Embryonic Stem Cells as a Source of Pancreatic Precursors and Islet Cells In Vitro 23

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The generation of unlimited supplies of islet stem cells or β cells from an abundant, renewable, and readily accessible source for transplantation would probably render current transplantation therapies obsolete. Although this ultimate goal is on the distant horizon, recent progress, repre- senting the first step, has been made in identify- ing pancreatic precursor cells and differentiated islet cells generated from both mouse and human embryonic stem (ES) cells. The next hur- dle, achieving enrichment of these cell types from ES cell cultures and isolating purified populations for functional testing, may be a more challenging step. It is becoming clear that a better understanding of the sequential genetic and epigenetic signals occurring during normal mouse and human development will be necessary. Particularly relevant is the need to understand the nature and identity of true embryonic pancreatic precursor cells and islet progenitor cells, and to identify conditions that allow their efficient, large-scale isolation. An ES cell-based in vitro differentiation system can facilitate these goals by providing a straightforward means to select and purify progenitor cells, and to investigate conditions that promote their expansion and differentiation ex vivo.

Specifically, a human ES cell-based in vitro model system would be invaluable for studying human islet development and for providing cells for transplantation.

The Clinical Problem

It is estimated that by 2010, more than 220 million people worldwide will be diagnosed with diabetes,¹of which approximately 10% will have the type 1 form characterized by profound insulin deficiency, requiring lifelong insulin injections.

Although administration of exogenous insulin remains a viable therapy for the disease, inade- quate glycemic control results in significant mor- bidity and premature mortality. For some patients, vascularized pancreas transplants or infusions of isolated islets of Langerhans can elim- inate or greatly reduce the amount of exogenous insulin required to maintain euglycemia, resulting in a reduction in long-term complications caused by repeated blood glucose excursions. However, cadaver donor shortages greatly limit the number of transplants that can be performed: Fewer than 100 islet cell transplants and approximately 2000 pancreas transplants are performed annually in the world.² Even with substantial efforts to increase donation rates, it is unlikely that the sup- ply will ever meet the demand. Thus, glucose- responsive, insulin-secreting β cells generated from a renewable source would be an ideal alter- native to tissue procured through organ donation.

Based on their unique capacities for self-renewal and multilineage differentiation, human ES cells may be such a source.^3–6

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Embryonic Stem Cells as a Source of Pancreatic Precursors and Islet Cells In Vitro

Victoria L. Browning, Brenda W. Kahan, and Jon S. Odorico

321

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ES Cells as Potential Therapy for Diabetes

ES cells are derived from the inner cell mass of a blastocyst, an embryo that is less than 1 week old. For derivation of human ES cell lines, the typical starting materials are discarded blastocyst-stage embryos from in vitro fertilization clinics; after the completion of in vitro fertilization procedures, residual embryos are donated by couples for ES cell derivations after informed consent.³ Because the derivation process ren- ders the embryo nonviable, ethical questions surrounding the use of human ES cell lines have arisen. However, the great promise of ES cell therapies is a mitigating factor in the minds of many. This promise lies in the unique biology of ES cells: They have both the capacity to replicate indefinitely in an undifferentiated state, and the ability to give rise to daughter cells capable of differentiating into any cell type of the body, a potential that has been demonstrated conclu- sively in vivo for murine ES cells,⁷and at least partially for both murine and human cells in vitro. It is thus the hope of scientists and non- scientists alike that vast quantities of specialized cells, such as β cells, can be generated from ES cells and used to treat a variety of diseases.

Differentiating in culture, ES cells can recapitulate many aspects of normal embryonic development, including the initial specification of lineage-restricted progenitor cells that can grow and differentiate into specialized postmitotic cell types including neurons, glia, chon- drocytes, adipocytes, osteoclasts, endothelial cells, hematopoietic cells, muscle lineages, keratinocytes, melanocytes, yolk sac, as well as endoderm-derived lineages such as hepatocytes and cells reminiscent of pancreatic β cells, among others.^8–31In order for ES cells to reach their full potential as therapeutic agents, protocols must be established that direct their differentiation into homogenous populations of specific cell types. This is important both to max- imize efficiency and also to prevent unwanted cell types and potentially tumorigenic undifferentiated cells from being transplanted into patients. One approach to create enriched populations of specific cell types is to create culture conditions in vitro that mimic the embryonic environment in which the specific cell types develop. This strategy has been used to generate cells of hematopoietic lineage,²⁸ cardiomy-

ocytes,³²and motor neurons.¹⁸For this approach to work for creation of pancreatic β cells in culture, an understanding of signals and cues that promote pancreas development in the embryo is required. Recent studies have advanced our understanding of the progressive stepwise commitment of endoderm to pancreatic epithelial progenitors, and then to islet-restricted progenitors ultimately leading to the formation of the four endocrine cell types of mature islets. Here, we will first review pancreaticogenesis and how islets form in the mouse embryo. In this context, we will then discuss the current state of research on the generation of pancreatic islet cells from murine and human ES cells in vitro and how our understanding of islet ontogeny might help achieve directed differentiation to pancreatic lineages in a more efficient manner.

Pancreaticogenesis

Pancreas formation is initiated as two distinct outgrowths of the foregut endoderm, starting first with the dorsal anlage at embryonic day (e) 9.5–10 in the mouse, and followed by the ventral bud approximately 1 day later. Shortly there- after, rapid growth and branching morphogenesis begin, and subsequently the ventral bud rotates and fuses with its dorsal counterpart, forming a single gland. Branching morphogenesis culminates in ductal differentiation and the formation of acini composed of digestive enzyme- and bicarbonate-secreting cells. Early endocrine cells expressing glucagon alone or glucagon and insulin can be found at approximately e9.5–10.5 within the early pancreatic duodenal homeobox 1-positive (PDX1⁺) pancreatic epithelium but these cells do not appear to be the lineage precursors of mature islet endocrine cells. Later in development, individual endocrine cells (α, β, δ, and PP) arising within the epithelium from neurogenin 3-positive (NGN3⁺) islet progenitors eventually migrate from the epithelium into the stroma of the gland and aggregate to form intact islets. Shortly after birth, the pancreas is able to secrete insulin when stimulated by glucose.

Numerous studies demonstrate that the exocrine and endocrine compartments are derived from the embryonic endoderm. Although the full complement of signals involved in the early formation of endoderm is currently unknown, several key transcription factors have emerged,

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including Sox17 and Mixl1. First identified as determinants of endoderm in Xenopus and zebrafish, inactivation of the murine homologs of Sox17 and Mixl1 demonstrate phenotypes consistent with their roles in development of the endoderm.^33,34. In addition, Nodal, a transform- ing growth factor β family member, which is expressed before and during gastrulation, is critically required for the formation of mesoderm and endoderm in mammals.³⁵ The forkhead box factor Foxa2 is essential for the development of anterior endoderm from which the pancreas and liver are formed.^36,37.

Once formed, the commitment of the endoderm toward a pancreatic fate is thought to occur as the result of expression of a particular combination of transcription factors, growth factors and growth factor receptors, and signaling molecules and their receptors, by the cells within a specific region of anterior embryonic endoderm. To better understand the molecular events that initiate this patterning, researchers have used tissue induction experiments to begin to define the embryonic tissue interactions that may guide pancreatic specification from the prepatterned endoderm. These studies suggest that factors derived from the notochord, such as fibroblast growth factor (FGF) 2 and activin βB are involved in the initial repression of Sonic hedgehog (Shh),^38,39a protein whose expression must be down-regulated in the presumptive dorsal pancreatic endoderm for correct development of the organ to ensue.⁴⁰In addition to the permissive signals from the notochord, the prepancreatic epithelium appears to respond to vascular epithelium and vascular endothelial growth factor, as determined through coculture studies with dissected dorsal aortae.^41,42 Other adjacent tissues, such as the lateral plate mesoderm (LPM) may also be involved. The LPM appears to be competent to signal endoderm not fated to become pancreas to initiate a pancreatic differentiation program, including expression of pdx1.⁴³ Bone morphogenetic protein (BMP)4, BMP7, and activin A are apparently capable of inducing expression of pdx1 in anterior chick endoderm when combined with the appropriate mesoderm, suggesting that perhaps these molecules are sufficient to mediate the inductive signals of the LPM.

After specification of the endoderm, proper signaling between the mesenchyme and the epithelium is also required for normal pancreatic development. For instance, FGF10, which is

normally expressed in the pancreatic mesenchyme, may mediate inductive interactions.⁴⁴ Norgaard and colleagues⁴⁵showed that ectopic expression of FGF10 in the foregut epithelium resulted in an increased proliferation of undifferentiated cells that went on to adopt a pancreatic fate, leading to a hyperplastic pancreas.

These studies provide insights into the early steps of embryonic endoderm patterning required for pancreas development.

In addition, the study of “knock-out” mice has revealed valuable information about the roles of a variety of transcription factors involved in pancreatic development^46–48(reviewed in Edlund⁴⁹).

For example, it is known that pdx1 (alias Ipf1) is absolutely required for pancreas development, although in its absence, pancreatic bud formation is initiated.^50,51Animals missing NGN3 lack mature endocrine cells, whereas exocrine development proceeds normally,⁵² placing NGN3 at an important branchpoint for pancreas development. Lineage tagging studies have also been used to determine which adult pancreatic cell types are formed from cells expressing certain gene products during development.^53–55 Together, these studies demonstrate the prove- nance of mature endocrine cells: They are derived from NGN3⁺progenitors present within the early pancreatic epithelium.

Although the roles of these and other important pancreatic transcription factors (Nkx6.1, Nkx2.2, Ptfla, HNF6, among others) have been described recently, their precise functions, i.e., interactions with other transcription factors and target genes, known and unknown, remain to be elucidated. In addition, how these key transcription factor proteins achieve tissue-specific functions, and exactly why cells in different positions in the embryo that express these proteins develop into distinctive tissue types, are questions that have not yet been answered.

Furthermore, the precise complement of homeobox transcription factors involved in the specification of pancreatic fate from uncommitted endoderm is not known. Lastly, characterization of unique cell surface markers of islet progenitors would aid in their efficient large-scale isolation from embryos or ES cells and would facilitate their identification in adult tissues.

Although significant insights into pancreas development have been achieved, it is clear we are far from a complete understanding of mammalian pancreaticogenesis. Yet, studies to date provide a rich framework of knowledge to begin

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to design experiments to more efficiently direct differentiation of ES cells to pancreatic lineages.

Pancreatic Lineage Specification in ES Cell Cultures

Although it was known that, in the context of whole animal development, murine ES cells were capable of differentiation into functional pancreatic cell types, many initial studies were aimed at determining whether the same was true for ES cells differentiating in vitro. We¹⁹ and others⁵⁶demonstrated that ES cells grown under nonselective conditions have the ability to differentiate into lineages of the endocrine pancreas (Figure 23.1). Our protocol includes a period of embryoid body (EB) formation. When ES cells are removed from conditions that prevent their differentiation and permit their growth under nonadherent conditions, they form multicellular aggregates of differentiated cells that are called EBs. To a certain extent, the initial morphogenesis of EB formation mim- ics that of morula- and blastula-stage embryos in vivo. After several days in culture, a layer of columnar epithelium ectoderm forms beneath the primitive endoderm, which often surrounds a fluid-filled cavity resembling a blastocoele.

At this point, EBs begin to express genes of primitive extraembryonic endoderm,^10,30 ectoderm,^20,57 and mesoderm lineages.^31,58 The cell–cell interactions afforded by EB formation

may promote inductive signals required for the formation of certain cell types or lineages. Our preliminary results suggest that, when grown under nonselective conditions, EBs express markers of definitive embryonic endoderm such as Sox17, Foxa2, and Pdx1. We also observed that under these conditions endoderm formation is greatly enhanced when a period of EB formation is included (unpublished results).

In studies of murine ES cells, we discovered that a culture protocol including a 5- to 7-day period of EB formation followed by further differentiation in the presence of fetal bovine serum after plating was sufficient to produce foci of cells expressing markers of pancreatic lineages. These include PDX1, a homeodomain protein that is absolutely required for development of the pancreas in humans and mice^50,51,59; peptide YY, a marker of early endocrine cells;

and insulin, glucagon, and somatostatin, hor- mones produced by the islet endocrine cells [Figure 23.2 (see color section)].¹⁹

In addition, it appears that these culture conditions provide an environment that facilitates the recreation of some classical developmental stages of islet cytodifferentiation, especially with regard to the timing of specific cell types that emerge in culture. Although lineage tracing experiments have yet to be performed, a series of cultures examined at different time points suggest that the primary to secondary transition that is seen during mouse development in vivo is recapitulated in vitro, because there are cells that are double-positive for both insulin and

Figure 23.1. Schematic diagram of in vitro ES cell differentiation focused on pancreatic lineage cells. The culture of both murine and human ES cells under conditions that permit the formation of embryoid bodies (EBs) result in a proportion of cells adopting a pancreatic fate. Pancreatic progenitor cells, which are PDX1⁺, as well as cells with the hallmarks of islet endocrine cells (α, β, δ, and PP) cells, have been identified (solid arrows). Other nonislet functional lineages of the pancreas, such as acinar tissue and ductal cells, are generally not found in serum-containing, non-growth factor supplemented cultures. Curved arrows denote cell division without further differentiation.

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glucagon in early stage cultures that appear to be replaced by single hormone-positive cells found in later cultures. At late stages, cells with characteristics similar to true β cells can be identified.¹⁹ We have obtained similar results differentiating human ES cells under nonselective conditions including an EB phase (unpublished results). As in the mouse cultures, discrete foci of PDX1⁺cells arise within the cultures during the EB stage and persist after plating. At later stages, hormone-positive cells can be identified.

It is important to note that although apparent pancreas progenitor cells can develop within the cultures grown under nonselective culture conditions, the conditions do not appear to promote growth of this cell type. Indeed, less than 0.1% of the cells in culture adopt a pancreas- specific fate, as judged by immunostaining with antibodies to PDX1, peptide YY, and/or insulin [Figure 23.2 (see color section)]. An obvious question is: Why are pancreatic lineage cells so rare in ES cell cultures? Although not precisely known, the answer may lie in the fact that islet endocrine cells are rare cell types in mammals that do not fully develop until late in gestation.

Islet endocrine cells are believed to constitute only approximately 1% of the cellular mass of the pancreas. In adult mammals, the function- ing endocrine cells of the islet maintain glycemic control. In the fetus, however, glycemic control is primarily regulated by the mother, and the embryo’s islet endocrine axis does not become functional until early postnatal development.

Another explanation for the low numbers of pancreas lineage cells observed in vitro is simply the nonselective conditions in which the cells were cultured. Clearly, culture conditions used to date do not precisely recapitulate the tempo- rally and spatially restricted developmental cues that occur in the embryo. Therefore, it is not unexpected that such cell types, although they arise under these conditions, do not dominate the cultures, nor do they preferentially expand or grow over time.

Indeed, it has proven to be a challenge to achieve directed differentiation of any specific cell lineage from ES cells in culture such that homogeneous populations of cells are generated. Several reports have emerged claiming to achieve directed differentiation of ES cells toward pancreatic β cells based on protocols that promote enrichment of nestin⁺ neuronal precursors.^60–62 The expression of many

neuronal cell markers in islet cells, such as synaptophysin and neuron-specific enolase, had led previously to the hypothesis that islets of Langerhans were of a neuroectodermal origin, and thus the idea that nestin might mark islet progenitor cells was put forth.⁶³ In 2001, Zulewski et al.⁶⁴ reported that nestin-positive cells derived from the adult rat pancreas could be expanded in vitro and had the apparent capa- bility to differentiate into cells that expressed insulin, glucagon, and PDX1. Researchers in several groups then used protocols that were originally designed to enrich for neural precursors to differentiate ES cells through a nestin- positive step and into pancreatic β cells.^60–62,65 These protocols typically involved growing cells in serum-free media containing a mixture of insulin, transferrin, selenium, and fibronectin (often abbreviated ITSFn). After this, and depending on the specific protocol, cells were exposed to bFGF, N2 medium, B27 medium, nicotinamide, and/or phosphoinositol-3 kinase inhibitors to first expand pancreatic progenitor cells, and then prompt their final differentiation.

Although cells resulting from this type of cultur- ing did often stain with anti-insulin antibodies, the cells typically functioned abnormally in in vitro glucose release assays, and failed to restore euglycemia is streptozotocin-treated animals after transplantation.

Further experimentation with protocols designed to enrich for nestin-positive cells demonstrated that the ES cell-derived clusters did not actually express insulin, but rather had taken up insulin from the medium.⁶⁶ Cells derived through such protocols did not robustly synthesize insulin or pdx1 gene products,^60,67nor did they stain with antibodies specific for C-peptide, a protein generated when proinsulin is cleaved to the functional insulin molecule.^68,69 Furthermore, many of the cells grown under these serum-free conditions that apparently stained with antibodies to insulin appeared to be undergoing apoptosis, as examined through a caspase-3 immunostaining⁶⁸ and TUNEL staining.^67,68 In addition, recent lineage-tracing experiments suggest that the endocrine pancreas is not composed of cells that express nestin during their course of development.^70–73Thus, there is no current evidence that selection of differentiated ES cells that express nestin will result in a population of glucose-responsive insulin-secreting β-like cells.

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Enhancement of Islet Differentiation from ES Cells by Lineage Selection and/or Reestablishment of

Developmental Pathways

The heterogeneity of differentiating ES cell cultures has hindered researchers’ abilities to study the function of ES cell-derived cells and tissues.

Therefore, a major goal of ES cell research is to generate purified populations of a desired cell type, in this case, pancreatic progenitors and/or β cells. To achieve this goal, some investigators have taken advantage of the relative ease with which ES cells can be genetically manipulated to create selection strategies. One strategy is to integrate lineage-specific promoter-driven fluorescent transgenes and then select marked cells by flow cytometric cell sorting.^74–77An alternate approach is to express a drug resistance gene under control of the chosen lineage-specific promoter. In either case, when the promoter is activated in a subset of cells among differentiating ES cell progeny, it is an indication that those cells have adopted the cell fate of interest. Cells activating the lineage-specific promoter can then be selected by virtue of their fluorescence or drug resistance. This technique has now been used to select for neural precursors,⁷⁸cardiomyocytes,⁷⁹and insulin-producing cells.^80,81

To purify insulin-producing cells derived from murine ES cells, Soria and colleagues⁸⁰ generated a cell line in which the βgeo fusion protein (β-galactosidase fused with neomycin resistance gene)⁸² was driven by the human insulin promoter. The ES cells were differentiated through a period of EB formation, and then cells that had activated the insulin promoter were selected by addition of G418 to the media.

Selected cells were then grown in a low glucose environment (5 mM) for 5 days in the presence of nicotinamide, after which the resulting cell clusters were analyzed in vitro and in vivo. In vitro, the cells appeared to respond appropri- ately in insulin secretion assays. When transplanted into diabetic mice to assess their ability to normalize hyperglycemia, the cells rescued several animals for a period of up to 16 weeks after transplantation. These successes are prom- ising, although some questions regarding the nature of the selected cells and their phenotypic stability remain. In addition, it is not clear that the animals were cured as a direct result of their

ES cell-derived graft as the donor cells (R1 line, 129 hybrid, primarily H-2^b) were allogeneic to the recipients (OF1, outbred) and should have been rejected, and studies to confirm that rever- sal of diabetes was dependent on the infused cells were not performed. In addition, euglycemia was not durable in a significant proportion of animals for reasons that remain unclear.

Nonetheless, this study in murine ES cells is the single study to date that demonstrates significant in vitro and in vivo function from ES cell- derived insulin-producing tissue.

An alternate, or complementary, strategy to achieve enhanced islet differentiation is to mod- ify culture conditions in order to mimic the in vivo growth environment and recapitulate normal developmental pathways in vitro. If embryonic inductive signals could be precisely reestablished, and the correct embryonic microenvironment provided to differentiating ES cells, directed differentiation into large numbers of PDX1-positive pancreatic progenitor cells and ultimately endocrine cells might be achieved. To begin to reconstruct pancreatic lineage differentiation from ES cells in culture, one needs to consider how this tissue forms in the embryo. Because the pancreas and islets are endoderm derived, it might be important to enrich for endodermal precursors by either selecting for cells that express genes required for endoderm development, such as Nodal, or overexpressing these genes in ES cell progeny. Then, to pattern the ES cell-derived endoderm and induce pancreas specification in a large proportion of cells, it might be important to inhibit the function of key developmental signaling molecules, such as Shh, which is normally down-regulated in the region of foregut endoderm that is specified to become pancreas.

Reconstructing combinations of intrinsic and extrinsic signals may also be important.

Overexpressing one or more key pancreatic transcription factor genes that are typically expressed in early committed pancreatic epithelium or are necessary for β cell formation may lead to a greater proportion of cells differentiating into endocrine cells or β cells. Indeed, early studies focusing on the overexpression of PDX1 and PAX4 in murine ES cell cultures suggest this strategy may be useful.^65,83. It is important to note, however, that simply because a transcription factor is expressed in a specific tissue at a particular time in development and is required for normal development does not necessarily

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imply that this protein will direct differentiation.

In addition, our knowledge of the cellular context in which these critical transcription factors act may be incomplete, but is undoubtedly important. Therefore, it may be difficult to recapitulate the actions of these genes precisely in ES cell progeny.

Mesenchymal-to-epithelial signaling, also critically important for proper growth and maturation and of the pancreatic epithelium, could be recapitulated by adding specific growth factors, such as BMP4 or FGF10, or by coculturing ES cells with stromal cell lines^28,84,85 or embryonic tissues. By providing the proper developmental signals, it may be possible to promote the differentiation of a proliferative precursor population that could be isolated and expanded.

However, some growth factors have pleiotropic effects or can function in multiple tissues.

Therefore, one needs to consider the context in which the cytokine is acting. The effect of growth factors in mixed populations of ES cell progeny may be unpredictable and not specific to the lineage of interest.⁸⁶ In addition, the presence of varied cell types may lead to unpredictable cell–cell interactions that could inhibit endoderm formation and/or pancreatic specification.

The correct cues for differentiating progenitors into postmitotic, single hormone-expressing differentiated islet cells or β cells remain to be determined. Also, little is known about the regulation of the later stages of islet development, including terminal differentiation, migra- tion of islet endocrine cells from the epithelium into the acinar lobules, or the mechanisms regu- lating their aggregation into micro-organs.

Growth factors that have been shown to acceler- ate the functional maturation of human fetal pancreas tissue may be worthwhile to test in the ES cell differentiation system at these late stages.

Clearly, the development of robust methods for enrichment of islet progenitor cell types and β cells from ES cells remains an important goal.

Our current knowledge of developmental mechanisms can be directly applied to ES cells as they differentiate in culture to promote lineage-specific differentiation. This framework serves as a useful starting point. For example, building on prior in vivo developmental studies, Wichterle et al.¹⁸demonstrated robust, directed differentiation of motor neurons from mouse ES cells. This, and other studies,⁸⁷ demonstrates how applying known developmental cues to ES

cells can direct differentiation to a desired phenotype and recapitulate normal ontogeny. As gaps in our knowledge of pancreatic islet development are filled in, we will be better able to faithfully reestablish important developmental signals in ES cell cultures.

Remaining Questions

In the quest to generate robustly functional insulin-producing cells from ES cells, numerous questions remain to be answered. For example, i) How do we determine if an insulin- staining cell is a true β cell, and what functional measures should be used? ii) Are all ES cell- derived insulin-staining cells equally mature or does a subpopulation retain a fetal phenotype?

iii) Are β cells alone sufficient for normal glucose-responsive insulin secretion in vivo, or are other islet cell types required? iv) Are human ES cell-derived insulin-producing cells able to secrete insulin in a glucose-responsive manner? These questions will be addressed below.

What should the benchmarks be for a “real” β cell? Not only is it important to correctly identify cells, but one needs to consider the fact that cells from a variety of tissues induced in ES cultures can potentially synthesize insulin, including extraplacental membranes⁸⁸ and neuronal cells,^89,90among others.^91,92Additionally, β cells at various stages of functional maturity might be expected to exist in ES cell cultures, because the differentiation process may be interrupted because of environmental deficiencies. Indeed, the response of islet tissue in standard assays may differ depending on the developmental stage.⁹³ Consequently, it will be important to evaluate function by a variety of parameters including studies at the single cell level.

Are other islet endocrine cell types, in addition to β cells, necessary for optimal function? It has been suggested that all islet subtypes are required to achieve regulated functionality in normal in situ conditions^94,95although there is some evidence that β cells alone can rescue streptozotocin-induced diabetes in mice.⁹⁶ The answer to this question may be different depending on the species and cause of diabetes.

Normal β-cell function has not yet been con- vincingly demonstrated for human ES-derived cells, and this should be carefully evaluated with

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dynamic in vitro perifusion assays once enriched populations of insulin-producing tissue are reli- ably generated. Ultimately, it is hoped that increased numbers of functional β cells can be obtained through a combination of new selective techniques and better culture conditions.

Acknowledgments

The authors thank Kathy Worrall for technical assistance in preparing the manuscript and Nick Weber for helping with production of the figures. This project was generously supported by grants from the NIH-Beta Cell Biology Consortium (U19-DK61244 and subcontract to Vanderbilt University U19- DK42502), JDRF (2001-191, 2004-145), and Roche Organ Transplant Research Foundation (221283847). This work was also supported in part from a grant to the University of Wisconsin Medical School under the Howard Hughes Medical Institute Research Resources Program for Medical Schools.

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