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The islets of Langerhans are highly organized structures that have species-specific, three-dimensional tissue architecture. Islet architecture is critical for proper hormone secretion in response to nutritional stimuli. Islet architecture is disrupted in all types of diabetes mellitus and in cadaveric islets for transplantation during isolation, culture, and perfusion, limiting patient outcomes. Moreover, recapitulating native islet architecture remains a key challenge for

SummaryPancreatic islets of Langerhans have a non-random spatial architecture, which is important to proper glucose homeostasis. Islet architecture forms during embryonic development, in a morphogenesis process partially involving expression of Roundabout (Robo) receptors in β cells, and their ligand, Slit, in the surrounding mesenchyme. Whether islet architecture is set during development and remains passive in adulthood, or whether it requires active maintenance throughout life, has not been determined. To distinguish between these two models, we conditionally deleted Robo2 in β cells of adult mice and observed their islet architecture following a two-month chase. Here, we show that deleting Robo2 in adult β cells causes significant loss of islet architecture without affecting β cell identity, maturation, or stress, indicating that Robo2 plays a role in actively maintaining adult islet architecture. Understanding the factors required to maintain islet architecture, and thus optimize islet function, is important for developing future diabetes therapies.

Loss of mature β cell function and identity, or β cell dedifferentiation, is seen in both type 1 and type 2 diabetes. Two competing models explain β cell dedifferentiation in diabetes. In the first model, β cells dedifferentiate in the reverse order of their developmental ontogeny. This model predicts that dedifferentiated β cells resemble β cell progenitors. In the second model, β cell dedifferentiation depends on the type of diabetogenic stress. This model, which we call the “Anna Karenina” model, predicts that in each type of diabetes, β cells dedifferentiate in their own way, depending on how their mature identity is disrupted by any particular diabetogenic stress. We directly tested the two models using a β cell-specific lineage-tracing system coupled with RNA-sequencing in mice. We constructed a multidimensional map of β cell transcriptional trajectories during the normal course of β cell postnatal development and during their dedifferentiation in models of both type 1 diabetes (NOD) and type 2 diabetes (BTBR-Lepob/ob). Using this unbiased approach, we show here that despite some similarities between immature and dedifferentiated β cells, β cells dedifferentiation in the two mouse models is not a reversal of developmental ontogeny and is different between different types of diabetes.

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among β cells, yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets have reduced synchronized intra-islet Ca2+ oscillations among β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

Diabetes is characterized by a loss of functional β-cell mass. With current rodent models of chemically-induced diabetes such as streptozotocin, it is difficult to distinguish the consequence of β-cell loss from the decline in individual β-cell function. Here, we generated a mouse model of severe β-cell mass loss by deleting β-cell CDK1 within the first week of life (Cdk1Ucn3Cre, Cdk1-floxed:Ucn3-Cre). At 4 weeks of age, islet mass in Cdk1Ucn3Cre islets is comparable to controls, although islet architecture is disrupted with α-cells having formed more heterotypic contacts with β-cells. By 12 weeks of age, Cdk1Ucn3Cre mice exhibit significantly reduced β-cell mass as well as α-cell mass, but maintain glucose tolerance and ex vivo insulin secretion in response to glucose, amino acids, and the gut incretin GIP. We therefore hypothesized that functional compensations in Cdk1Ucn3Cre islets arise from both β- and α-cells, which are insulinotropic in the fed state. Interestingly, α-cell glucagon secretion from Cdk1Ucn3Cre islets exhibits hypersensitivity to suppression by glucose, including when glucagonotropic amino acids are present. In parallel, we observed a reduction in amino acid-stimulated cAMP in β‑cells from Cdk1Ucn3Cre islets. We also found a left-shift in the glucose dependence of β-cell calcium oscillations in Cdk1Ucn3Cre islets, identifying β-cell recruitment as one mechanism offsetting the loss of α-cell paracrine signaling. Importantly, GIP elicited a much larger glucagon response from glucose- and amino-acid stimulated Cdk1Ucn3Cre islets, implying that α-cell compensation contributes significantly to the maintained insulin secretory output in vivo. In sum, the Cdk1Ucn3Cre model reveals the interdependence of α- and β-cell mass, and function, and will be useful to define the temporal sequence of islet compensation for chronic loss of β-cell mass under stressed conditions. Disclosure S. Sdao: None. M. Adams: None. H. R. Foster: None. B. Blum: None. M. J. Merrins: None.

Hallmarks of mature β cells are restricted proliferation and a highly energetic secretory state. Paradoxically, cyclin-dependent kinase 2 (CDK2) is synthesized throughout adulthood, its cytosolic localization raising the likelihood of cell cycle-independent functions. In the absence of any changes in β cell mass, maturity, or proliferation, genetic deletion of Cdk2 in adult β cells enhanced insulin secretion from isolated islets and improved glucose tolerance in vivo. At the single β cell level, CDK2 restricts insulin secretion by increasing KATP conductance, raising the set point for membrane depolarization in response to activation of the phosphoenolpyruvate (PEP) cycle with mitochondrial fuels. In parallel with reduced β cell recruitment, CDK2 restricts oxidative glucose metabolism while promoting glucose-dependent amplification of insulin secretion. This study provides evidence of essential, non-canonical functions of CDK2 in the secretory pathways of quiescent β cells.

The spatial architecture of the islets of Langerhans is vitally important for their correct function, and alterations in islet morphogenesis often result in diabetes mellitus. We have previously reported that Roundabout (Robo) receptors are required for proper islet morphogenesis. As part of the Slit-Robo signaling pathway, Robo receptors function in conjunction with Slit ligands to mediate axon guidance, cell migration, and cell positioning in development. However, the role of Slit ligands in islet morphogenesis has not yet been determined. Here, we report that Slit ligands are expressed in overlapping and distinct patterns in both endocrine and nonendocrine tissues in late pancreas development. We show that the function of either Slit2 or Slit3, which are predominantly expressed in the pancreatic mesenchyme, is required and sufficient for islet morphogenesis, while Slit1, which is predominantly expressed in the β cells, is dispensable for islet morphogenesis. We further show that Slit functions as a repellent signal to β cells. These data suggest that clustering of endocrine cells during islet morphogenesis is guided, at least in part, by repelling Slit2/3 signals from the pancreatic mesenchyme.

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among β cells, yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets have reduced synchronized intra-islet Ca2+ oscillations among β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

The spatial architecture of the islets of Langerhans is vitally important for their correct function, and alterations in islet morphogenesis often result in diabetes mellitus. We have previously reported that Roundabout (Robo) receptors are required for proper islet morphogenesis. As part of the Slit-Robo signaling pathway, Robo receptors work in conjunction with Slit ligands to mediate axon guidance, cell migration, and cell positioning in development. However, the role of Slit ligands in islet morphogenesis has not yet been determined. Here we report that Slit ligands are expressed in overlapping and distinct patterns in both endocrine and non-endocrine tissues in late pancreas development. We show that function of either Slit2 or Slit3, which are predominantly expressed in the pancreatic mesenchyme, is required and sufficient for islet morphogenesis, while Slit1, which is predominantly expressed in the β cells, is dispensable for islet morphogenesis. We further show that Slit functions as a repellent signal to β cells. These data suggest that clustering of endocrine cells during islet morphogenesis is guided, at least in part, by repelling Slit2/3 signals from the pancreatic mesenchyme.

SUMMARY Hallmarks of mature β cells are restricted proliferation and a highly energetic secretory state. Paradoxically, cyclin-dependent kinase 2 (CDK2) is synthesized throughout adulthood, its cytosolic localization raising the likelihood of cell cycle-independent functions. In the absence of any changes in β cell mass, maturity, or proliferation, genetic deletion of Cdk2 in adult β cells enhanced insulin secretion from isolated islets and improved glucose tolerance in vivo. At the single β cell level, CDK2 restricts insulin secretion by increasing KATP conductance, raising the set point for membrane depolarization in response to activation of the phosphoenolpyruvate (PEP) cycle with mitochondrial fuels. In parallel with reduced β cell recruitment, CDK2 restricts oxidative glucose metabolism while promoting glucose-dependent amplification of insulin secretion. This study provides evidence of essential, non-canonical functions of CDK2 in the secretory pathways of quiescent β cells., Graphical Abstract, In Brief Despite loss of proliferative capacity with age, mature β cells continually synthesize CDK2. Sdao et al. demonstrate that CDK2 depletion in adult β cells improves glucose tolerance in vivo. By augmenting PEP cycle-dependent KATP channel closure, CDK2 inactivation lowers the set point for membrane depolarization, augmenting oxidative metabolism and insulin secretion.

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion between β cells, yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets lose synchronized intra-islet Ca2+ oscillations between β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, loss of Connexin36 gap junctions, or changes in islet vascularization, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results will have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion between β cells, yet testing thisin vivoin the intact pancreas is challenging.Robo βKOmice, in which the genesRobo1andRobo2are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. CombiningRobo βKOmice with intravital microscopy, we show here thatRobo βKOislets lose synchronized intra-islet Ca2+oscillations between β cellsin vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, loss of Connexin36 gap junctions, or changes in islet vascularization, suggesting that the islet architecture itself is required for synchronized Ca2+oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

Pancreatic islets of Langerhans display characteristic spatial architecture of their endocrine cell types. This architecture is critical for cell-cell communication and coordinated hormone secretion. Islet architecture is disrupted in type-2 diabetes. Moreover, the generation of architecturally correct islets in vitro remains a challenge in regenerative approaches to type-1 diabetes. Although the characteristic islet architecture is well documented, the mechanisms controlling its formation remain obscure. Here, we report that correct endocrine cell type sorting and the formation of mature islet architecture require the expression of Roundabout (Robo) receptors in β cells. Mice with whole-body deletion of Robo1 and conditional deletion of Robo2 either in all endocrine cells or selectively in β cells show complete loss of endocrine cell type sorting, highlighting the importance of β cells as the primary organizer of islet architecture. Conditional deletion of Robo in mature β cells subsequent to islet formation results in a similar phenotype. Finally, we provide evidence to suggest that the loss of islet architecture in Robo KO mice is not due to β cell transdifferentiation, cell death or loss of β cell differentiation or maturation.

This article looks into the fate of Ovid's works in the wake of his relegation. Were they expelled from the public libraries of Rome as is commonly thought? A re-examination of pertinent passages in the Tristia and Epistulae ex Ponto , as well as of what is known of the workings of ancient libraries and the history of Roman book censorship, presents an alternative. The investigation concludes with an account of the factors which may have given rise to the communis opinio .

At the beginning ofTristia2, a single long apologetic elegy, Ovid struggles with making sense of his continued occupation with poetry, despite the disaster it has brought upon him. In lines 7–8 he broods over Augustus’ displeasure, roused by theArs Amatoria, which led to his reproach. The passage is also sometimes adduced as a reference to the removal of theArsfrom Rome's public libraries.1

Islet β-cells from newborn mammals exhibit high basal insulin secretion and poor glucose-stimulated-insulin-secretion (GSIS). Here we show that β cells of newborns secrete more insulin than adults in response to similar intracellular Ca(2+) concentrations, suggesting differences in the Ca(2+) sensitivity of insulin secretion. Synaptotagmin-4 (Syt4), a non-Ca(2+) binding paralogue of the β-cell Ca(2+) sensor Syt7, increased by ~8-fold during β-cell maturation. Syt4 ablation increased basal insulin secretion and compromised GSIS. Precocious Syt4 expression repressed basal insulin secretion but also impaired islet morphogenesis and GSIS. Syt4 was localized on insulin granules and Syt4 levels inversely related to the number of readily releasable vesicles. Thus, transcriptional regulation of Syt4 impacts insulin secretion; Syt4 expression is regulated in part by Myt transcription factors, which repress Syt4 transcription. Finally, human SYT4 regulated GSIS in EndoC-βH1 cells, a human β-cell line. These findings reveal the role that altered Ca(2+) sensing plays in regulating β-cell maturation.

Insulin-expressing cells that have been differentiated from human pluripotent stem cells in vitro lack the glucose responsiveness characteristic of mature beta cells. Beta-cell maturation in mice was studied to find genetic markers that enable screens for factors that induce bona fide beta cells in vitro. We find that functional beta-cell maturation is marked by an increase in the glucose threshold for insulin secretion and by expression of the gene urocortin 3.

Type 2 diabetes is characterized by a reduction in insulin function and an increase in glucagon activity that together result in hyperglycemia. Glucagon receptor antagonists have been developed as drugs for diabetes; however, they often increase glucagon plasma levels and induce the proliferation of glucagon-secreting α-cells. We find that the secreted protein Angiopoietin-like 4 (Angptl4) is up-regulated via Pparγ activation in white adipose tissue and plasma following an acute treatment with a glucagon receptor antagonist. Induction of adipose angptl4 and Angptl4 supplementation promote α-cell proliferation specifically. Finally, glucagon receptor antagonist improves glycemia in diet-induced obese angptl4 knockout mice without increasing glucagon levels or α-cell proliferation, underscoring the importance of this protein. Overall, we demonstrate that triglyceride metabolism in adipose tissue regulates α-cells in the endocrine pancreas.

This article explores an issue of some ongoing controversy in Ovidian scholarship: the identity of the anonymous person addressed in the epilogue of the “Tristia”’s third book. The exploration combines an analysis of the text with an examination of salient aspects of Hellenistic and Roman literary culture. These in turn inform a systematic prosopographical reassessment of four hypotheses. Evidence converges in the conclusion that the addressee was likely a high-level library administrator, most probably C. Iulius Hyginus.

Human embryonic stem cells (HESCs) and induced pluripotent stem cells (HiPSCs) offer an immense potential as a source of cells for regenerative medicine. However, the ability of undifferentiated HESCs and HiPSCs to produce tumors in vivo presents a major obstacle for the translation of this potential into clinical reality. Therefore, characterizing the nature of HESC- and HiPSC-derived tumors, especially their malignant potential, is extremely important in order to evaluate the risk involved in their clinical use. Here we review recent observations on the tumorigenicity of human pluripotent stem cells. We argue that diploid, early passage, HESCs produce benign teratomas without undergoing genetic modifications. Conversely, HESCs that acquired genetic or epigenetic changes upon adaptation to in vitro culture can produce malignant teratocarcinomas. We discuss the molecular mechanisms of HESC tumorigenicity and suggest approaches to prevent tumor formation from these cells. We also discuss the differences in the tumorigenicity between mouse embryonic stem cells (MESCs) and HESCs, and suggest methodologies that may help to identify cellular markers for culture adapted HESCs.

CpG island-like sequences are commonly thought to provide the sole signals for designating constitutively unmethylated regions in the genome, thus generating open chromatin domains within a sea of global repression. Using a new database obtained from comprehensive microarray analysis, we show that unmethylated regions (UMRs) seem to be formed during early embryogenesis, not as a result of CpG-ness, but rather through the recognition of specific sequence motifs closely associated with transcription start sites. This same system probably brings about the resetting of pluripotency genes during somatic cell reprogramming. The data also reveal a new class of nonpromoter UMRs that become de novo methylated in a tissue-specific manner during development, and this process may be involved in gene regulation. In short, we show that UMRs are an important aspect of genome structure that have a dynamic role in development.

Teratomas derived from human embryonic stem (hES) cells are unique among oncogenic phenomena as they are polyclonal and develop from apparently normal cells. A deeper understanding of this process should aid in the development of safer cell therapies and may help elucidate the basic principles of tumor initiation. We find that transplantation of diploid hES cells from four independent cell lines generates benign teratomas with no sign of malignancy or persisting embryonal carcinoma-like cells. In contrast, mouse embryonic stem (mES) cells from four cell lines consistently generate malignant teratocarcinomas. Global gene expression analysis shows that survivin (BIRC5), an anti-apoptotic oncofetal gene, is highly expressed in hES cells and teratomas but not in embryoid bodies. Genetic and pharmacological ablation of survivin induces apoptosis in hES cells and in teratomas both in vitro and in vivo. We suggest that continued expression of survivin upon differentiation in vivo may contribute to teratoma formation by hES cells.

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue- nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

The cytosolic form of the phosphoenolpyruvate carboxykinase (PEPCK-C) gene is selectively expressed in several tissues, primarily in the liver, kidney, and adipose tissue. The transcription of the gene is reciprocally regulated by glucocorticoids in these tissues. It is induced in the liver and kidney but repressed in the white adipose tissue. To elucidate which adipocyte-specific transcription factors participate in the repression of the gene, DNase I footprinting analyses of nuclear proteins from 3T3-F442A adipocytes and transient transfection experiments in NIH3T3 cells were utilized. Glucocorticoid treatment slightly reduced the nuclear C/EBP alpha concentration but prominently diminished the binding of adipocyte-derived nuclear proteins to CCAAT/enhancer-binding protein (C/EBP) recognition sites, without affecting the binding to nuclear receptor sites in the PEPCK-C gene promoter. Of members of the C/EBP family of transcription factors, C/EBP alpha was the strongest trans-activator of the PEPCK-C gene promoter in the NIH3T3 cell line. The glucocorticoid receptor (GR), in the presence of its hormone ligand, inhibited the activation of the PEPCK-C gene promoter by C/EBP alpha or C/EBP beta but not by the adipocyte-specific peroxisome proliferator-activated receptor gamma 2. This inhibition effect was similar using the wild type or mutant GR and did not depend on GR binding to the DNA. The glucocorticoid response unit (GRU) in the PEPCK-C gene promoter (-2000 to +73) restrained C/EBP alpha-mediated trans-activation, because mutation of each single GRU element increased this activation by 3-4-fold. This series of GRU mutations were repressed by wild type GR to the same percent as was the nonmutated PEPCK-C gene promoter. In contrast, the repression by mutant GR depended on the intact AF1 site in the gene promoter, whereby mutation of the AF1 element abolished the repression.

Dysfunction or death of pancreatic β cells underlies both types of diabetes. This functional decline begins with β cell stress and de-differentiation. Current drugs for type 2 diabetes (T2D) lower blood glucose levels but they do not directly alleviate β cell stress nor prevent, let alone reverse, β cell de-differentiation. We show here that Urocortin 3 (Ucn3), a marker for mature β cells, is down-regulated in the early stages of T2D in mice and when β cells are stressed in vitro. Using an insulin expression-coupled lineage tracer, with Ucn3 as a reporter for the mature β cell state, we screen for factors that reverse β cell de-differentiation. We find that a small molecule inhibitor of TGFβ receptor I (Alk5) protects cells from the loss of key β cell transcription factors and restores a mature β cell identity even after exposure to prolonged and severe diabetes. DOI: http://dx.doi.org/10.7554/eLife.02809.001, eLife digest Diabetes is a condition that develops when the body does not produce or use a hormone called insulin effectively. Insulin helps fat and muscle cells absorb glucose from the blood, and so diabetes can result in high levels of blood glucose, which can cause strokes, blindness, and heart disease. In healthy individuals, beta cells in the pancreas (a large gland located behind the stomach) produce insulin. The beta cells develop from endocrine progenitor cells, which are unspecialised cells that can either duplicate themselves or ‘differentiate’ to form one of the specialised cell types found in the pancreas. In diabetic patients, however, certain stresses (such as an immune attack in type-1 diabetics or insulin-resistance due to obesity, pregnancy, or ageing in type-2 diabetics) can cause mature beta cells to lose their identity in a process known as ‘de-differentiation’. This means that beta cells either revert back to an earlier stage in their development or adopt a new dysfunctional identity. When this occurs, the body loses beta cells and is unable to produce insulin. It was not known whether de-differentiated beta cells in diabetic patients can recover to form mature beta cells that are capable of producing insulin. Additionally, the drugs currently used to treat diabetes are able to lower blood glucose levels, but these drugs do not replace the lost beta cells. Blum et al. now show that mice stop expressing a gene called Urocortin 3 when beta cells first start to de-differentiate. Only functional beta cells express Urocortin 3, so this gene is a useful ‘marker’ that can be used to tell if a cell is a mature, functional beta cell or not. Using this system, Blum et al. found that if de-differentiated cells are transplanted into a non-diabetic mouse, they are able to revert back into mature beta cells that can produce insulin. This happens even if the cells have been de-differentiated for a long time. Blum et al. then used this system to investigate ways of protecting against or reversing beta cell de-differentiation. Using small molecules to block the activity of a protein called TGF beta receptor 1 was found to protect against beta cell de-differentiation and to restore the identity of mature beta cells. The findings of Blum et al. represent a first step towards the possible development of new drugs to prevent or even restore the loss of healthy, mature beta cells in diabetic patients. DOI: http://dx.doi.org/10.7554/eLife.02809.002

Human embryonic stem cells (HESCs) are the in vitro descendants of the pluripotent inner cell mass (ICM) of human blastocyst stage embryos. HESCs can be kept undifferentiated in culture or be differentiated to tissues representing all three germ layers, both in vivo and in vitro. These properties make HESC-based therapy remarkably appealing for the treatment of various disorders. Upon transplantation in vivo, undifferentiated HESCs rapidly generate the formation of large tumors called teratomas. These are benign masses of haphazardly differentiated tissues. Teratomas also appear spontaneously in humans and in mice. When they also encompass a core of malignant undifferentiated cells, these tumors are defined as teratocarcinomas. These malignant undifferentiated cells are termed embryonic carcinoma (EC), and are the malignant counterparts of embryonic stem cells. Here we review the history of experimental teratomas and teratocarcinomas, from spontaneous teratocarcinomas in mice to induced teratomas by HESC transplantation. We then discuss cellular and molecular aspects of the tumorigenicity of HESCs. We also describe the utilization of HESC-induced teratomas for the modeling of early human embryogenesis and for modeling developmental diseases. The problem of HESC-induced teratomas may also impede or prevent future HESC-based therapies. We thus conclude with a survey of approaches to evade HESC-induced tumor formation.

Human embryonic stem cells (HESCs) are the in vitro descendants of the pluripotent inner cell mass (ICM) of human blastocyst stage embryos. HESCs can be kept undifferentiated in culture or be differentiated to tissues representing all three germ layers, both in vivo and in vitro. These properties make HESC-based therapy remarkably appealing for the treatment of various disorders. Upon transplantation in vivo, undifferentiated HESCs rapidly generate the formation of large tumors called teratomas. These are benign masses of haphazardly differentiated tissues. Teratomas also appear spontaneously in humans and in mice. When they also encompass a core of malignant undifferentiated cells, these tumors are defined as teratocarcinomas. These malignant undifferentiated cells are termed embryonic carcinoma (EC), and are the malignant counterparts of embryonic stem cells. Here we review the history of experimental teratomas and teratocarcinomas, from spontaneous teratocarcinomas in mice to induced teratomas by HESC transplantation. We then discuss cellular and molecular aspects of the tumorigenicity of HESCs. We also describe the utilization of HESC-induced teratomas for the modeling of early human embryogenesis and for modeling developmental diseases. The problem of HESC-induced teratomas may also impede or prevent future HESC-based therapies. We thus conclude with a survey of approaches to evade HESC-induced tumor formation.

Differentiation of human embryonic stem cells (HESCs) can be studied in vivo through the induction of teratomas in immune-deficient mice. Cells within the teratomas differentiate into all three embryonic germ layers. However, the exact nature of the proliferation and differentiation of HESCs within the teratoma is not fully characterized, and it is not clear whether the differentiation is cell autonomous or affected by neighboring cells. Here, we establish a genetic approach to study the clonality of differentiation in teratomas using a mixture of HESC lines. We first demonstrate, by means of 5-bromo-2′-deoxyuridine incorporation, that cell proliferation occurs throughout the teratoma, and that there are no clusters of undifferentiated-proliferating cells. Using a combination of laser capture microdissection and DNA fingerprinting analysis, we show that different cell lines contribute mutually to the same distinctive tissue structures. Further support for the nonclonal differentiation within the teratoma was achieved by fluorescence in situ hybridization analysis of sex chromosomes. We therefore suggest that in vivo differentiation of HESCs is polyclonal and, thus, may not be cell autonomous, stressing the need for a three-dimensional growth in order to achieve complex differentiation of HESCs. Disclosure of potential conflicts of interest is found at the end of this article.

The hepatic transcriptional regulation by glucocorticoids of the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C) gene is coordinated by interactions of specific transcription factors at the glucocorticoid regulatory unit (GRU). We propose an extended GRU that consists of four accessory sites, two proximal AF1 and AF2 sites and their distal counterpart dAF1 (-993) and a new site, dAF2 (-1365); together, these four sites form a palindrome. Sequencing and gel shift binding assays of hepatic nuclear proteins interacting with these sites indicated similarity of dAF1 and dAF2 sites to the GRU proximal AF1 and AF2 sites. Chromatin immunoprecipitation assays demonstrated that glucocorticoids enhanced the binding of FOXO1 and peroxisome proliferator-activated receptor-alpha to AF2 and dAF2 sites and not to dAF1 site but enhanced the binding of hepatic nuclear transcription factor-4alpha only to the dAF1 site. Insulin inhibited the binding of these factors to their respective sites but intensified the binding of phosphorylated FOXO1. Transient transfections in HepG2 human hepatoma cells showed that glucocorticoid receptor interacts with several non-steroid nuclear receptors, yielding a synergistic response of the PEPCK-C gene promoter to glucocorticoids. The synergistic stimulation by glucocorticoid receptor together with peroxisome proliferator-activated receptor-alpha or hepatic nuclear transcription factor-4alpha requires all four accessory sites, i.e. a mutation of each of these markedly affects the synergistic response. Mice with a targeted mutation of the dAF1 site confirmed this requirement. This mutation inhibited the full response of hepatic PEPCK-C gene to diabetes by reducing PEPCK-C mRNA level by 3.5-fold and the level of circulating glucose by 25%.