Person: Melton, Douglas
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Melton, Douglas
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Publication MARIS: Method for Analyzing RNA following Intracellular Sorting(Public Library of Science, 2014) Hrvatin, Sinisa; Deng, Francis; O'Donnell, Charles W.; Gifford, David K.; Melton, DouglasTranscriptional profiling is a key technique in the study of cell biology that is limited by the availability of reagents to uniquely identify specific cell types and isolate high quality RNA from them. We report a Method for Analyzing RNA following Intracellular Sorting (MARIS) that generates high quality RNA for transcriptome profiling following cellular fixation, intracellular immunofluorescent staining and FACS. MARIS can therefore be used to isolate high quality RNA from many otherwise inaccessible cell types simply based on immunofluorescent tagging of unique intracellular proteins. As proof of principle, we isolate RNA from sorted human embryonic stem cell-derived insulin-expressing cells as well as adult human β cells. MARIS is a basic molecular biology technique that could be used across several biological disciplines.Publication Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3(Nature Publishing Group, 2012) Blum, Barak; Hrvatin, Sinisa; Schuetz, Christian; Bonal, Claire; Rezania, Alireza; Melton, DouglasInsulin-expressing cells that have been differentiated from human pluripotent stem cells in vitro lack the glucose responsiveness characteristic of mature beta cells. Beta-cell maturation in mice was studied to find genetic markers that enable screens for factors that induce bona fide beta cells in vitro. We find that functional beta-cell maturation is marked by an increase in the glucose threshold for insulin secretion and by expression of the gene urocortin 3.Publication Adenosine kinase inhibition selectively promotes rodent and porcine islet β-cell replication(Proceedings of the National Academy of Sciences, 2012) Annes, J. P.; Ryu, J. H.; Lam, K.; Carolan, Peter; Utz, K.; Hollister-Lock, J.; Arvanites, Anthony C.; Rubin, Lee; Weir, Gordon; Melton, DouglasDiabetes is a pathological condition characterized by relative insulin deficiency, persistent hyperglycemia, and, consequently, diffuse micro- and macrovascular disease. One therapeutic strategy is to amplify insulin-secretion capacity by increasing the number of the insulin-producing β cells without triggering a generalized proliferative response. Here, we present the development of a small-molecule screening platform for the identification of molecules that increase β-cell replication. Using this platform, we identify a class of compounds [adenosine kinase inhibitors (ADK-Is)] that promote replication of primary β cells in three species (mouse, rat, and pig). Furthermore, the replication effect of ADK-Is is cell type-selective: treatment of islet cell cultures with ADK-Is increases replication of β cells but not that of α cells, PP cells, or fibroblasts. Short-term in vivo treatment with an ADK-I also increases β-cell replication but not exocrine cell or hepatocyte replication. Therefore, we propose ADK inhibition as a strategy for the treatment of diabetes.Publication Self-renewal of embryonic-stem-cell-derived progenitors by organ-matched mesenchyme(Nature Publishing Group, 2012) Sneddon, Julie Beth; Borowiak, Malgorzata; Melton, DouglasOne goal of regenerative medicine, to use stem cells to replace cells lost by injury or disease, depends on producing an excess of the relevant cell for study or transplantation. To this end, the stepwise differentiation of stem cells into specialized derivatives has been successful for some cell types, but a major problem remains the inefficient conversion of cells from one stage of differentiation to the next. If specialized cells are to be produced in large numbers it will be necessary to expand progenitor cells, without differentiation, at some steps of the process. Using the pancreatic lineage as a model for embryonic-stem-cell differentiation, we demonstrate that this is a solvable problem. Co-culture with organ-matched mesenchyme permits proliferation and self-renewal of progenitors, without differentiation, and enables an expansion of more than a million-fold for human endodermal cells with full retention of their developmental potential. This effect is specific both to the mesenchymal cell and to the progenitor being amplified. Progenitors that have been serially expanded on mesenchyme give rise to glucose-sensing, insulin-secreting cells when transplanted in vivo. Theoretically, the identification of stage-specific renewal signals can be incorporated into any scheme for the efficient production of large numbers of differentiated cells from stem cells and may therefore have wide application in regenerative biology.Publication In vivo reprogramming of pancreatic acinar cells to three islet endocrine subtypes(eLife Sciences Publications, Ltd, 2014) Li, Weida; Nakanishi, Mio; Zumsteg, Adrian; Shear, Matthew; Wright, Christopher; Melton, Douglas; Zhou, QiaoDirect lineage conversion of adult cells is a promising approach for regenerative medicine. A major challenge of lineage conversion is to generate specific cell subtypes. The pancreatic islets contain three major hormone-secreting endocrine subtypes: insulin+ β-cells, glucagon+ α-cells, and somatostatin+ δ-cells. We previously reported that a combination of three transcription factors, Ngn3, Mafa, and Pdx1, directly reprograms pancreatic acinar cells to β-cells. We now show that acinar cells can be converted to δ-like and α-like cells by Ngn3 and Ngn3+Mafa respectively. Thus, three major islet endocrine subtypes can be derived by acinar reprogramming. Ngn3 promotes establishment of a generic endocrine state in acinar cells, and also promotes δ-specification in the absence of other factors. δ-specification is in turn suppressed by Mafa and Pdx1 during α- and β-cell induction. These studies identify a set of defined factors whose combinatorial actions reprogram acinar cells to distinct islet endocrine subtypes in vivo. DOI: http://dx.doi.org/10.7554/eLife.01846.001Publication Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal(Cold Spring Harbor Laboratory Press, 2010) Niakan, K. K.; Ji, H.; Maehr, R.; Vokes, S. A.; Rodolfa, K. T.; Sherwood, Richard; Yamaki, M.; Dimos, J. T.; Chen, A. E.; Melton, Douglas; McMahon, Andrew P.; Eggan, KevinIn embryonic stem (ES) cells, a well-characterized transcriptional network promotes pluripotency and represses gene expression required for differentiation. In comparison, the transcriptional networks that promote differentiation of ES cells and the blastocyst inner cell mass are poorly understood. Here, we show that Sox17 is a transcriptional regulator of differentiation in these pluripotent cells. ES cells deficient in Sox17 fail to differentiate into extraembryonic cell types and maintain expression of pluripotency-associated transcription factors, including Oct4, Nanog, and Sox2. In contrast, forced expression of Sox17 down-regulates ES cell-associated gene expression and directly activates genes functioning in differentiation toward an extraembryonic endoderm cell fate. We show these effects of Sox17 on ES cell gene expression are mediated at least in part through a competition between Sox17 and Nanog for common DNA-binding sites. By elaborating the function of Sox17, our results provide insight into how the transcriptional network promoting ES cell self-renewal is interrupted, allowing cellular differentiation.Publication Notch signaling promotes airway mucous metaplasia and inhibits alveolar development(The Company of Biologists, 2009) Guseh, James; Bores, S. A.; Stanger, B. Z.; Zhou, Qiao; Anderson, William; Melton, Douglas; Rajagopal, JayarajThe airways are conduits that transport atmospheric oxygen to the distal alveolus. Normally, airway mucous cells are rare. However, diseases of the airway are often characterized by mucous metaplasia, in which there are dramatic increases in mucous cell numbers. As the Notch pathway is known to regulate cell fate in many contexts, we misexpressed the active intracellular domain of the mouse Notch1 receptor in lung epithelium. Notch misexpression resulted in an increase in mucous cells and a decrease in ciliated cells in the airway. Similarly, mouse embryonic tracheal explants and adult human airway epithelium treated with Notch agonists displayed increased mucous cell numbers and decreased ciliated cell numbers. Notch antagonists had the opposite effect. Notably, Notch antagonists blocked IL13-induced mucous metaplasia. IL13 has a well-established role as an inflammatory mediator of mucous metaplasia and functions through Stat6-mediated gene transcription. We found that Notch ligands, however, are able to cause mucous metaplasia in Stat6-null cultured trachea, thus identifying a novel pathway that stimulates mucous metaplasia. Notch signaling may therefore play an important role in airway disease and, by extension, Notch antagonists may have therapeutic value. Conversely, in the distal lung, Notch misexpression prevented the differentiation of alveolar cell types. Instead, the distal lung formed cysts composed of cells that were devoid of alveolar markers but that expressed some, but not all, markers of proximal airway epithelium. Occasional distal cystic cells appeared to differentiate into normal proximal airway cells, suggesting that ectopic Notch signaling arrests the normal differentiation of distal lung progenitors before they initiate an alveolar program.Publication How to make beta cells?(Elsevier, 2009) Borowiak, Malgorzata; Melton, DouglasInsulin-producing beta cells are lost or insufficient in diabetic patients, presenting the medical challenge for new beta cells. Currently, there are three strategies that offer promise. One involves the generation of beta cells de novo by directing the differentiation of either embryonic stem cells or induced pluripotent cells to the beta cell lineage. The second is based on the conversion of another terminally differentiated cell to beta cells in a process called reprogramming. The third approach is to promote the replication of existing beta cells either in vivo or in vitro. Significant progress is evident for each strategy, but it remains unclear which approach will ultimately prove successful.Publication Small Molecules Efficiently Direct Endodermal Differentiation of Mouse and Human Embryonic Stem Cells(Elsevier, 2009) Borowiak, Malgorzata; Maehr, René; Chen, Shuibing; Chen, Alice E.; Tang, Weiping; Fox, Julia L.; Schreiber, Stuart; Melton, DouglasAn essential step for therapeutic and research applications of stem cells is the ability to differentiate them into specific cell types. Endodermal cell derivatives, including lung, liver, and pancreas, are of interest for regenerative medicine, but efforts to produce these cells have been met with only modest success. In a screen of 4000 compounds, two cell-permeable small molecules were indentified that direct differentiation of ESCs into the endodermal lineage. These compounds induce nearly 80% of ESCs to form definitive endoderm, a higher efficiency than that achieved by Activin A or Nodal, commonly used protein inducers of endoderm. The chemically induced endoderm expresses multiple endodermal markers, can participate in normal development when injected into developing embryos, and can form pancreatic progenitors. The application of small molecules to differentiate mouse and human ESCs into endoderm represents a step toward achieving a reproducible and efficient production of desired ESC derivatives.Publication Reprogramming within Hours Following Nuclear Transfer into Mouse but not Human Zygotes(Nature Publishing Group, 2011) Egli, Dieter; Chen, Alice E.; Saphier Belfer, Genevieve; Ichida, Justin; Fitzgerald, Claire; Go, Kathryn J.; Acevedo, Nicole; Patel, Jay; Baetscher, Manfred; Kearns, William G.; Goland, Robin; Leibel, Rudolph L.; Melton, Douglas; Eggan, KevinFertilized mouse zygotes can reprogram somatic cells to a pluripotent state. Human zygotes might therefore be useful for producing patient-derived pluripotent stem cells. However, logistical, legal and social considerations have limited the availability of human eggs for research. Here we show that a significant number of normal fertilized eggs (zygotes) can be obtained for reprogramming studies. Using these zygotes, we found that when the zygotic genome was replaced with that of a somatic cell, development progressed normally throughout the cleavage stages, but then arrested before the morula stage. This arrest was associated with a failure to activate transcription in the transferred somatic genome. In contrast to human zygotes, mouse zygotes reprogrammed the somatic cell genome to a pluripotent state within hours after transfer. Our results suggest that there may be a previously unappreciated barrier to successful human nuclear transfer, and that future studies could focus on the requirements for genome activation.