Director, Stanford Diabetes Research Center (2016 - Present)
Director, Stanford MSTP (2008 - 2013)
Member, Medical Science Review Board, Juvenile Diabetes Research Foundation (2002 - Present)
Associate Director, Stanford Medical Scientist Training Program (2000 - 2008)
Honors & Awards
Living and Giving Award, Juvenile Diabetes Research Foundation Northern California Chapter (2004)
Faculty Scholar Award, SmithKline Beecham-Stanford University School of Medicine (1999-2001)
Junior Faculty Scholar, Howard Hughes Medical Institute/Stanford University School of Medicine (1999-2001)
Henry J. Kaiser Family Foundation Award for Excellence in Preclinical Teaching, Stanford University School of Medicine (2002)
Faculty Scholar Award, Donald E. and Delia B. Baxter Foundation (1999-2001)
Career Development Award, American Diabetes Association (1999-2003)
Named Investigator Award, Stanford-NIH Digestive Diseases Center (2000)
Pew Biomedical Research Scholar, The Pew Charitable Trusts (1999-2003)
Investigator, Howard Hughes Medical Institute (2008-2016)
Gerald and Kayla Grodsky Basic Science Research Award, Juvenile Diabetes Research Foundation (JDRF) (2013)
Ho-Am Prize in Medicine, Ho-Am Foundation (2014)
Faculty Award for Excellence in Mentoring and Service, Office of Graduate Education, Stanford University School of Medicine (2015)
A.B., Harvard University, Biochemical Sciences (1985)
M.D., Stanford University, Medicine (1992)
Ph.D., Stanford University, Biochemistry (1992)
Current Research and Scholarly Interests
Organ development requires mechanisms to establish an integrated, stereotyped tissue pattern from multiple distinct cellular components. Many vital organs derive from the endodermal and mesodermal germ layers to form the gastrointestinal and respiratory tracts, yet little is known about the genetic programs that coordinate steps culminating in proper organ morphogenesis and axial position, cell differentiation and physiologic function. Our goal is to identify and understand the pathways that govern organogenesis of the pancreas, a vital organ with endocrine and exocrine functions.
We are using Drosophila, chicks and mice, organisms accessible to embryological, genetic and molecular methods, to identify cell interactions and signaling pathways that regulate early steps in pancreatic islet development. Some of the pathways active during ontogeny also regulate pancreatic growth during adulthood, and we are studying the role of these genetic pathways in growth control and function of the mature pancreas in mice. Armed with an understanding of the mechanisms regulating normal development of insulin-producing cells and other islet cells, we have been able to differentiate functional glucose-responsive islets from embryonic stem cells and other cell lines. These are capable of rescuing glucose regulation and survival in experimental animal models of diabetes mellitus. We are now using this in vitro culture system to isolate candidate islet stem/precursor populations from adult human stem cell populations. We are also using Drosophila to study neuroendocrine cells that govern metabolism. We have discovered that two cell types, one which produces insulin, the other which produces a glucagon-like peptide called AKH, are crucial regulators of glucose homeostasis in Drosophila. Genetic, biochemical, and electrophysiologic studies are being used to elucidate the programs that control development and function of these cells, which comprise the Drosophila endocrine 'pancreas'. In turn, we expect that these studies will identify important conserved functions that govern islet cell biology.
Dakin's Solution in Preventing Radiation Dermatitis in Patients With Breast Cancer Undergoing Radiation Therapy
This pilot clinical trial studies Dakin's solution in preventing radiation dermatitis in patients with breast cancer undergoing radiation therapy. Radiation dermatitis is a skin condition in which the affected skin becomes painful, red, itchy, and blistered. Dakin's solution may help reduce dermatitis caused by radiation therapy.
Stanford is currently not accepting patients for this trial. For more information, please contact Amanda Simmons, 650-724-4606.
- Elements of Grant Writing
DBIO 234 (Aut)
Independent Studies (10)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Developmental Biology
DBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Neurosciences
NEPR 299 (Aut, Sum)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
DBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
NEPR 399 (Aut, Sum)
- Medical Scholars Research
DBIO 370 (Aut, Win, Spr, Sum)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Sum)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Undergraduate Research
DBIO 199 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
- Prior Year Courses
Reconstituting development of pancreatic intraepithelial neoplasia from primary human pancreas duct cells.
2017; 8: 14686-?
Development of systems that reconstitute hallmark features of human pancreatic intraepithelial neoplasia (PanINs), the precursor to pancreatic ductal adenocarcinoma, could generate new strategies for early diagnosis and intervention. However, human cell-based PanIN models with defined mutations are unavailable. Here, we report that genetic modification of primary human pancreatic cells leads to development of lesions resembling native human PanINs. Primary human pancreas duct cells harbouring oncogenic KRAS and induced mutations in CDKN2A, SMAD4 and TP53 expand in vitro as epithelial spheres. After pancreatic transplantation, mutant clones form lesions histologically similar to native PanINs, including prominent stromal responses. Gene expression profiling reveals molecular similarities of mutant clones with native PanINs, and identifies potential PanIN biomarker candidates including Neuromedin U, a circulating peptide hormone. Prospective reconstitution of human PanIN development from primary cells provides experimental opportunities to investigate pancreas cancer development, progression and early-stage detection.
View details for DOI 10.1038/ncomms14686
View details for PubMedID 28272465
Converting Adult Pancreatic Islet a Cells into ß Cells by Targeting Both Dnmt1 and Arx.
Insulin-producing pancreatic β cells in mice can slowly regenerate from glucagon-producing α cells in settings like β cell loss, but the basis of this conversion is unknown. Moreover, it remains unclear if this intra-islet cell conversion is relevant to diseases like type 1 diabetes (T1D). We show that the α cell regulators Aristaless-related homeobox (Arx) and DNA methyltransferase 1 (Dnmt1) maintain α cell identity in mice. Within 3 months of Dnmt1 and Arx loss, lineage tracing and single-cell RNA sequencing revealed extensive α cell conversion into progeny resembling native β cells. Physiological studies demonstrated that converted α cells acquire hallmark β cell electrophysiology and show glucose-stimulated insulin secretion. In T1D patients, subsets of glucagon-expressing cells show loss of DNMT1 and ARX and produce insulin and other β cell factors, suggesting that DNMT1 and ARX maintain α cell identity in humans. Our work reveals pathways regulated by Arx and Dnmt1 that are sufficient for achieving targeted generation of β cells from adult pancreatic α cells.
View details for DOI 10.1016/j.cmet.2017.01.009
View details for PubMedID 28215845
View details for PubMedCentralID PMC5358097
A Drosophila LexA Enhancer-Trap Resource for Developmental Biology and Neuroendocrine Research.
G3 (Bethesda, Md.)
2016; 6 (10): 3017-3026
Novel binary gene expression tools like the LexA-LexAop system could powerfully enhance studies of metabolism, development, and neurobiology in Drosophila However, specific LexA drivers for neuroendocrine cells and many other developmentally relevant systems remain limited. In a unique high school biology course, we generated a LexA-based enhancer trap collection by transposon mobilization. The initial collection provides a source of novel LexA-based elements that permit targeted gene expression in the corpora cardiaca, cells central for metabolic homeostasis, and other neuroendocrine cell types. The collection further contains specific LexA drivers for stem cells and other enteric cells in the gut, and other developmentally relevant tissue types. We provide detailed analysis of nearly 100 new LexA lines, including molecular mapping of insertions, description of enhancer-driven reporter expression in larval tissues, and adult neuroendocrine cells, comparison with established enhancer trap collections and tissue specific RNAseq. Generation of this open-resource LexA collection facilitates neuroendocrine and developmental biology investigations, and shows how empowering secondary school science can achieve research and educational goals.
View details for DOI 10.1534/g3.116.031229
View details for PubMedID 27527793
Age-Dependent Pancreatic Gene Regulation Reveals Mechanisms Governing Human beta Cell Function
2016; 23 (5): 909-920
Intensive efforts are focused on identifying regulators of human pancreatic islet cell growth and maturation to accelerate development of therapies for diabetes. After birth, islet cell growth and function are dynamically regulated; however, establishing these age-dependent changes in humans has been challenging. Here, we describe a multimodal strategy for isolating pancreatic endocrine and exocrine cells from children and adults to identify age-dependent gene expression and chromatin changes on a genomic scale. These profiles revealed distinct proliferative and functional states of islet α cells or β cells and histone modifications underlying age-dependent gene expression changes. Expression of SIX2 and SIX3, transcription factors without prior known functions in the pancreas and linked to fasting hyperglycemia risk, increased with age specifically in human islet β cells. SIX2 and SIX3 were sufficient to enhance insulin content or secretion in immature β cells. Our work provides a unique resource to study human-specific regulators of islet cell maturation and function.
View details for DOI 10.1016/j.cmet.2016.04.002
View details for PubMedID 27133132
Suppression of insulin production and secretion by a decretin hormone.
2015; 21 (2): 323-333
Decretins, hormones induced by fasting that suppress insulin production and secretion, have been postulated from classical human metabolic studies. From genetic screens, we identified Drosophila Limostatin (Lst), a peptide hormone that suppresses insulin secretion. Lst is induced by nutrient restriction in gut-associated endocrine cells. limostatin deficiency led to hyperinsulinemia, hypoglycemia, and excess adiposity. A conserved 15-residue polypeptide encoded by limostatin suppressed secretion by insulin-producing cells. Targeted knockdown of CG9918, a Drosophila ortholog of Neuromedin U receptors (NMURs), in insulin-producing cells phenocopied limostatin deficiency and attenuated insulin suppression by purified Lst, suggesting CG9918 encodes an Lst receptor. NMUR1 is expressed in islet β cells, and purified NMU suppresses insulin secretion from human islets. A human mutant NMU variant that co-segregates with familial early-onset obesity and hyperinsulinemia fails to suppress insulin secretion. We propose Lst as an index member of an ancient hormone class called decretins, which suppress insulin output.
View details for DOI 10.1016/j.cmet.2015.01.006
View details for PubMedID 25651184
View details for PubMedCentralID PMC4349554
Neonatal beta Cell Development in Mice and Humans Is Regulated by Calcineurin/NFAT
2012; 23 (1): 21-34
Little is known about the mechanisms governing neonatal growth and maturation of organs. Here we demonstrate that calcineurin/Nuclear Factor of Activated T cells (Cn/NFAT) signaling regulates neonatal pancreatic development in mouse and human islets. Inactivation of calcineurin b1 (Cnb1) in mouse islets impaired dense core granule biogenesis, decreased insulin secretion, and reduced cell proliferation and mass, culminating in lethal diabetes. Pancreatic β cells lacking Cnb1 failed to express genes revealed to be direct NFAT targets required for replication, insulin storage, and secretion. In contrast, glucokinase activation stimulated Cn-dependent expression of these genes. Calcineurin inhibitors, such as tacrolimus, used for human immunosuppression, induce diabetes. Tacrolimus exposure reduced Cn/NFAT-dependent expression of factors essential for insulin dense core granule formation and secretion and neonatal β cell proliferation, consistent with our genetic studies. Discovery of conserved pathways regulating β cell maturation and proliferation suggests new strategies for controlling β cell growth or replacement in human islet diseases.
View details for DOI 10.1016/j.devcel.2012.05.014
View details for Web of Science ID 000306583800007
View details for PubMedID 22814600
View details for PubMedCentralID PMC3587727
PDGF signalling controls age-dependent proliferation in pancreatic beta-cells
2011; 478 (7369): 349-?
Determining the signalling pathways that direct tissue expansion is a principal goal of regenerative biology. Vigorous pancreatic β-cell replication in juvenile mice and humans declines with age, and elucidating the basis for this decay may reveal strategies for inducing β-cell expansion, a long-sought goal for diabetes therapy. Here we show that platelet-derived growth factor receptor (Pdgfr) signalling controls age-dependent β-cell proliferation in mouse and human pancreatic islets. With age, declining β-cell Pdgfr levels were accompanied by reductions in β-cell enhancer of zeste homologue 2 (Ezh2) levels and β-cell replication. Conditional inactivation of the Pdgfra gene in β-cells accelerated these changes, preventing mouse neonatal β-cell expansion and adult β-cell regeneration. Targeted human PDGFR-α activation in mouse β-cells stimulated Erk1/2 phosphorylation, leading to Ezh2-dependent expansion of adult β-cells. Adult human islets lack PDGF signalling competence, but exposure of juvenile human islets to PDGF-AA stimulated β-cell proliferation. The discovery of a conserved pathway controlling age-dependent β-cell proliferation indicates new strategies for β-cell expansion.
View details for DOI 10.1038/nature10502
View details for Web of Science ID 000296021100038
View details for PubMedID 21993628
View details for PubMedCentralID PMC3503246
Calcineurin/NFAT signalling regulates pancreatic beta-cell growth and function
2006; 443 (7109): 345-349
The growth and function of organs such as pancreatic islets adapt to meet physiological challenges and maintain metabolic balance, but the mechanisms controlling these facultative responses are unclear. Diabetes in patients treated with calcineurin inhibitors such as cyclosporin A indicates that calcineurin/nuclear factor of activated T-cells (NFAT) signalling might control adaptive islet responses, but the roles of this pathway in beta-cells in vivo are not understood. Here we show that mice with a beta-cell-specific deletion of the calcineurin phosphatase regulatory subunit, calcineurin b1 (Cnb1), develop age-dependent diabetes characterized by decreased beta-cell proliferation and mass, reduced pancreatic insulin content and hypoinsulinaemia. Moreover, beta-cells lacking Cnb1 have a reduced expression of established regulators of beta-cell proliferation. Conditional expression of active NFATc1 in Cnb1-deficient beta-cells rescues these defects and prevents diabetes. In normal adult beta-cells, conditional NFAT activation promotes the expression of cell-cycle regulators and increases beta-cell proliferation and mass, resulting in hyperinsulinaemia. Conditional NFAT activation also induces the expression of genes critical for beta-cell endocrine function, including all six genes mutated in hereditary forms of monogenic type 2 diabetes. Thus, calcineurin/NFAT signalling regulates multiple factors that control growth and hallmark beta-cell functions, revealing unique models for the pathogenesis and therapy of diabetes.
View details for DOI 10.1038/nature05097
View details for PubMedID 16988714
Pathways to clinical CLARITY: volumetric analysis of irregular, soft, and heterogeneous tissues in development and disease.
2017; 7 (1): 5899
Three-dimensional tissue-structural relationships are not well captured by typical thin-section histology, posing challenges for the study of tissue physiology and pathology. Moreover, while recent progress has been made with intact methods for clearing, labeling, and imaging whole organs such as the mature brain, these approaches are generally unsuitable for soft, irregular, and heterogeneous tissues that account for the vast majority of clinical samples and biopsies. Here we develop a biphasic hydrogel methodology, which along with automated analysis, provides for high-throughput quantitative volumetric interrogation of spatially-irregular and friable tissue structures. We validate and apply this approach in the examination of a variety of developing and diseased tissues, with specific focus on the dynamics of normal and pathological pancreatic innervation and development, including in clinical samples. Quantitative advantages of the intact-tissue approach were demonstrated compared to conventional thin-section histology, pointing to broad applications in both research and clinical settings.
View details for PubMedID 28724969
A radial axis defined by semaphorin-to-neuropilin signaling controls pancreatic islet morphogenesis.
Development (Cambridge, England)
2017; 144 (20): 3744–54
The islets of Langerhans are endocrine organs characteristically dispersed throughout the pancreas. During development, endocrine progenitors delaminate, migrate radially and cluster to form islets. Despite the distinctive distribution of islets, spatially localized signals that control islet morphogenesis have not been discovered. Here, we identify a radial signaling axis that instructs developing islet cells to disperse throughout the pancreas. A screen of pancreatic extracellular signals identified factors that stimulated islet cell development. These included semaphorin 3a, a guidance cue in neural development without known functions in the pancreas. In the fetal pancreas, peripheral mesenchymal cells expressed Sema3a, while central nascent islet cells produced the semaphorin receptor neuropilin 2 (Nrp2). Nrp2 mutant islet cells developed in proper numbers, but had defects in migration and were unresponsive to purified Sema3a. Mutant Nrp2 islets aggregated centrally and failed to disperse radially. Thus, Sema3a-Nrp2 signaling along an unrecognized pancreatic developmental axis constitutes a chemoattractant system essential for generating the hallmark morphogenetic properties of pancreatic islets. Unexpectedly, Sema3a- and Nrp2-mediated control of islet morphogenesis is strikingly homologous to mechanisms that regulate radial neuronal migration and cortical lamination in the developing mammalian brain.
View details for PubMedID 28893946
Single-Cell Analysis of Human Pancreas Reveals Transcriptional Signatures of Aging and Somatic Mutation Patterns.
2017; 171 (2): 321–30.e14
As organisms age, cells accumulate genetic and epigenetic errors that eventually lead to impaired organ function or catastrophic transformation such as cancer. Because aging reflects a stochastic process of increasing disorder, cells in an organ will be individually affected in different ways, thus rendering bulk analyses of postmitotic adult cells difficult to interpret. Here, we directly measure the effects of aging in human tissue by performing single-cell transcriptome analysis of 2,544 human pancreas cells from eight donors spanning six decades of life. We find that islet endocrine cells from older donors display increased levels of transcriptional noise and potential fate drift. By determining the mutational history of individual cells, we uncover a novel mutational signature in healthy aging endocrine cells. Our results demonstrate the feasibility of using single-cell RNA sequencing (RNA-seq) data from primary cells to derive insights into genetic and transcriptional processes that operate on aging human tissue.
View details for PubMedID 28965763
Age-dependent human β cell proliferation induced by glucagon-like peptide 1 and calcineurin signaling.
The Journal of clinical investigation
2017; 127 (10): 3835–44
Inadequate pancreatic β cell function underlies type 1 and type 2 diabetes mellitus. Strategies to expand functional cells have focused on discovering and controlling mechanisms that limit the proliferation of human β cells. Here, we developed an engraftment strategy to examine age-associated human islet cell replication competence and reveal mechanisms underlying age-dependent decline of β cell proliferation in human islets. We found that exendin-4 (Ex-4), an agonist of the glucagon-like peptide 1 receptor (GLP-1R), stimulates human β cell proliferation in juvenile but not adult islets. This age-dependent responsiveness does not reflect loss of GLP-1R signaling in adult islets, since Ex-4 treatment stimulated insulin secretion by both juvenile and adult human β cells. We show that the mitogenic effect of Ex-4 requires calcineurin/nuclear factor of activated T cells (NFAT) signaling. In juvenile islets, Ex-4 induced expression of calcineurin/NFAT signaling components as well as target genes for proliferation-promoting factors, including NFATC1, FOXM1, and CCNA1. By contrast, expression of these factors in adult islet β cells was not affected by Ex-4 exposure. These studies reveal age-dependent signaling mechanisms regulating human β cell proliferation, and identify elements that could be adapted for therapeutic expansion of human β cells.
View details for PubMedID 28920919
View details for PubMedCentralID PMC5617654
A p53 Super-tumor Suppressor Reveals a Tumor Suppressive p53-Ptpn14-Yap Axis in Pancreatic Cancer.
2017; 32 (4): 460–73.e6
The p53 transcription factor is a critical barrier to pancreatic cancer progression. To unravel mechanisms of p53-mediated tumor suppression, which have remained elusive, we analyzed pancreatic cancer development in mice expressing p53 transcriptional activation domain (TAD) mutants. Surprisingly, the p5353,54 TAD2 mutant behaves as a "super-tumor suppressor," with an enhanced capacity to both suppress pancreatic cancer and transactivate select p53 target genes, including Ptpn14. Ptpn14 encodes a negative regulator of the Yap oncoprotein and is necessary and sufficient for pancreatic cancer suppression, like p53. We show that p53 deficiency promotes Yap signaling and that PTPN14 and TP53 mutations are mutually exclusive in human cancers. These studies uncover a p53-Ptpn14-Yap pathway that is integral to p53-mediated tumor suppression.
View details for PubMedID 29017057
T cells expressing chimeric antigen receptor promote immune tolerance.
2017; 2 (20)
Cellular therapies based on permanent genetic modification of conventional T cells have emerged as a promising strategy for cancer. However, it remains unknown if modification of T cell subsets, such as Tregs, could be useful in other settings, such as allograft transplantation. Here, we use a modular system based on a chimeric antigen receptor (CAR) that binds covalently modified mAbs to control Treg activation in vivo. Transient expression of this mAb-directed CAR (mAbCAR) in Tregs permitted Treg targeting to specific tissue sites and mitigated allograft responses, such as graft-versus-host disease. mAbCAR Tregs targeted to MHC class I proteins on allografts prolonged islet allograft survival and also prolonged the survival of secondary skin grafts specifically matched to the original islet allograft. Thus, transient genetic modification to produce mAbCAR T cells led to durable immune modulation, suggesting therapeutic targeting strategies for controlling alloreactivity in settings such as organ or tissue transplantation.
View details for PubMedID 29046484
Gestational Diabetes Mellitus From Inactivation of Prolactin Receptor and MafB in Islet ß-Cells.
2016; 65 (8): 2331-2341
β-Cell proliferation and expansion during pregnancy are crucial for maintaining euglycemia in response to increased metabolic demands placed on the mother. Prolactin and placental lactogen signal through the prolactin receptor (PRLR) and contribute to adaptive β-cell responses in pregnancy; however, the in vivo requirement for PRLR signaling specifically in maternal β-cell adaptations remains unknown. We generated a floxed allele of Prlr, allowing conditional loss of PRLR in β-cells. In this study, we show that loss of PRLR signaling in β-cells results in gestational diabetes mellitus (GDM), reduced β-cell proliferation, and failure to expand β-cell mass during pregnancy. Targeted PRLR loss in maternal β-cells in vivo impaired expression of the transcription factor Foxm1, both G1/S and G2/M cyclins, tryptophan hydroxylase 1 (Tph1), and islet serotonin production, for which synthesis requires Tph1. This conditional system also revealed that PRLR signaling is required for the transient gestational expression of the transcription factor MafB within a subset of β-cells during pregnancy. MafB deletion in maternal β-cells also produced GDM, with inadequate β-cell expansion accompanied by failure to induce PRLR-dependent target genes regulating β-cell proliferation. These results unveil molecular roles for PRLR signaling in orchestrating the physiologic expansion of maternal β-cells during pregnancy.
View details for DOI 10.2337/db15-1527
View details for PubMedID 27217483
View details for PubMedCentralID PMC4955982
Using Drosophila to discover mechanisms underlying type 2 diabetes
DISEASE MODELS & MECHANISMS
2016; 9 (4): 365-376
Mechanisms of glucose homeostasis are remarkably well conserved between the fruit flyDrosophila melanogasterand mammals. From the initial characterization of insulin signaling in the fly came the identification of downstream metabolic pathways for nutrient storage and utilization. Defects in these pathways lead to phenotypes that are analogous to diabetic states in mammals. These discoveries have stimulated interest in leveraging the fly to better understand the genetics of type 2 diabetes mellitus in humans. Type 2 diabetes results from insulin insufficiency in the context of ongoing insulin resistance. Although genetic susceptibility is thought to govern the propensity of individuals to develop type 2 diabetes mellitus under appropriate environmental conditions, many of the human genes associated with the disease in genome-wide association studies have not been functionally studied. Recent advances in the phenotyping of metabolic defects have positionedDrosophilaas an excellent model for the functional characterization of large numbers of genes associated with type 2 diabetes mellitus. Here, we examine results from studies modeling metabolic disease in the fruit fly and compare findings to proposed mechanisms for diabetic phenotypes in mammals. We provide a systematic framework for assessing the contribution of gene candidates to insulin-secretion or insulin-resistance pathways relevant to diabetes pathogenesis.
View details for DOI 10.1242/dmm.023887
View details for Web of Science ID 000373491000002
View details for PubMedID 27053133
Research Resource: Genetic Labeling of Human Islet Alpha Cells.
2016; 30 (2): 248-253
The 2 most abundant human pancreatic islet cell types are insulin-producing β-cells and glucagon-producing α-cells. Defined cis-regulatory elements from rodent Insulin genes have permitted genetic labeling of human islet β-cells, enabling lineage tracing and generation of human β-cell lines, but analogous elements for genetically labeling human α-cells with high specificity do not yet exist. To identify genetic elements that specifically direct reporter expression to human α-cells, we investigated noncoding sequences adjacent to the human GLUCAGON and ARX genes, which are expressed in islet α-cells. Elements with high evolutionary conservation were cloned into lentiviral vectors to direct fluorescent reporter expression in primary human islets. Based on the specificity of reporter expression for α- and β-cells, we found that rat glucagon promoter was not specific for human α-cells but that addition of human GLUCAGON untranslated region sequences substantially enhanced specificity of labeling in both cultured and transplanted islets to a degree not previously reported, to our knowledge. Specific transgene expression from these cis-regulatory sequences in human α-cells should enable targeted genetic modification and lineage tracing.
View details for DOI 10.1210/me.2015-1220
View details for PubMedID 26745668
- iPSCs: 10 Years and Counting. Cell 2016; 165 (5): 1041–42
Efficient generation of pancreatic β-like cells from the mouse gallbladder.
Stem cell research
2016; 17 (3): 587–96
Direct reprogramming is a promising approach for the replacement of β cells in diabetes. Reprogramming of cells originating from the endodermal lineage, such as acinar cells in the pancreas, liver cells and gallbladder cells has been of particular interest because of their developmental proximity to β cells. Our previous work showed that mouse gallbladder epithelium can be partially reprogrammed in vitro to generate islet-like cells (rGBC1). Here, the reprogramming protocol was substantially improved, yielding cells (rGBC2) closer to functional β cells than the 1st generation method with higher conversion efficiency and insulin expression. In addition to insulin synthesis and processing, rGBC2 presented many hallmark features of β cells, including insulin secretion in response to high glucose stimulation. Gene expression analysis indicated that rGBC2 clustered closer with β cells and had a metabolic gene expression profile resembling neonatal β cells. When transplanted into immune-deficient animals, rGBC2 were stable for at least 5months and further matured in vivo. Taken together, this approach provides further understanding of endodermal lineage conversion and potential for development of cell replacement therapy for type 1 diabetes patients.
View details for PubMedID 27833043
A cellular, molecular, and pharmacological basis for appendage regeneration in mice.
Genes & development
2015; 29 (20): 2097-2107
Regenerative medicine aims to restore normal tissue architecture and function. However, the basis of tissue regeneration in mammalian solid organs remains undefined. Remarkably, mice lacking p21 fully regenerate injured ears without discernable scarring. Here we show that, in wild-type mice following tissue injury, stromal-derived factor-1 (Sdf1) is up-regulated in the wound epidermis and recruits Cxcr4-expressing leukocytes to the injury site. In p21-deficient mice, Sdf1 up-regulation and the subsequent recruitment of Cxcr4-expressing leukocytes are significantly diminished, thereby permitting scarless appendage regeneration. Lineage tracing demonstrates that this regeneration derives from fate-restricted progenitor cells. Pharmacological or genetic disruption of Sdf1-Cxcr4 signaling enhances tissue repair, including full reconstitution of tissue architecture and all cell types. Our findings identify signaling and cellular mechanisms underlying appendage regeneration in mice and suggest new therapeutic approaches for regenerative medicine.
View details for DOI 10.1101/gad.267724.115
View details for PubMedID 26494786
Dissecting Human Gene Functions Regulating Islet Development With Targeted Gene Transduction
2015; 64 (8): 3037-3049
During pancreas development, endocrine precursors and their progeny differentiate, migrate, and cluster to form nascent islets. The transcription factor Neurogenin 3 (Neurog3) is required for islet development in mice, but its role in these dynamic morphogenetic steps has been inferred from fixed tissues. Moreover, little is known about the molecular genetic functions of NEUROG3 in human islet development. We developed methods for gene transduction by viral microinjection in the epithelium of cultured Neurog3-null mutant fetal pancreas, permitting genetic complementation in a developmentally relevant context. In addition, we developed methods for quantitative assessment of live-cell phenotypes in single developing islet cells. Delivery of wild-type NEUROG3 rescued islet differentiation, morphogenesis, and live cell deformation, whereas the patient-derived NEUROG3(R107S) allele partially restored indicators of islet development. NEUROG3(P39X), a previously unreported patient allele, failed to restore islet differentiation or morphogenesis and was indistinguishable from negative controls, suggesting that it is a null mutation. Our systems also permitted genetic suppression analysis and revealed that targets of NEUROG3, including NEUROD1 and RFX6, can partially restore islet development in Neurog3-null mutant mouse pancreata. Thus, advances described here permitted unprecedented assessment of gene functions in regulating crucial dynamic aspects of islet development in the fetal pancreas.
View details for DOI 10.2337/db15-0042
View details for Web of Science ID 000358671300041
View details for PubMedCentralID PMC4512220
Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing
GENES & DEVELOPMENT
2015; 29 (14): 1576-1585
Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.
View details for DOI 10.1101/gad.264861.115
View details for Web of Science ID 000358596300010
View details for PubMedCentralID PMC4526740
Novel GATA6 mutations in patients with pancreatic agenesis and congenital heart malformations.
2015; 10 (2)
Patients with pancreatic agenesis are born without a pancreas, causing permanent neonatal diabetes and pancreatic enzyme insufficiency. These patients require insulin and enzyme replacement therapy to survive, grow, and maintain normal blood glucose levels. Pancreatic agenesis is an uncommon condition but high-throughput sequencing methods provide a rare opportunity to identify critical genes that are necessary for human pancreas development. Here we present the clinical history, evaluation, and the genetic and molecular analysis from two patients with pancreatic agenesis. Both patients were born with intrauterine growth restriction, minor heart defects and neonatal diabetes. In both cases, pancreatic agenesis was confirmed by imaging studies. The patients are clinically stable with pancreatic enzymes and insulin therapy. In order identify the etiology for their disease, we performed whole exome sequencing on both patients. For each proband we identified a de novo heterozygous mutation in the GATA6 gene. GATA6 is a homeobox containing transcription factor involved in both early development of the pancreas and heart. In vitro functional analysis of one of the variants revealed that the mutation creates a premature stop codon in the coding sequence resulting in the production of a truncated protein with loss of activity. These results show how genetic mutations in GATA6 may lead to functional inactivity and pancreatic agenesis in humans.
View details for DOI 10.1371/journal.pone.0118449
View details for PubMedID 25706805
Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa.
Science translational medicine
2014; 6 (264): 264ra163-?
Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen owing to mutations in the gene COL7A1 and suffer severe blistering and chronic wounds that ultimately lead to infection and development of lethal squamous cell carcinoma. The discovery of induced pluripotent stem cells (iPSCs) and the ability to edit the genome bring the possibility to provide definitive genetic therapy through corrected autologous tissues. We generated patient-derived COL7A1-corrected epithelial keratinocyte sheets for autologous grafting. We demonstrate the utility of sequential reprogramming and adenovirus-associated viral genome editing to generate corrected iPSC banks. iPSC-derived keratinocytes were produced with minimal heterogeneity, and these cells secreted wild-type type VII collagen, resulting in stratified epidermis in vitro in organotypic cultures and in vivo in mice. Sequencing of corrected cell lines before tissue formation revealed heterogeneity of cancer-predisposing mutations, allowing us to select COL7A1-corrected banks with minimal mutational burden for downstream epidermis production. Our results provide a clinical platform to use iPSCs in the treatment of debilitating genodermatoses, such as RDEB.
View details for DOI 10.1126/scitranslmed.3009540
View details for PubMedID 25429056
An integrated cell purification and genomics strategy reveals multiple regulators of pancreas development.
2014; 10 (10)
The regulatory logic underlying global transcriptional programs controlling development of visceral organs like the pancreas remains undiscovered. Here, we profiled gene expression in 12 purified populations of fetal and adult pancreatic epithelial cells representing crucial progenitor cell subsets, and their endocrine or exocrine progeny. Using probabilistic models to decode the general programs organizing gene expression, we identified co-expressed gene sets in cell subsets that revealed patterns and processes governing progenitor cell development, lineage specification, and endocrine cell maturation. Purification of Neurog3 mutant cells and module network analysis linked established regulators such as Neurog3 to unrecognized gene targets and roles in pancreas development. Iterative module network analysis nominated and prioritized transcriptional regulators, including diabetes risk genes. Functional validation of a subset of candidate regulators with corresponding mutant mice revealed that the transcription factors Etv1, Prdm16, Runx1t1 and Bcl11a are essential for pancreas development. Our integrated approach provides a unique framework for identifying regulatory genes and functional gene sets underlying pancreas development and associated diseases such as diabetes mellitus.
View details for DOI 10.1371/journal.pgen.1004645
View details for PubMedID 25330008
A genetic strategy to measure circulating Drosophila insulin reveals genes regulating insulin production and secretion.
2014; 10 (8)
Insulin is a major regulator of metabolism in metazoans, including the fruit fly Drosophila melanogaster. Genome-wide association studies (GWAS) suggest a genetic basis for reductions of both insulin sensitivity and insulin secretion, phenotypes commonly observed in humans with type 2 diabetes mellitus (T2DM). To identify molecular functions of genes linked to T2DM risk, we developed a genetic tool to measure insulin-like peptide 2 (Ilp2) levels in Drosophila, a model organism with superb experimental genetics. Our system permitted sensitive quantification of circulating Ilp2, including measures of Ilp2 dynamics during fasting and re-feeding, and demonstration of adaptive Ilp2 secretion in response to insulin receptor haploinsufficiency. Tissue specific dissection of this reduced insulin signaling phenotype revealed a critical role for insulin signaling in specific peripheral tissues. Knockdown of the Drosophila orthologues of human T2DM risk genes, including GLIS3 and BCL11A, revealed roles of these Drosophila genes in Ilp2 production or secretion. Discovery of Drosophila mechanisms and regulators controlling in vivo insulin dynamics should accelerate functional dissection of diabetes genetics.
View details for DOI 10.1371/journal.pgen.1004555
View details for PubMedID 25101872
Insight into insulin secretion from transcriptome and genetic analysis of insulin-producing cells of Drosophila.
2014; 197 (1): 175-192
Insulin-producing cells (IPCs) in the Drosophila brain produce and release insulin-like peptides (ILPs) to the hemolymph. ILPs are crucial for growth and regulation of metabolic activity in flies, functions analogous to those of mammalian insulin and insulin-like growth factors (IGFs). To identify components functioning in IPCs to control ILP production, we employed genomic and candidate gene approaches. We used laser microdissection and messenger RNA sequencing to characterize the transcriptome of larval IPCs. IPCs highly express many genes homologous to genes active in insulin-producing β-cells of the mammalian pancreas. The genes in common encode ILPs and proteins that control insulin metabolism, storage, secretion, β-cell proliferation, and some not previously linked to insulin production or β-cell function. Among these novelties is unc-104, a kinesin 3 family gene, which is more highly expressed in IPCs compared to most other neurons. Knockdown of unc-104 in IPCs impaired ILP secretion and reduced peripheral insulin signaling. Unc-104 appears to transport ILPs along axons. As a complementary approach, we tested dominant-negative Rab genes to find Rab proteins required in IPCs for ILP production or secretion. Rab1 was identified as crucial for ILP trafficking in IPCs. Inhibition of Rab1 in IPCs increased circulating sugar levels, delayed development, and lowered weight and body size. Immunofluorescence labeling of Rab1 showed its tight association with ILP2 in the Golgi of IPCs. Unc-104 and Rab1 join other proteins required for ILP transport in IPCs.
View details for DOI 10.1534/genetics.113.160663
View details for PubMedID 24558258
View details for PubMedCentralID PMC4012477
Dicer Regulates Differentiation and Viability during Mouse Pancreatic Cancer Initiation
2014; 9 (5)
miRNA levels are altered in pancreatic ductal adenocarcinoma (PDA), the most common and lethal pancreatic malignancy, and intact miRNA processing is essential for lineage specification during pancreatic development. However, the role of miRNA processing in PDA has not been explored. Here we study the role of miRNA biogenesis in PDA development by deleting the miRNA processing enzyme Dicer in a PDA mouse model driven by oncogenic Kras. We find that loss of Dicer accelerates Kras driven acinar dedifferentiation and acinar to ductal metaplasia (ADM), a process that has been shown to precede and promote the specification of PDA precursors. However, unconstrained ADM also displays high levels of apoptosis. Dicer loss does not accelerate development of Kras driven PDA precursors or PDA, but surprisingly, we observe that mouse PDA can develop without Dicer, although at the expense of proliferative capacity. Our data suggest that intact miRNA processing is involved in both constraining pro-tumorigenic changes in pancreatic differentiation as well as maintaining viability during PDA initiation.
View details for DOI 10.1371/journal.pone.0095486
View details for PubMedID 24788257
Topical hypochlorite ameliorates NF-kappa B-mediated skin diseases in mice
JOURNAL OF CLINICAL INVESTIGATION
2013; 123 (12): 5361-5370
Nuclear factor-κB (NF-κB) regulates cellular responses to inflammation and aging, and alterations in NF-κB signaling underlie the pathogenesis of multiple human diseases. Effective clinical therapeutics targeting this pathway remain unavailable. In primary human keratinocytes, we found that hypochlorite (HOCl) reversibly inhibited the expression of CCL2 and SOD2, two NF-κB-dependent genes. In cultured cells, HOCl inhibited the activity of inhibitor of NF-κB kinase (IKK), a key regulator of NF-κB activation, by oxidizing cysteine residues Cys114 and Cys115. In NF-κB reporter mice, topical HOCl reduced LPS-induced NF-κB signaling in skin. We further evaluated topical HOCl use in two mouse models of NF-κB-driven epidermal disease. For mice with acute radiation dermatitis, topical HOCl inhibited the expression of NF-κB-dependent genes, decreased disease severity, and prevented skin ulceration. In aged mice, topical HOCl attenuated age-dependent production of p16INK4a and expression of the DNA repair gene Rad50. Additionally, skin of aged HOCl-treated mice acquired enhanced epidermal thickness and proliferation, comparable to skin in juvenile animals. These data suggest that topical HOCl reduces NF-κB-mediated epidermal pathology in radiation dermatitis and skin aging through IKK modulation and motivate the exploration of HOCl use for clinical aims.
View details for DOI 10.1172/JCI70895
View details for Web of Science ID 000327826100039
View details for PubMedID 24231355
View details for PubMedCentralID PMC3859383
Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells
Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001.
View details for DOI 10.7554/eLife.00940
View details for Web of Science ID 000328641800001
View details for PubMedID 24252877
View details for PubMedCentralID PMC3826580
Reconstituting pancreas development from purified progenitor cells reveals genes essential for islet differentiation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (31): 12691-12696
Developmental biology is challenged to reveal the function of numerous candidate genes implicated by recent genome-scale studies as regulators of organ development and diseases. Recapitulating organogenesis from purified progenitor cells that can be genetically manipulated would provide powerful opportunities to dissect such gene functions. Here we describe systems for reconstructing pancreas development, including islet β-cell and α-cell differentiation, from single fetal progenitor cells. A strict requirement for native genetic regulators of in vivo pancreas development, such as Ngn3, Arx, and Pax4, revealed the authenticity of differentiation programs in vitro. Efficient genetic screens permitted by this system revealed that Prdm16 is required for pancreatic islet development in vivo. Discovering the function of genes regulating pancreas development with our system should enrich strategies for regenerating islets for treating diabetes mellitus.
View details for DOI 10.1073/pnas.1304507110
View details for PubMedID 23852729
Gene regulatory networks governing pancreas development.
2013; 25 (1): 5-13
Elucidation of cellular and gene regulatory networks (GRNs) governing organ development will accelerate progress toward tissue replacement. Here, we have compiled reference GRNs underlying pancreas development from data mining that integrates multiple approaches, including mutant analysis, lineage tracing, cell purification, gene expression and enhancer analysis, and biochemical studies of gene regulation. Using established computational tools, we integrated and represented these networks in frameworks that should enhance understanding of the surging output of genomic-scale genetic and epigenetic studies of pancreas development and diseases such as diabetes and pancreatic cancer. We envision similar approaches would be useful for understanding the development of other organs.
View details for DOI 10.1016/j.devcel.2013.03.016
View details for PubMedID 23597482
View details for PubMedCentralID PMC3645877
Combined modulation of polycomb and trithorax genes rejuvenates β cell replication.
The Journal of clinical investigation
2013; 123 (11): 4849–58
Inadequate functional β cell mass underlies both type 1 and type 2 diabetes. β Cell growth and regeneration also decrease with age through mechanisms that are not fully understood. Age-dependent loss of enhancer of zeste homolog 2 (EZH2) prevents adult β cell replication through derepression of the gene encoding cyclin-dependent kinase inhibitor 2a (INK4a). We investigated whether replenishing EZH2 could reverse the age-dependent increase of Ink4a transcription. We generated an inducible pancreatic β cell-specific Ezh2 transgenic mouse model and showed that transgene expression of Ezh2 was sufficient to increase β cell replication and regeneration in young adult mice. In mice older than 8 months, induction of Ezh2 was unable to repress Ink4a. Older mice had an enrichment of a trithorax group (TrxG) protein complex at the Ink4a locus. Knockdown of TrxG complex components, in conjunction with expression of Ezh2, resulted in Ink4a repression and increased replication of β cells in aged mice. These results indicate that combined modulation of polycomb group proteins, such as EZH2, along with TrxG proteins to repress Ink4a can rejuvenate the replication capacity of aged β cells. This study provides potential therapeutic targets for expansion of adult β cell mass.
View details for DOI 10.1172/JCI69468
View details for PubMedID 24216481
A Molecular Signature for Purified Definitive Endoderm Guides Differentiation and Isolation of Endoderm from Mouse and Human Embryonic Stem Cells
STEM CELLS AND DEVELOPMENT
2012; 21 (12): 2273-2287
Embryonic definitive endoderm (DE) generates the epithelial compartment of vital organs such as liver, pancreas, and intestine. However, purification of DE in mammals has not been achieved, limiting the molecular "definition" of endoderm, and hindering our understanding of DE development and attempts to produce endoderm from sources such as embryonic stem (ES) cells. Here, we describe purification of mouse DE using fluorescence-activated cell sorting (FACS) and mice harboring a transgene encoding enhanced green fluorescent protein (eGFP) inserted into the Sox17 locus, which is expressed in the embryonic endoderm. Comparison of patterns of signaling pathway activation in native mouse DE and endoderm-like cells generated from ES cells produced novel culture modifications that generated Sox17-eGFP⁺ progeny whose gene expression resembled DE more closely than achieved with standard methods. These studies also produced new FACS methods for purifying DE from nontransgenic mice and mouse ES cell cultures. Parallel studies of a new human SOX17-eGFP ES cell line allowed analysis of endoderm differentiation in vitro, leading to culture modifications that enhanced expression of an endoderm-like signature. This work should accelerate our understanding of mechanisms regulating DE development in mice and humans, and guide further use of ES cells for tissue replacement.
View details for DOI 10.1089/scd.2011.0416
View details for PubMedID 22236333
Deconstructing Pancreas Developmental Biology
COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY
2012; 4 (6)
The relentless nature and increasing prevalence of human pancreatic diseases, in particular, diabetes mellitus and adenocarcinoma, has motivated further understanding of pancreas organogenesis. The pancreas is a multifunctional organ whose epithelial cells govern a diversity of physiologically vital endocrine and exocrine functions. The mechanisms governing the birth, differentiation, morphogenesis, growth, maturation, and maintenance of the endocrine and exocrine components in the pancreas have been discovered recently with increasing tempo. This includes recent studies unveiling mechanisms permitting unexpected flexibility in the developmental potential of immature and mature pancreatic cell subsets, including the ability to interconvert fates. In this article, we describe how classical cell biology, genetic analysis, lineage tracing, and embryological investigations are being complemented by powerful modern methods including epigenetic analysis, time-lapse imaging, and flow cytometry-based cell purification to dissect fundamental processes of pancreas development.
View details for DOI 10.1101/cshperspect.a012401
View details for Web of Science ID 000308028500015
View details for PubMedID 22587935
View details for PubMedCentralID PMC3367550
Gut insulin from Foxo1 loss
2012; 44 (4): 363-364
Neuroendocrine cells, including those in the gut, have a vast array of functions. A new study shows that conditional inactivation of the gene encoding Foxo1 in mouse intestinal endocrine cells converts them into cells synthesizing and secreting insulin. Ectopic gut insulin production was sufficient to ameliorate glucose control in mice with conditional pancreatic β-cell loss and diabetes mellitus.
View details for DOI 10.1038/ng.2226
View details for Web of Science ID 000302130600005
View details for PubMedID 22456735
Specification of Drosophila Corpora Cardiaca Neuroendocrine Cells from Mesoderm Is Regulated by Notch Signaling
2011; 7 (8)
Drosophila neuroendocrine cells comprising the corpora cardiaca (CC) are essential for systemic glucose regulation and represent functional orthologues of vertebrate pancreatic α-cells. Although Drosophila CC cells have been regarded as developmental orthologues of pituitary gland, the genetic regulation of CC development is poorly understood. From a genetic screen, we identified multiple novel regulators of CC development, including Notch signaling factors. Our studies demonstrate that the disruption of Notch signaling can lead to the expansion of CC cells. Live imaging demonstrates localized emergence of extra precursor cells as the basis of CC expansion in Notch mutants. Contrary to a recent report, we unexpectedly found that CC cells originate from head mesoderm. We show that Tinman expression in head mesoderm is regulated by Notch signaling and that the combination of Daughterless and Tinman is sufficient for ectopic CC specification in mesoderm. Understanding the cellular, genetic, signaling, and transcriptional basis of CC cell specification and expansion should accelerate discovery of molecular mechanisms regulating ontogeny of organs that control metabolism.
View details for DOI 10.1371/journal.pgen.1002241
View details for Web of Science ID 000294297000036
View details for PubMedID 21901108
View details for PubMedCentralID PMC3161926
Extensive and coordinated transcription of noncoding RNAs within cell-cycle promoters
2011; 43 (7): 621-U196
Transcription of long noncoding RNAs (lncRNAs) within gene regulatory elements can modulate gene activity in response to external stimuli, but the scope and functions of such activity are not known. Here we use an ultrahigh-density array that tiles the promoters of 56 cell-cycle genes to interrogate 108 samples representing diverse perturbations. We identify 216 transcribed regions that encode putative lncRNAs, many with RT-PCR-validated periodic expression during the cell cycle, show altered expression in human cancers and are regulated in expression by specific oncogenic stimuli, stem cell differentiation or DNA damage. DNA damage induces five lncRNAs from the CDKN1A promoter, and one such lncRNA, named PANDA, is induced in a p53-dependent manner. PANDA interacts with the transcription factor NF-YA to limit expression of pro-apoptotic genes; PANDA depletion markedly sensitized human fibroblasts to apoptosis by doxorubicin. These findings suggest potentially widespread roles for promoter lncRNAs in cell-growth control.
View details for DOI 10.1038/ng.848
View details for PubMedID 21642992
Targeting SOX17 in Human Embryonic Stem Cells Creates Unique Strategies for Isolating and Analyzing Developing Endoderm
CELL STEM CELL
2011; 8 (3): 335-346
Human embryonic stem cells (hESCs) can provide insights into development of inaccessible human tissues such as embryonic endoderm. Progress in this area has been hindered by a lack of methods for isolating endodermal cells and tracing fates of their differentiated progeny. By using homologous recombination in human ESCs, we inserted an enhanced green fluorescent protein (eGFP) transgene into the SOX17 locus, a postulated marker of human endoderm. FACS purification and gene expression profiling confirmed that SOX17(+)-hESC progeny expressed endodermal markers and unveiled specific cell surface protein combinations that permitted FACS-based isolation of primitive gut tube endodermal cells produced from unmodified human ESCs and from induced pluripotent stem cells (iPSC). Differentiating SOX17(+) endodermal cells expressed markers of liver, pancreas, and intestinal epithelium in vitro and gave rise to endodermal progeny in vivo. Thus, prospective isolation, lineage tracing, and developmental studies of SOX17(+) hESC progeny have revealed fundamental aspects of human endodermal biology.
View details for DOI 10.1016/j.stem.2011.01.017
View details for Web of Science ID 000288404400015
View details for PubMedID 21362573
View details for PubMedCentralID PMC3063711
Deconstructing Pancreas Development to Reconstruct Human Islets from Pluripotent Stem Cells
CELL STEM CELL
2010; 6 (4): 300-308
There is considerable excitement about harnessing the potential of human stem cells to replace pancreatic islets that are destroyed in type 1 diabetes mellitus. However, our current understanding of the mechanisms underlying pancreas and islet ontogeny has come largely from the powerful genetic, developmental, and embryological approaches available in nonhuman organisms. Successful islet reconstruction from human pluripotent cells will require greater attention to "deconstructing" human pancreas and islet developmental biology and consistent application of conditional genetics, lineage tracing, and cell purification to stem cell biology.
View details for DOI 10.1016/j.stem.2010.03.003
View details for Web of Science ID 000276823300009
View details for PubMedID 20362535
View details for PubMedCentralID PMC3148083
Polycomb protein Ezh2 regulates pancreatic beta-cell Ink4a/Arf expression and regeneration in diabetes mellitus
GENES & DEVELOPMENT
2009; 23 (8): 975-985
Proliferation of pancreatic islet beta cells is an important mechanism for self-renewal and for adaptive islet expansion. Increased expression of the Ink4a/Arf locus, which encodes the cyclin-dependent kinase inhibitor p16(INK4a) and tumor suppressor p19(Arf), limits beta-cell regeneration in aging mice, but the basis of beta-cell Ink4a/Arf regulation is poorly understood. Here we show that Enhancer of zeste homolog 2 (Ezh2), a histone methyltransferase and component of a Polycomb group (PcG) protein complex, represses Ink4a/Arf in islet beta cells. Ezh2 levels decline in aging islet beta cells, and this attrition coincides with reduced histone H3 trimethylation at Ink4a/Arf, and increased levels of p16(INK4a) and p19(Arf). Conditional deletion of beta-cell Ezh2 in juvenile mice also reduced H3 trimethylation at the Ink4a/Arf locus, leading to precocious increases of p16(INK4a) and p19(Arf). These mutant mice had reduced beta-cell proliferation and mass, hypoinsulinemia, and mild diabetes, phenotypes rescued by germline deletion of Ink4a/Arf. beta-Cell destruction with streptozotocin in controls led to increased Ezh2 expression that accompanied adaptive beta-cell proliferation and re-establishment of beta-cell mass; in contrast, mutant mice treated similarly failed to regenerate beta cells, resulting in lethal diabetes. Our discovery of Ezh2-dependent beta-cell proliferation revealed unique epigenetic mechanisms underlying normal beta-cell expansion and beta-cell regenerative failure in diabetes pathogenesis.
View details for DOI 10.1101/gad.1742509
View details for Web of Science ID 000265449900010
View details for PubMedID 19390090
View details for PubMedCentralID PMC2675862
Fluorescence-activated cell sorting purification of pancreatic progenitor cells
DIABETES OBESITY & METABOLISM
2008; 10: 179-185
Here we review progress on isolation and characterization of progenitor cells in the pancreas. We discuss advantages and current limitations of experiments with purified pancreatic cells, and areas where future growth in our understanding is needed to advance experiments in pancreas biology based on cell purification.
View details for DOI 10.1111/j.1463-13262008.00954.x
View details for Web of Science ID 000262726300019
View details for PubMedID 18834445
Characterization of six new human embryonic stem cell lines (HSF7, -8, -9, -10, -12, and -13) derived under minimal-animal component conditions
STEM CELLS AND DEVELOPMENT
2008; 17 (3): 535-546
Human embryonic stem cells (hESCs) provide a renewable source of a variety of cell types with the potential for use in both scientific research and clinical cell-based therapy. Several hESC lines have previously been isolated and characterized, however, the majority of these lines were generated in the presence of animal serum and animal-derived feeder cells. Therefore, the exposure of the hESC to animal products may have induced phenotypic and/or genomic changes in the hESC lines not characteristic of normal hESC. Moreover, those hESC lines exposed to animal components may not be used for therapeutic applications due to the risk of graft rejection and pathogenic transmission from animal sources. In this study, we characterized six new hESC lines derived from human blastocysts under minimal-animal component conditions and cultured with human fetal lung fibroblasts. The hESC lines retained the ability to self-renew, are karytopically normal, and express stage-specific embryonic antigen-3 (SSEA-3), SSEA-4, TRA-1-60, and TRA-1-81, but not SSEA-1, markers of pluripotent hESC. In addition, we show that telomerase activity decreased in each of the hESC lines following differentiation into embryoid bodies, albeit to different degrees. Finally, we demonstrate that the hESC lines are capable of differentiating into the three embryonic germ layers in vitro and form complex teratomas in vivo. This suggests that the hESC lines described here are valuable models for both future in vitro and in vivo studies, which may aid in the progression toward clinical-grade cell therapy.
View details for DOI 10.1089/scd.2007.0216
View details for Web of Science ID 000257115600014
View details for PubMedID 18513167
Menin controls growth of pancreatic beta-cells in pregnant mice and promotes gestational diabetes mellitus
2007; 318 (5851): 806-809
During pregnancy, maternal pancreatic islets grow to match dynamic physiological demands, but the mechanisms regulating adaptive islet growth in this setting are poorly understood. Here we show that menin, a protein previously characterized as an endocrine tumor suppressor and transcriptional regulator, controls islet growth in pregnant mice. Pregnancy stimulated proliferation of maternal pancreatic islet beta-cells that was accompanied by reduced islet levels of menin and its targets. Transgenic expression of menin in maternal beta-cells prevented islet expansion and led to hyperglycemia and impaired glucose tolerance, hallmark features of gestational diabetes. Prolactin, a hormonal regulator of pregnancy, repressed islet menin levels and stimulated beta-cell proliferation. These results expand our understanding of mechanisms underlying diabetes pathogenesis and reveal potential targets for therapy in diabetes.
View details for DOI 10.1126/science.1146812
View details for PubMedID 17975067
Menin-mediated caspase 8 expression in suppressing multiple endocrine neoplasia type 1
JOURNAL OF BIOLOGICAL CHEMISTRY
2007; 282 (43): 31332-31340
Multiple endocrine neoplasia type 1 (MEN1) is a familial tumor syndrome linked to mutation of the MEN1 gene, which encodes a tumor suppressor, menin. We previously reported that menin up-regulates the caspase 8 expression and promotes TNF-alpha-induced apoptosis. However, it remains unclear how menin up-regulates caspase 8 expression and whether menin-mediated caspase 8 expression plays a role in repressing MEN1 development. Here we show that menin binds the 5'-untranslated region (5'-UTR) of the Caspase 8 locus in vivo and activates transcription of a reporter gene through the 5'-UTR. Menin directly binds the 5'-UTR in a sequence-independent manner in vitro. Moreover, Men1 ablation in cells reduces acetylation of histones H3 and H4 at the 5'-UTR of the caspase 8 locus bound by menin in vivo. Notably, the MEN1-derived menin point mutants lose their ability to bind the caspase 8 locus and fail to induce caspase 8 expression and TNF-alpha-mediated apoptosis. Consistent with these observations, the expression level of caspase 8 is markedly reduced in insulinomas from Men1(+/-) mice. Together, our results indicate that menin enhances the caspase 8 expression by binding the caspase 8 locus, and suggest that menin suppresses MEN1 tumorigenesis, at least in part, by up-regulating caspase 8 expression.
View details for DOI 10.1074/jbc.M609555200
View details for Web of Science ID 000250309200022
View details for PubMedID 17766243
Glucose infusion in mice - A new model to induce beta-cell replication
2007; 56 (7): 1792-1801
Developing new techniques to induce beta-cells to replicate is a major goal in diabetes research. Endogenous beta-cells replicate in response to metabolic changes, such as obesity and pregnancy, which increase insulin requirement. Mouse genetic models promise to reveal the pathways responsible for compensatory beta-cell replication. However, no simple, short-term, physiological replication stimulus exists to test mouse models for compensatory replication. Here, we present a new tool to induce beta-cell replication in living mice. Four-day glucose infusion is well tolerated by mice as measured by hemodynamics, body weight, organ weight, food intake, and corticosterone level. Mild sustained hyperglycemia and hyperinsulinemia induce a robust and significant fivefold increase in beta-cell replication. Glucose-induced beta-cell replication is dose and time dependent. Beta-cell mass, islet number, beta-cell size, and beta-cell death are not altered by glucose infusion over this time frame. Glucose infusion increases both the total protein abundance and nuclear localization of cyclin D2 in islets, which has not been previously reported. Thus, we have developed a new model to study the regulation of compensatory beta-cell replication, and we describe important novel characteristics of mouse beta-cell responses to glucose in the living pancreas.
View details for DOI 10.2337/db06-1513
View details for Web of Science ID 000247768000005
View details for PubMedID 17400928
Wnt signaling regulates pancreatic beta cell proliferation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (15): 6247-6252
There is widespread interest in defining factors and mechanisms that stimulate proliferation of pancreatic islet cells. Wnt signaling is an important regulator of organ growth and cell fates, and genes encoding Wnt-signaling factors are expressed in the pancreas. However, it is unclear whether Wnt signaling regulates pancreatic islet proliferation and differentiation. Here we provide evidence that Wnt signaling stimulates islet beta cell proliferation. The addition of purified Wnt3a protein to cultured beta cells or islets promoted expression of Pitx2, a direct target of Wnt signaling, and Cyclin D2, an essential regulator of beta cell cycle progression, and led to increased beta cell proliferation in vitro. Conditional pancreatic beta cell expression of activated beta-catenin, a crucial Wnt signal transduction protein, produced similar phenotypes in vivo, leading to beta cell expansion, increased insulin production and serum levels, and enhanced glucose handling. Conditional beta cell expression of Axin, a potent negative regulator of Wnt signaling, led to reduced Pitx2 and Cyclin D2 expression by beta cells, resulting in reduced neonatal beta cell expansion and mass and impaired glucose tolerance. Thus, Wnt signaling is both necessary and sufficient for islet beta cell proliferation, and our study provides previously unrecognized evidence of a mechanism governing endocrine pancreas growth and function.
View details for DOI 10.1073/pnas.0701509104
View details for Web of Science ID 000245737500029
View details for PubMedID 17404238
View details for PubMedCentralID PMC1847455
Conserved markers of fetal pancreatic epithelium permit prospective isolation of islet progenitor cells by FACS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (1): 175-180
Prospective isolation and characterization of progenitor cells is a paradigmatic strategy for studies of organ development. However, extraction of viable cells, fractionation of lineages, and in vitro analysis of progenitors from the fetal pancreas in experimental organisms like mice has proved challenging and has not yet been reported for human fetal pancreas. Here, we report isolation of pancreatic islet progenitor cells from fetal mice by FACS. Monoclonal antibodies that recognize cell-surface proteins on candidate stem cells in brain, skin, and other organs enabled separation of major pancreatic cell lineages and isolation of native pancreatic cells expressing neurogenin 3, an established marker of islet progenitors. New in vitro cell culture methods permitted isolated mouse islet progenitors to develop into hormone-expressing endocrine cells. Insulin-producing cells derived in vitro required or expressed factors that regulate fetal beta cell differentiation; thus, the genetic programs normally controlling in vivo mouse islet development are similarly required in our system. Moreover, antibodies that recognize conserved orthologous cell-surface epitopes in human fetal pancreas allowed FACS-based enrichment of candidate islet progenitor cells expressing neurogenin 3. Our studies reveal previously undescribed strategies for prospective purification and analysis of pancreatic endocrine progenitor cells that should accelerate studies of islet development and replacement.
View details for DOI 10.1073/pnas.0609490104
View details for Web of Science ID 000243456300033
View details for PubMedID 17190805
View details for PubMedCentralID PMC1765430
The ATP-sensitive potassium (K-ATP) channel-encoded dSUR gene is required for Drosophila heart function and is regulated by tinman
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2006; 103 (32): 11999-12004
The homeobox transcription factor Tinman plays an important role in the initiation of heart development. Later functions of Tinman, including the target genes involved in cardiac physiology, are less well studied. We focused on the dSUR gene, which encodes an ATP-binding cassette transmembrane protein that is expressed in the heart. Mammalian SUR genes are associated with K(ATP) (ATP-sensitive potassium) channels, which are involved in metabolic homeostasis. We provide experimental evidence that Tinman directly regulates dSUR expression in the developing heart. We identified a cis-regulatory element in the first intron of dSUR, which contains Tinman consensus binding sites and is sufficient for faithful dSUR expression in the fly's myocardium. Site-directed mutagenesis of this element shows that these Tinman sites are critical to dSUR expression, and further genetic manipulations suggest that the GATA transcription factor Pannier is synergistically involved in cardiac-restricted dSUR expression in vivo. Physiological analysis of dSUR knock-down flies supports the idea that dSUR plays a protective role against hypoxic stress and pacing-induced heart failure. Because dSUR expression dramatically decreases with age, it is likely to be a factor involved in the cardiac aging phenotype of Drosophila. dSUR provides a model for addressing how embryonic regulators of myocardial cell commitment can contribute to the establishment and maintenance of cardiac performance.
View details for DOI 10.1073/pnas.0603098103
View details for Web of Science ID 000239701900033
View details for PubMedID 16882722
NFAT dysregulation by increased dosage of DSCR1 and DYRK1A on chromosome 21
2006; 441 (7093): 595-600
Trisomy 21 results in Down's syndrome, but little is known about how a 1.5-fold increase in gene dosage produces the pleiotropic phenotypes of Down's syndrome. Here we report that two genes, DSCR1 and DYRK1A , lie within the critical region of human chromosome 21 and act synergistically to prevent nuclear occupancy of NFATc transcription factors, which are regulators of vertebrate development. We use mathematical modelling to predict that autoregulation within the pathway accentuates the effects of trisomy of DSCR1 and DYRK1A, leading to failure to activate NFATc target genes under specific conditions. Our observations of calcineurin-and Nfatc-deficient mice, Dscr1- and Dyrk1a-overexpressing mice, mouse models of Down's syndrome and human trisomy 21 are consistent with these predictions. We suggest that the 1.5-fold increase in dosage of DSCR1 and DYRK1A cooperatively destabilizes a regulatory circuit, leading to reduced NFATc activity and many of the features of Down's syndrome. More generally, these observations suggest that the destabilization of regulatory circuits can underlie human disease.
View details for DOI 10.1038/nature04678
View details for PubMedID 16554754
Conditional expression of Smad7 in pancreatic beta cells disrupts TGF-beta signaling and induces reversible diabetes mellitus
2006; 4 (2): 200-209
Identification of signaling pathways that maintain and promote adult pancreatic islet functions will accelerate our understanding of organogenesis and improve strategies for treating diseases like diabetes mellitus. Previous work has implicated transforming growth factor-beta (TGF-beta) signaling as an important regulator of pancreatic islet development, but has not established whether this signaling pathway is required for essential islet functions in the adult pancreas. Here we describe a conditional system for expressing Smad7, a potent inhibitor of TGF-beta signaling, to identify distinct roles for this pathway in adult and embryonic beta cells. Smad7 expression in Pdx1+ embryonic pancreas cells resulted in striking embryonic beta cell hypoplasia and neonatal lethality. Conditional expression of Smad7 in adult Pdx1+ cells reduced detectable beta cell expression of MafA, menin, and other factors that regulate beta cell function. Reduced pancreatic insulin content and hypoinsulinemia produced overt diabetes that was fully reversed upon resumption of islet TGF-beta signaling. Thus, our studies reveal that TGF-beta signaling is crucial for establishing and maintaining defining features of mature pancreatic beta cells.
View details for DOI 10.1371/journal.pbio.0040039
View details for Web of Science ID 000235342900007
View details for PubMedID 16435884
View details for PubMedCentralID PMC1351925
Intrinsic regulators of pancreatic beta-cell proliferation
ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY
2006; 22: 311-338
Once thought incapable of significant proliferation, the pancreatic beta-cell has recently been shown to harbor immense powers of self-renewal. Pancreatic beta-cells, the sole source of insulin in vertebrate animals, can grow facultatively to a degree unmatched by other organs in experimental animals. beta-cell growth matches changes in systemic insulin demand, which increase during common physiologic states such as aging, obesity, and pregnancy. Compensatory changes in beta-cell mass are controlled by beta-cell proliferation. Here we review recent advances in our understanding of the intrinsic factors and mechanisms that control beta-cell cycle progression. Dysregulation of beta-cell proliferation is emerging as a fundamental feature in the pathogenesis of human disease states such as cancer and diabetes mellitus. New experimental observations and studies of these diseases suggest that beta-cell fate and expansion are coordinately regulated. We speculate on how these advances may accelerate the discovery of new strategies for the treatment of diseases characterized by a deficiency or excess of beta-cells.
View details for DOI 10.1146/annurev.cellbio.22.010305.104425
View details for PubMedID 16824015
Menin regulates pancreatic islet growth by promoting histone methylation and expression of genes encoding p27(Kip1) and p18(INK4c)
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2005; 102 (41): 14659-14664
Menin, the product of the Men1 gene mutated in familial multiple endocrine neoplasia type 1 (MEN1), regulates transcription in differentiated cells. Menin associates with and modulates the histone methyltransferase activity of a nuclear protein complex to activate gene expression. However, menin-dependent histone methyltransferase activity in endocrine cells has not been demonstrated, and the mechanism of endocrine tumor suppression by menin remains unclear. Here, we show that menin-dependent histone methylation maintains the in vivo expression of cyclin-dependent kinase (CDK) inhibitors to prevent pancreatic islet tumors. In vivo expression of CDK inhibitors, including p27 and p18, and other cell cycle regulators is disrupted in mouse islet tumors lacking menin. Chromatin immunoprecipitation studies reveal that menin directly associates with regions of the p27 and p18 promoters and increases methylation of lysine 4 (Lys-4) in histone H3 associated with these promoters. Moreover, H3 Lys-4 methylation associated with p27 and p18 is reduced in islet tumors from Men1 mutant mice. Thus, H3 Lys-4 methylation is a crucial function of menin in islet tumor suppression. These studies suggest an epigenetic mechanism of tumor suppression: by promoting histone modifications, menin maintains transcription at multiple loci encoding cell cycle regulators essential for endocrine growth control.
View details for DOI 10.1073/pnas.0503484102
View details for Web of Science ID 000232603600034
View details for PubMedID 16195383
View details for PubMedCentralID PMC1253549
Differentiation of insulin-producing cells from human neural progenitor cells
2005; 2 (4): 347-356
Success in islet-transplantation-based therapies for type 1 diabetes, coupled with a worldwide shortage of transplant-ready islets, has motivated efforts to develop renewable sources of islet-replacement tissue. Islets and neurons share features, including common developmental programs, and in some species brain neurons are the principal source of systemic insulin.Here we show that brain-derived human neural progenitor cells, exposed to a series of signals that regulate in vivo pancreatic islet development, form clusters of glucose-responsive insulin-producing cells (IPCs). During in vitro differentiation of neural progenitor cells with this novel method, genes encoding essential known in vivo regulators of pancreatic islet development were expressed. Following transplantation into immunocompromised mice, IPCs released insulin C-peptide upon glucose challenge, remained differentiated, and did not form detectable tumors.Production of IPCs solely through extracellular factor modulation in the absence of genetic manipulations may promote strategies to derive transplantable islet-replacement tissues from human neural progenitor cells and other types of multipotent human stem cells.
View details for DOI 10.1371/journal.pmed.0020103
View details for Web of Science ID 000229163300017
View details for PubMedID 15839736
View details for PubMedCentralID PMC1087208
GDF11 modulates NGN3(+) islet progenitor cell number and promotes beta-cell differentiation in pancreas development
2004; 131 (24): 6163-6174
Identification of endogenous signals that regulate expansion and maturation of organ-specific progenitor cells is a major goal in studies of organ development. Here we provide evidence that growth differentiation factor 11 (GDF11), a member of the TGF-beta ligand family, governs the number and maturation of islet progenitor cells in mouse pancreas development. Gdf11 is expressed in embryonic pancreatic epithelium during formation of islet progenitor cells that express neurogenin 3. Mice deficient for Gdf11 harbor increased numbers of NGN3+ cells, revealing that GDF11 negatively regulates production of islet progenitor cells. Despite a marked expansion of these NGN3+ islet progenitors, mice lacking Gdf11 have reduced beta-cell numbers and evidence of arrested beta-cell development, indicating that GDF11 is also required for beta-cell maturation. Similar precursor and islet cell phenotypes are observed in mice deficient for SMAD2, an intracellular signaling factor activated by TGF-beta signals. Our data suggest that Gdf11 and Smad2 regulate islet cell differentiation in parallel to the Notch pathway, which previously has been shown to control development of NGN3+ cells. Thus, our studies reveal mechanisms by which GDF11 regulates the production and maturation of islet progenitor cells in pancreas development.
View details for DOI 10.1242/dev.01535
View details for Web of Science ID 000226324200014
View details for PubMedID 15548585
Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells
2004; 431 (7006): 316-320
Antagonistic activities of glucagon and insulin control metabolism in mammals, and disruption of this balance underlies diabetes pathogenesis. Insulin-producing cells (IPCs) in the brain of insects such as Drosophila also regulate serum glucose, but it remains unclear whether insulin is the sole hormonal regulator of glucose homeostasis and whether mechanisms of glucose-sensing and response in IPCs resemble those in pancreatic islets. Here we show, by targeted cell ablation, that Drosophila corpora cardiaca (CC) cells of the ring gland are also essential for larval glucose homeostasis. Unlike IPCs, CC cells express Drosophila cognates of sulphonylurea receptor (Sur) and potassium channel (Ir), proteins that comprise ATP-sensitive potassium channels regulating hormone secretion by islets and other mammalian glucose-sensing cells. They also produce adipokinetic hormone, a polypeptide with glucagon-like functions. Glucose regulation by CC cells is impaired by exposure to sulphonylureas, drugs that target the Sur subunit. Furthermore, ubiquitous expression of an akh transgene reverses the effect of CC ablation on serum glucose. Thus, Drosophila CC cells are crucial regulators of glucose homeostasis and they use glucose-sensing and response mechanisms similar to islet cells.
View details for DOI 10.1038/nature02897
View details for Web of Science ID 000223864000043
View details for PubMedID 15372035
Embryonic stem cells and islet replacement in diabetes mellitus
2004; 5: 5-15
Transplantation of functional islets of Langerhans may emerge as a useful therapy for some patients with type 1 diabetes mellitus (DM), but donor islet shortages motivate the search for new sources of transplantable islets. Pluripotent embryonic stem (ES) cells are expandable in culture and have the potential to give rise to all cell types in the body. The recent isolation of pluripotent ES cells from humans has generated excitement over the possibility of engineering glucose-responsive islet replacement tissue from these cells in large quantities. In this study, we review the recent advances in generating insulin-producing cells (IPC) from mouse and human ES (hES) cells.
View details for PubMedID 15601369
Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2002; 99 (25): 16105-16110
The use of embryonic stem cells for cell-replacement therapy in diseases like diabetes mellitus requires methods to control the development of multipotent cells. We report that treatment of mouse embryonic stem cells with inhibitors of phosphoinositide 3-kinase, an essential intracellular signaling regulator, produced cells that resembled pancreatic beta cells in several ways. These cells aggregated in structures similar, but not identical, to pancreatic islets of Langerhans, produced insulin at levels far greater than previously reported, and displayed glucose-dependent insulin release in vitro. Transplantation of these cell aggregates increased circulating insulin levels, reduced weight loss, improved glycemic control, and completely rescued survival in mice with diabetes mellitus. Graft removal resulted in rapid relapse and death. Graft analysis revealed that transplanted insulin-producing cells remained differentiated, enlarged, and did not form detectable tumors. These results provide evidence that embryonic stem cells can serve as the source of insulin-producing replacement tissue in an experimental model of diabetes mellitus. Strategies for producing cells that can replace islet functions described here can be adapted for similar uses with human cells.
View details for DOI 10.1073/pnas.252618999
View details for PubMedID 12441403
Signaling and transcriptional control of pancreatic organogenesis
CURRENT OPINION IN GENETICS & DEVELOPMENT
2002; 12 (5): 540-547
The results of several new studies encourage a revision of fundamental hypotheses concerning the cellular and molecular mechanisms underlying pancreatic morphogenesis and cell differentiation in the embryo. The roles of FGF- and BMP-signaling indicate a fundamental difference in the induction of the dorsal and the ventral pancreatic anlage. Final commitment to the pancreatic fate requires the action of several transcriptional regulators including IPF1/PDX1, PBX1 and PTF1-P48 after the onset of pancreatic bud formation. Two, largely independent endocrine cell lineages develop during the formation of the embryonic pancreas. Lineage tracing has begun to refine our understanding of the origins of the acinar, ductal and islet cells.
View details for Web of Science ID 000177898200007
View details for PubMedID 12200159
Ablation of insulin-producing neurons in flies: Growth and diabetic phenotypes
2002; 296 (5570): 1118-1120
In the fruit fly Drosophila, four insulin genes are coexpressed in small clusters of cells [insulin-producing cells (IPCs)] in the brain. Here, we show that ablation of these IPCs causes developmental delay, growth retardation, and elevated carbohydrate levels in larval hemolymph. All of the defects were reversed by ectopic expression of a Drosophila insulin transgene. On the basis of these functional data and the observation that IPCs release insulin into the circulatory system, we conclude that brain IPCs are the main systemic supply of insulin during larval growth. We propose that IPCs and pancreatic islet beta cells are functionally analogous and may have evolved from a common ancestral insulin-producing neuron. Interestingly, the phenotype of flies lacking IPCs includes certain features of diabetes mellitus.
View details for Web of Science ID 000175565000053
View details for PubMedID 12004130
Pbx1 inactivation disrupts pancreas development and in Ipf1-deficient mice promotes diabetes mellitus
2002; 30 (4): 430-435
Pbx1 is a member of the TALE (three-amino acid loop extension) class of homeodomain transcription factors, which are components of hetero-oligomeric protein complexes thought to regulate developmental gene expression and to maintain differentiated cell states. In vitro studies have shown that Pbx1 regulates the activity of Ipf1 (also known as Pdx1), a ParaHox homeodomain transcription factor required for the development and function of the pancreas in mice and humans. To investigate in vivo roles of Pbx1 in pancreatic development and function, we examined pancreatic Pbx1 expression, and morphogenesis, cell differentiation and function in mice deficient for Pbx1. Pbx1-/- embryos had pancreatic hypoplasia and marked defects in exocrine and endocrine cell differentiation prior to death at embryonic day (E) 15 or E16. In these embryos, expression of Isl1 and Atoh5, essential regulators of pancreatic morphogenesis and differentiation, was severely reduced. Pbx1+/- adults had pancreatic islet malformations, impaired glucose tolerance and hypoinsulinemia. Thus, Pbx1 is essential for normal pancreatic development and function. Analysis of trans-heterozygous Pbx1+/- Ipf1+/- mice revealed in vivo genetic interactions between Pbx1 and Ipf1 that are essential for postnatal pancreatic function; these mice developed age-dependent overt diabetes mellitus, unlike Pbx1+/- or Ipf1+/- mice. Mutations affecting the Ipf1 protein may promote diabetes mellitus in mice and humans. This study suggests that perturbation of Pbx1 activity may also promote susceptibility to diabetes mellitus.
View details for DOI 10.1038/ng860
View details for PubMedID 11912494
Hedgehog signaling in gastrointestinal development and disease.
Current molecular medicine
2002; 2 (1): 67-82
The development of the gastrointestinal (GI) tract and its associated parenchymal organs depends on Hedgehog signals from the endoderm to the surrounding mesoderm. During development, Hedgehog signaling is essential for patterning the GI tract along anterior-posterior (A-P), dorsal-ventral (D-V), and radial axes, as well as in maintenance of stem cells. Our knowledge about these roles for Hedgehog signaling is derived from studies of developmental defects that result from disrupted or activated Hedgehog signaling in model organisms including mouse, chick, and frog. These studies provide evidence for distinct roles of specific Hedgehog ligands in GI development. Studies in model organisms have also elucidated how Hedgehog signaling may function in development and function of the GI tract in humans. Several diseases and congenital syndromes are known to result from genetic defects in Hedgehog signaling components, and this pathway may ultimately prove to be an important target for future diagnostic and therapeutic tools.
View details for PubMedID 11898849
- Pancreatic islet cell replacement - Successes and opportunities Symposium on Reparative Medicine - Growing Tissues and Organs NEW YORK ACAD SCIENCES. 2002: 41–43
- Intercellular signals regulating pancreas development and function GENES & DEVELOPMENT 2001; 15 (2): 111-127
Regulation of pancreas development by hedgehog signaling
2000; 127 (22): 4905-4913
Pancreas organogenesis is regulated by the interaction of distinct signaling pathways that promote or restrict morphogenesis and cell differentiation. Previous work has shown that activin, a TGF(beta+) signaling molecule, permits pancreas development by repressing expression of Sonic hedgehog (Shh), a member of the hedgehog family of signaling molecules that antagonize pancreas development. Here we show that Indian hedgehog (Ihh), another hedgehog family member, and Patched 1 (Ptc1), a receptor and negative regulator of hedgehog activity, are expressed in pancreatic tissue. Targeted inactivation of Ihh in mice allows ectopic branching of ventral pancreatic tissue resulting in an annulus that encircles the duodenum, a phenotype frequently observed in humans suffering from a rare disorder known as annular pancreas. Shh(-)(/)(-) and Shh(-)(/)(-) Ihh(+/)(-) mutants have a threefold increase in pancreas mass, and a fourfold increase in pancreatic endocrine cell numbers. In contrast, mutations in Ptc1 reduce pancreas gene expression and impair glucose homeostasis. Thus, islet cell, pancreatic mass and pancreatic morphogenesis are regulated by hedgehog signaling molecules expressed within and adjacent to the embryonic pancreas. Defects in hedgehog signaling may lead to congenital pancreatic malformations and glucose intolerance.
View details for Web of Science ID 000165754100015
View details for PubMedID 11044404
Activin receptor patterning of foregut organogenesis
GENES & DEVELOPMENT
2000; 14 (15): 1866-1871
Foregut development produces a characteristic sequence of gastrointestinal and respiratory organs, but the signaling pathways that ensure this developmental order remain largely unknown. Here, mutations of activin receptors ActRIIA and ActRIIB are shown to disrupt the development of posterior foregut-derived organs, including the stomach, pancreas, and spleen. Foregut expression of genes including Shh and Isl1 is shifted in mutant mice. The endocrine pancreas is particularly sensitive to the type and extent of receptor inactivation. ActRIIA(+/-)B(+/-) animals lack axial defects, but have hypoplastic pancreatic islets, hypoinsulinemia, and impaired glucose tolerance. Thus, activin receptor-mediated signaling regulates axial patterning, cell differentiation, and function of foregut-derived organs.
View details for PubMedID 10921901
Screening for novel pancreatic genes expressed during embryogenesis
1999; 48 (8): 1550-1556
We have combined suppressive subtractive hybridization with in situ hybridization to identify genes expressed at early stages of pancreas development. By using polymerase chain reaction amplification and subtractive hybridization, this protocol for screening can be applied when the amount of RNA is limited. Seven genes expressed in or adjacent to the pancreas anlage were isolated, three of which show similarity to known genes. The expression pattern and sequence information indicate that some of the genes could govern pancreas development.
View details for Web of Science ID 000081577400008
View details for PubMedID 10426372
Pancreas development is promoted by cyclopamine, a Hedgehog signaling inhibitor
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1998; 95 (22): 13036-13041
Exposure to cyclopamine, a steroid alkaloid that blocks Sonic hedgehog (Shh) signaling, promotes pancreatic expansion in embryonic chicks. Heterotopic development of pancreatic endocrine and exocrine structures occurs in regions adjacent to the pancreas including stomach and duodenum, and insulin-producing islets in the pancreas are enlarged. The homeodomain transcription factor PDX1, required for pancreas development, is expressed broadly in the posterior foregut but pancreas development normally initiates only in a restricted region of PDX1-expressing posterior foregut where endodermal Shh expression is repressed. The results suggests that cyclopamine expands the endodermal region where Shh signaling does not occur, resulting in pancreatic differentiation in a larger region of PDX1-expressing foregut endoderm. Cyclopamine reveals the capacity of a broad region of the posterior embryonic foregut to form pancreatic cells and provides a means for expanding embryonic pancreas development.
View details for Web of Science ID 000076757300058
View details for PubMedID 9789036
Notochord repression of endodermal Sonic hedgehog permits pancreas development
GENES & DEVELOPMENT
1998; 12 (11): 1705-1713
Notochord signals to the endoderm are required for development of the chick dorsal pancreas. Sonic hedgehog (SHH) is normally absent from pancreatic endoderm, and we provide evidence that notochord, in contrast to its effects on adjacent neuroectoderm where SHH expression is induced, represses SHH expression in adjacent nascent pancreatic endoderm. We identify activin-betaB and FGF2 as notochord factors that can repress endodermal SHH and thereby permit expression of pancreas genes including Pdx1 and insulin. Endoderm treatment with antibodies that block hedgehog activity also results in pancreatic gene expression. Prevention of SHH expression in prepancreatic dorsal endoderm by intercellular signals, like activin and FGF, may be critical for permitting early steps of chick pancreatic development.
View details for Web of Science ID 000074149000013
View details for PubMedID 9620856
Notochord to endoderm signaling is required for pancreas development
1997; 124 (21): 4243-4252
The role of the notochord in inducing and patterning adjacent neural and mesodermal tissues is well established. We provide evidence that the notochord is also required for one of the earliest known steps in the development of the pancreas, an endodermally derived organ. At a developmental stage in chick embryos when the notochord touches the endoderm, removal of notochord eliminates subsequent expression of several markers of dorsal pancreas bud development, including insulin, glucagon and carboxypeptidase A. Pancreatic gene expression can be initiated and maintained in prepancreatic chick endoderm grown in vitro with notochord. Non-pancreatic endoderm, however, does not express pancreatic genes when recombined with the same notochord. The results suggest that the notochord provides a permissive signal to endoderm to specify pancreatic fate in a stepwise manner.
View details for Web of Science ID A1997YG86200007
View details for PubMedID 9334273
- Pancreas development in the chick embryo Cold Spring Harbor Symposium on Quantitative Biology - Pattern Formation During Development COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 1997: 377–383
Chemotherapy and neutropenia
HEMATOLOGY-ONCOLOGY CLINICS OF NORTH AMERICA
1996; 10 (2): 377-?
Myelosuppression is the most common toxicity associated with the administration of dose-intensive cytotoxic chemotherapy. The basic understanding of neutrophil biology and the physiology of chemotherapy-induced neutropenia has advanced tremendously in the past 2 decades. Concordantly, the ability to reduce the morbidity associated with neutropenia has improved. Adjunctive cytokine and progenitor cell support of hematologic recovery after myelosuppressive therapy have proved to be models of translational research and have led to novel therapeutic initiatives for patients with cancer and hematologic malignancies. In this article, fundamental aspects of neutrophil production are discussed, and the clinical development of hematopoietic cytokines active on cells of the leukocyte lineages is presented.
View details for Web of Science ID A1996UC64900006
View details for PubMedID 8707761
CONTROL OF CELL-DENSITY AND PATTERN BY INTERCELLULAR SIGNALING IN MYXOCOCCUS DEVELOPMENT
ANNUAL REVIEW OF MICROBIOLOGY
1992; 46: 117-139
Myxococcus xanthus cells feed, move, and develop cooperatively. Genetic, biochemical, and cell mosaic studies demonstrate that cells coordinate their multicellular behavior by transmission of intercellular signals. Starvation for amino acids at sufficiently high density on a solid surface initiates a series of events culminating in the formation of a multicellular structure called a fruiting body filled with dormant, environmentally resistant spores. This review discusses how myxobacteria use extracellular signals to sequentially check the density and arrangement of cells at different stages during development. For at least one early and one late developmental signal, cell density determines the efficiency of intercellular signaling. In turn, proper signaling insures that the appropriate cell density exists, thus controlling the progress of multicellular development in M. xanthus.
View details for Web of Science ID A1992JQ92900005
View details for PubMedID 1444251
INTERCELLULAR SIGNALING IN MYXOCOCCUS DEVELOPMENT - THE ROLE OF C-FACTOR
TRENDS IN GENETICS
1991; 7 (11-12): 361-365
Cell communication governs differentiation and morphogenesis in fruiting body formation by Myxococcus xanthus. Transmission of a small short-range intercellular signal by a protein called C factor directs multicellular pattern formation and coordinates the timing of major developmental events.
View details for Web of Science ID A1991GN92700005
View details for PubMedID 1668187
C-FACTOR HAS DISTINCT AGGREGATION AND SPORULATION THRESHOLDS DURING MYXOCOCCUS DEVELOPMENT
JOURNAL OF BACTERIOLOGY
1991; 173 (5): 1722-1728
C-factor, the protein product of the csgA gene, acts as a short-range morphogenetic signal. It is required for fruiting body development of the gram-negative bacterium Myxococcus xanthus. Aggregation, sporulation, and expression of a set of genes that are C-factor dependent, all of which fail in csgA mutant cells, are completely restored by addition of purified C-factor. We report here that, depending on its concentration, C-factor can elicit two distinct morphogenetic and transcriptional responses from csgA cells. Low levels of C-factor bring about aggregation and expression of an early C-dependent gene, whereas higher levels lead to the same effects plus expression of a late C-dependent gene and spore formation. C-factor positively regulates its own transcription. An approximately fourfold net increase in csgA transcription and C-factor levels during development was measured. We propose that autoregulation and the two distinct activity thresholds allow C-factor to act as a timer, first triggering aggregation, then sporulation, thereby producing the appropriate developmental order.
View details for Web of Science ID A1991EZ17000022
View details for PubMedID 1847908
CELL ALIGNMENT REQUIRED IN DIFFERENTIATION OF MYXOCOCCUS-XANTHUS
1990; 249 (4971): 926-928
During fruiting body morphogenesis of Myxococcus xanthus, cell movement is required for transmission of C-factor, a short range intercellular signaling protein necessary for sporulation and developmental gene expression. Nonmotile cells fail to sporulate and to express C-factor-dependent genes, but both defects were rescued by a simple manipulation of cell position that oriented the cells in aligned, parallel groups. A similar pattern of aligned cells normally results from coordinated recruitment of wildtype cells into multicellular aggregates, which later form mature fruiting bodies. It is proposed that directed cell movement establishes critical contacts between adjacent cells, which are required for efficient intercellular C-factor transmission.
View details for Web of Science ID A1990DV75100048
View details for PubMedID 2118274
CELL MOTILITY IS REQUIRED FOR THE TRANSMISSION OF C-FACTOR, AN INTERCELLULAR SIGNAL THAT COORDINATES FRUITING BODY MORPHOGENESIS OF MYXOCOCCUS-XANTHUS
GENES & DEVELOPMENT
1990; 4 (6): 896-904
There are striking similarities between the developmental phenotypes of two different mutant classes of Myxococcus xanthus. The first class, mglA mutants, are nonmotile under all conditions tested. The second class, csgA mutants, are motile but belong to a class of signal-defective developmental mutants that cannot develop alone but will develop when mixed with intact wild-type cells. Nevertheless, both csgA and mglA mutants fail to aggregate properly or to sporulate when induced to form fruiting bodies. An mglA mutation and a csgA mutation affect expression of a panel of lacZ fusions to developmental genes in the same way, indicating that nonmotile cells and csgA cells arrest development at a similar stage. One explanation for the similarity of developmental phenotypes between these mutants is that motility is required for the csgA-mediated cell interaction. In support of this hypothesis, we report that C-factor, a protein purified from nascent wild-type fruiting bodies based on its ability to rescue csgA mutant fruiting body development, also rescues sporulation and expression of beta-galactosidase from developmentally controlled lacZ fusions in mglA strains, apparently without restoring their motility. Wild-type levels of active C-factor can be purified from mglA cells, yet intact mglA cells do not rescue csgA cells upon cell-cell mixing. Intact wild-type cells are unable to restore the sporulation and beta-galactosidase expression of mglA mutants. These results support the hypothesis that donor and responder cell motility is required for C-factor transmission between cells during development.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1990DJ09500002
View details for PubMedID 2116988
PURIFICATION AND PROPERTIES OF MYXOCOCCUS-XANTHUS C-FACTOR, AN INTERCELLULAR SIGNALING PROTEIN
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1990; 87 (10): 3635-3639
C-factor, a Myxococcus xanthus protein that restores the developmental defects of a class of nonautonomous mutants resulting from mutation of the csgA gene, has been purified approximately 1000-fold from starved wild-type cells. The monomeric form of C-factor is a single polypeptide with a molecular mass of 17 kDa that can be solubilized by detergent from membrane components. Characterization by gel filtration and denaturing gel electrophoresis suggests that biologically active C-factor is a dimer composed of two 17-kDa monomers. Antibodies against a form of the M. xanthus csgA gene product overexpressed in Escherichia coli react with purified C-factor.
View details for Web of Science ID A1990DD87300001
View details for PubMedID 2111012
C-FACTOR - A CELL CELL SIGNALING PROTEIN REQUIRED FOR FRUITING BODY MORPHOGENESIS OF M-XANTHUS
1990; 61 (1): 19-26
During fruiting body development, the product of the csgA gene is necessary for cellular aggregation, for spore differentiation, and for gene expression that is initiated after 6 hr of starvation. From nascent wild-type fruiting bodies we have purified a polypeptide of 17 kd called C-factor, which, at approximately 1 to 2 nM, restores normal development to csgA mutant cells. C-factor activity is not recovered from extracts of unstarved, growing cells or csgA mutant cells. The amino acid sequence from purified C-factor demonstrates that it is the product of the csgA gene. C-factor is active over a narrow range of concentration and has properties of a morphogenetic paracrine signal.
View details for Web of Science ID A1990CY38200006
View details for PubMedID 2107980
PARA-DEPENDENT TRANSCRIPTION TERMINATION OF A BACTERIAL OPERON IS ANTAGONIZED BY AN EXTRACHROMOSOMAL GENE-PRODUCT
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1986; 83 (24): 9561-9565
The psu gene product of "phasmid" (phage-plasmid) P4 acts as a transcription antitermination factor in trans and in cis, respectively, within the morphogenic operons of its P2 phage helper during lytic viral development and on P4 itself during the establishment stage of its alternative mode of propagation as a plasmid. Here we show that psu also antagonizes activity of the Escherichia coli transcription termination factor rho at the terminator of the trp operon. Such a finding provides to our knowledge the first direct evidence for antitermination activity at a known rho-dependent site by the psu gene product. It also reveals an example of an extrachromosomal gene product that acts on specific sites of three different genomes to regulate expression of unlinked families of genes.
View details for Web of Science ID A1986F500600061
View details for PubMedID 3540944