through inhibition of PI3K activity (Zhang et al., 2009), and the replacement of Activin A with Activin B further increases the number of Pdx1-expressing cells in hESC-derived embryoid bodies (Frandsen et al., 2007). Recently, a screen for small molecule inducers of endoderm identified two components, termed IDE1 and IDE2, with efficiencies similar to that of Activin A (Borowiak et al., 2009).

Exogenous expression of pancreas-associated transcription factors The timely expression of Pdx1 during early development of endocrine cells, as well as its sustained presence in mature islets (Jonsson et al., 1994; Offield et al., 1996), has been difficult to accomplish in vitro. An inducible system to control the expression of a Pdx1-VP16 fusion protein (Kaneto

D. Van Hoof et al. et al., 2005) stimulated the differentiation of hESC-derived definitive endoderm cells into endocrine pancreas-like cells (Bernardo et al., 2008). However, their endocrine-like identity was based primarily on mRNA analyses and limited immunocytochemistry; C-peptide expression was not demonstrated, and no functional studies were conducted. Constitutive overexpression of Pax4 (Smith et al., 2003) also enhanced the differentiation of hESCs into cells when subjected to an embryoid body-based procedure, as evidenced by upregulated expression of endogenous β-cellassociated transcripts (Liew et al., 2008). The differentiated cells were enriched in Pdx1 and insulin transcripts, and stained positive for pro-insulin, indicating de novo synthesis. Although these cells released marginal levels of C-peptide on exposure to the insulin secretagogue tolbutamide (Sturgess et al., 1986), they did not respond to glucose stimuli, indicating that the cells had not gained characteristics of mature β-cells (Liew et al., 2008).

Directed differentiation into insulin-producing cells Mimicking the environmental conditions during pancreagenesis in vivo by the successive exposure of pluripotent hESCs to different growth factors and other signaling molecules in vitro has proven most successful for generating hESC-derived endocrine-like tissue (Table 1). Guiding differentiation of hESCs into pancreatic cells by a stepwise process was exploited shortly after achieving efficient formation of definitive endoderm. Most of these multistage procedures take 2 to 3 weeks (Fig. 2), a period much shorter than the in vivo development of the human islet cells from the pluripotent cells of the inner cell mass. The exposure of hESCs to extracellular stimuli and inhibitors in a consecutive manner has proven to be an efficient method for generating pancreatic hormone-expressing endocrine cells, even though the resulting insulinexpressing cells still differ from mature β-cells (D'Amour et al., 2006). After formation of definitive endoderm (D'Amour et al., 2005), removal of Activin A allows the cells to adopt gut endoderm-like characteristics. This can be enhanced by the addition of FGF10 to stimulate proliferation (Hart et al., 2002; Bhushan et al., 2001; Ye et al., 2005), KAADcyclopamine to inhibit hedgehog signaling (Lau et al., 2006), and indolactam V to activate PKC signaling (Chen et al., 2009). Following this protocol, and growth in medium supplemented with IGF1 and HGF, all five pancreatic hormones are readily detected. The insulin content of dithizone-stained cell clusters (Latif et al., 1988) generated with this protocol exceeded the ∼ 2 pmol/μg DNA of human fetal islets (Hayek and Beattie, 1997; Demeterco et al., 2000) by 7- to 100-fold, thereby approximating levels of mature islets, which range from 50 to 310 pmol/μg DNA. However, many of the resulting endocrine cells express multiple hormones simultaneously, thus resembling the immature polyhormonal cells that arise during the primary transition (Polak et al., 2000; De Krijger et al., 1992; Teitelman et al., 1993). This protocol, optimized primarily for the CyT203 hESC cell line, also produced endocrine cells from five additional cell lines (CyT25, CyT49, BG01, BG02, and BG03), albeit at different efficiencies (D'Amour et al., 2006). Despite these differences between hESC lines, Activin A induction of

Studies reporting the directed differentiation of hESCs into insulin-producing cells.

Reference

Cell lines

Differentiation conditions

Endocrine hormones

human Glucose responsive Transplant recipient C-peptide insulin release

Transplantation site

Amelioration of hyperglycemia

(D'Amour et al., 2006) (Cho et al., 2008)

CyT203

GCG,INS, SST, PPY, GHRL GCG, INS

Yes

No

n.a

n.a

n.d.

SNUhES3

Yes

n.d.

n.a

n.a

n.d.

(Jiang et al., 2007a)

H1, H7, H9

GCG, INS, SST

Yes

No

n.a

n.a

n.d.

(Zhang et al., 2009)

H1, H9

Adherent on low density MEFs Adherent on low density MEFs Adherent on Matrigel Adherent on Matrigel Adherent on Matrigel Suspension

INS, SST

Yes

In vitro

n.a.

n.a.

n.d.

GCG, INS, SST

Yes

C57BL6 Rag-1/-1 mice

Kidney capsule

No

GCG, INS, SST

Yes

In vitro (moderate) In vitro (marginal)

Male SCID mice

Intraperitoneal injection

No

GCG, INS, SST, PPY Yes

n.d.

Male SCID mice

GCG, INS, SST GCG, INS, SST

Yes Yes

GCG, INS, SST, PPY, GHRL

Yes

In vivo In vitro (marginal) and in vivo In vivo

Male SCID-Beige mice

(Eshpeter et al., 2008) H1 (Phillips et al., 2007)

(Mao et al., 2009) (Shim et al., 2007) (Jiang et al., 2007b) (Kroon et al., 2008)

HES-1, HES-2, HES-3, HES-4, HUES-7, HUES-9 PKU1.1

Adherent on Gelatin Miz-hES4, Miz-hES6 Suspension H1, H9 Adherent on Matrigel CyT49 Adherent on low density MEFs

Kidney capsule and subcutaneous Male BALB/c nude mice Kidney capsule Male BALB/c nude mice Kidney capsule

Derivation of insulin-producing cells from human embryonic stem cells

Table 1

Only when fasting Yes Yes

Epididymal fat pad Yes

79

80 D. Van Hoof et al.

Figure 2 Time lines of the various multistep procedures to induce differentiation of hESCs into insulin-secreting cells. Abbreviations: ActA, Activin A; bFGF, basic fibroblast growth factor; BMP4, bone morphogenetic protein 4; CM, conditioned medium; DAPT, γ-secretase inhibitor; EGF, epidermal growth factor; FBS, fetal bovine serum; FGF10, fibroblast growth factor 10; HGF, hepatocyte growth factor; IGF1, insulin-like growth factor 1; IGF2, insulin-like growth factor 2; KGF, keratinocyte growth factor; MEFs, mouse embryonic fibroblast feeders; Nic, nicotinamide; RA, all-trans retinoic acid.

Derivation of insulin-producing cells from human embryonic stem cells definitive endoderm has subsequently been applied in multiple studies with a variety of hESC lines cultured and derived under diverse conditions (Kroon et al., 2008; Jiang et al., 2007a,b; Phillips et al., 2007; Shim et al., 2007; Cho et al., 2008). The five-stage protocol was slightly modified for the hESC line SNUhES3 with the inclusion of nicotinamide and βcellulin during the last two stages, which resulted in sustained expression of Pdx1 (Cho et al., 2008). In combination with Activin A, β-cellulin promotes the conversion of amylase-secreting exocrine pancreatic cells into insulin-producing cells (Mashima et al., 1996), and stimulates β-cell regeneration when injected into pancreatectomized rats (Li et al., 2001). In addition to inducing endocrine differentiation of human fetal pancreatic cells in culture (Otonkoski et al., 1993), nicotinamide increases the mitotic index of β-cells after pancreatectomy (Sugiyama et al., 1991). However, even though the resulting endocrine cells retain Pdx1 expression, the insulin concentration in the culture medium after stimulation with various secretagogues does not increase significantly in the cells derived with use of nicotinamide and β-cellulin (Cho et al., 2008). A combination of Activin A and the histone deacetylase inhibitor Na-butyrate (Candido et al., 1978) at the first stage increases the expression levels of the endoderm markers Sox17, Foxa2, and Hnf4a (Jiang et al., 2007a). Furthermore, addition of Noggin during the second and third stage of the protocol enhances differentiation of definitive endoderm into pancreatic endoderm (Kroon et al., 2008; Zhang et al., 2009; Jiang et al., 2007a), possibly by antagonizing BMP signaling (Winnier et al., 1995; Solloway and Robertson, 1999), thus excluding a hepatic fate (Rossi et al., 2001). Under these growth conditions, islet-like clusters can be generated that contain glucagon-, somatostatin-, and insulin-producing cells, the latter of which increase Cpeptide release 3.5-fold on glucose stimulation (Jiang et al., 2007a). Although transplantation of these clusters under the kidney capsule of streptozotocin-induced diabetic mice did not reduce blood glucose levels significantly, human Cpeptide could be detected in serum after an oral glucose bolus (Eshpeter et al., 2008). EGF treatment can increase the number of Pdx1-positive cells in vitro to nearly 40%, suggesting that these cells might function as an expandable population of progenitor cells for generating the large numbers of endocrine cells required for transplantations (Zhang et al., 2009). Pancreatic endocrine cells that arise at the final stage of this differentiation procedure do not coexpress insulin with glucagon or somatostatin and give a 2-fold increase in insulin release on glucose stimulation, but have not been tested for amelioration of hyperglycemia in vivo (Zhang et al., 2009). Interestingly, human iPS cells in place of hESCs give similar results. Furthermore, the small molecule indolactam V can produce similar effects as those observed for EGF, but only when combined with FGF10 (Chen et al., 2009). As noted before, growing hESCs as aggregates in suspension results in their spontaneous differentiation into various lineages, including the extraembryonic lineages such as visceral endoderm (Mitsui et al., 2003). BMP4 also promotes differentiation of hESCs into extraembryonic endoderm (Pera et al., 2004). In contrast, growth on Matrigel suppresses visceral endoderm formation in embryoid bodies

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(Rust et al., 2006). When combined with Activin A and Matrigel, BMP4 induces expression of Sox17 and Foxa2 in hESC-derived embryoid bodies (Phillips et al., 2007). Although the precise role of BMP4 in this process is not clear it has been argued that this represents differentiation of hESCs into definitive endoderm rather than the extraembryonic lineage. Irrespectively, the resulting islet-like clusters studied at the end of this differentiation protocol contained 5–20% Pdx1-positive cells, and these clusters secreted C-peptide in response to glucose stimulation, albeit at very low levels.

Glucose-responsive insulin-producing cells that ameliorate hyperglycemia Although several of the studies discussed above describe increased secretion of insulin and C-peptide by hESC-derived pancreatic endocrine cells on glucose stimulation in vitro, none produced cells capable of restoring euglycemia in diabetic animal models in vivo (e.g., streptozotocin-treated mice). Recently, several independent groups used distinct differentiation conditions to develop hESC-derived insulinsecreting cells that were glucose responsive in vivo (Fig. 2) (Kroon et al., 2008; Jiang et al., 2007b; Shim et al., 2007). The first two of these studies were published almost simultaneously, one of which relied on the formation of embryoid bodies at the onset of the differentiation procedure (Shim et al., 2007), whereas the other applied an aggregation step at the very end of the protocol (Jiang et al., 2007b). Both differentiation methodologies included Activin A at the start to generate definitive endoderm (De Caestecker, 2001), and retinoic acid during the second stage to stimulate the formation of pancreatic endoderm, as determined by the expression of specific transcription factors, among which were Foxa2, Sox17, Pdx1, Gata4, and Mnx1. Though only one of the two studies showed a meager 2fold increase in insulin secretion by their differentiated cells on glucose stimulation in vitro (Jiang et al., 2007b), both groups transplanted their cell clusters under the renal capsule of streptozotocin-treated diabetic mice. Surprisingly, this resulted in a significant reversal of the hyperglycemic state in 30% (Jiang et al., 2007b) or all of the recipients within one week, and was associated with a gain in weight (Shim et al., 2007), suggesting that the insulin-producing cells in the grafts had matured into functional β-cell-like cells. This conclusion was further supported by the immediate increase in blood glucose levels on removal of the transplantation site after 4 (Shim et al., 2007) or 6 weeks (Jiang et al., 2007b). Immunofluorescence microscopic analysis indicated that many human cells in the grafts coexpressed Pdx1 and insulin (Shim et al., 2007) or Cpeptide (Jiang et al., 2007b), though some cells resembled more immature endocrine cells, as they were immunoreactive for both insulin and glucagon (Shim et al., 2007). Interestingly, no teratomas were found at 4 weeks (Shim et al., 2007) or even 3 months (Jiang et al., 2007b) when the studies were concluded, suggesting that all pluripotent cells had either differentiated into nonproliferative cells, or had not survived. The finding that only 6 out of 19 of the recipient mice sustained long-term restoration of euglycemia was ascribed to

82 the physiological difference between human and mouse, and the impurity of the grafts, which may account for poor or delayed revascularization after implantation (Jiang et al., 2007b). However, the most remarkable finding was that, although transplantation of these islet-like structures rapidly reversed diabetes, no C-peptide could be detected in the serum at fasting conditions or after feeding, yet cells containing Cpeptide were present in the grafts (Jiang et al., 2007b). This dichotomy may be explained by the limited sensitivity of the method used to measure serum C-peptide levels. Recently, a new strategy was adopted to generate glucose-responsive insulin-producing cells (Kroon et al., 2008). The five-stage protocol developed previously (D'Amour et al., 2006) was improved empirically as based on expression levels of pancreatic progenitor and endocrine cell markers. Furthermore, the protocol was limited to the first four stages, at the end of which hESCs have differentiated into pancreatic endoderm-like tissue that contains few hormone-expressing cells (Kroon et al., 2008). This tissue, resembling the 6- to 9-week-old human pancreas, was cut into small chunks and wrapped in Gelfoam overlaid with Matrigel before transplantation into the epididymal fat pads of immune-compromised normoglycemic mice (Figs. 3A–C) to allow further development in vivo. In contrast to the studies described above, these grafts become fully responsive to glucose 3 months posttransplantation. Serum human C-peptide levels in mice with these hESC-derived grafts were ∼660 pM at fasting, and ∼ 2180 pM on intraperitoneal glucose administration, approximating that of mice with human islet implants (∼ 330 pM at fasting, and ∼ 3130 pM after glucose stimulation). Secretion of mature human insulin showed a similar pattern and was similar to that found for mice that had received human islets (Gaber et al., 2004); (Kroon et al., 2008). Immunohistochemical analysis showed that the hESCderived grafts exhibited features associated typically with

D. Van Hoof et al. mature pancreatic islets, including their overall structural organization, and the mutually exclusive expression of all endocrine hormones (Kroon et al., 2008). Furthermore, the transcription factors Pdx1, Nkx6.1, and MafA as well as islet amyloid polypeptide were expressed in insulin-positive cells, whereas glucagon-positive cells expressed the α-cell-specific transcription factor Arx, but were devoid of islet amyloid polypeptide and MafA. In addition to releasing human Cpeptide and insulin in response to a glucose challenge, the grafts protected these mice from becoming diabetic after streptozotocin treatment. When the grafts were removed 2.5 to 3 months later, blood glucose levels increased promptly to hyperglycemic levels, which further corroborates that the hESC-derived endocrine cells had matured and had become fully functional in vivo. Interestingly, these insulin-expressing implants remained viable for more than a year, while sustaining expression of Pdx1 (Fig. 3D), which is critical for the proper functioning of β-cells, but had not been reported for in vitro-derived grafts before.

Conclusions and prospectives Formation of the pancreas, and the clusters of endocrine cells therein, depends on growth factor-induced signaling as well as the interaction with neighboring cells and tissues that are tightly regulated in timely manner in vivo (Murtaugh, 2007). Mimicking this complex developmental process to drive the differentiation of hESCs into β-cell-like cells in vitro has proven challenging, but several important breakthroughs have been published over the past few years. In contrast to the other primary germ layers, endoderm proved particularly difficult to produce, until the use of Activin A yielded the formation of definitive endoderm from hESCs with efficiencies exceeding 80% (D'Amour et al., 2005); (D'Amour et al., 2006). Since then, the most successful

Figure 3 Transplantation of hESC-derived pancreatic endoderm into the epididymal fat pad. (A) The epididymal fat pad exposed. (B) Pancreatic endoderm layered on the epididymal fat pad. (C) Epididymal fat pad wrapped around the pancreatic endoderm. (D) hESC-derived pancreatic endoderm graft 377 days postimplantation, stained with immunofluorescence for Pdx1 (red) and insulin (green); scale bar, 100 μm.

Derivation of insulin-producing cells from human embryonic stem cells multistep protocols commonly include Activin A during the initial stage, but vary widely at the subsequent stages. Some incorporate an aggregation step through suspension growth to form multicellular clusters (Phillips et al., 2007; Shim et al., 2007). However, the assembly process itself may initiate spontaneous differentiation into undesired types of cells, and the clusters might also reduce the diffusion efficiency of growth factors and small molecules, thus forming a concentration gradient from the periphery to the core of the cluster. An aggregation step might contribute to the generation of the classical islet-like shape, but it is unclear whether the insulinproducing cells have to reside in clusters for their proper functioning and survival after transplantation. Even though independent groups have shown repeatedly that insulin-producing cells can be derived from hESCs in vitro, none of these end-stage products responded adequately to exogenous glucose stimuli to meet the requirements for surrogate β-cells. Based on the observed expression of multiple endocrine hormones per cell, it has been argued that these cells may represent immature endocrine cells that might develop eventually into fully functional glucose-responsive cells. One successful approach to the treatment of experimental diabetes with hESC-derived cells did not use fully differentiated insulin-producing cells, but a heterogeneous population of cells containing pancreatic endoderm-like tissue (Kroon et al., 2008). The additional weeks of development in vivo that were required before endocrine cells fully differentiated and normoglycemia was achieved in these grafts of pancreatic endoderm suggest that the final differentiation process may take longer than allowed for the in vitro protocols. Thus final maturation may simply depend on time. Alternatively, other undefined factors that are difficult to reproduce in vitro, such as vascularization, and the interaction with adjacent tissues, may contribute to the final steps of maturation of functional endocrine cells. The source of such signals remains unknown, although they do not appear to depend on the normal pancreatic location, since the epididymal fat pad appeared apt at providing those signals. It also remains possible that other undefined hESCderived cells included with the engrafted pancreatic endoderm tissue have assisted the differentiation of progenitors into mature endocrine cells. Further progress on defining these signals may aid efforts to complete the final differentiation steps of mature β-cells in vitro. The different strategies developed to derive β-cell-like cells from hESCs described thus far have yielded relatively small numbers of pancreatic endocrine cells, and the heterogeneous nature of the resulting cell populations could prove problematic. We do not know whether cotransplanting such nonendocrine cells or undifferentiated cells represents a risk to patients. Interestingly, transplantation of a mixed population of human cells into mice showed a strong bias toward the eventual formation of endocrine cells (Kroon et al., 2008), suggesting that the heterogeneous grafts experienced either a positive selection (i.e., a preference for endocrine cells to proliferate and differentiate) or a negative selection (i.e., a higher death rate of nonendocrine cells). However, teratomas can arise in recipient mice that receive unpurified hESC grafts, indicating that the inclusion of a purification step prior to

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transplantation is desired to dispose of potentially hazardous mitotically active cells. Despite all the hurdles that need to be taken before hESCderived differentiated cells reach the clinics, stem cell research has proven its potential repeatedly in animal model systems. Moreover, the recent approval of the world's first clinical trial involving hESC-derived oligodendrocyte progenitor cells that had been shown to remyelinate and restore locomotion in rats after spinal cord injury (Keirstead et al., 2005) will pave the way for future trials. Although as of now, only the symptoms of diabetes can be treated, the investigations discussed here suggest that a definitive cure for diabetes has come closer.

Acknowledgments We thank Dr. T. Miyatsuka for helpful suggestions. This work was financially supported by the Juvenile Diabetes Research Foundation (JDRF Grants 3-2008-477 and 35-2008-628).

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