Harvard Stem Cell Institute Research Newsletter
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| | | |  | Research Commentary Genomic approaches provide insights into the molecular basis of pluripotency by Lisa Girard, PhD
HSCI Science Editor Nuclear reprogramming demonstrates the potential for reversability in cellular differentiation. Reprogramming introduces a small number of transcription factors into somatic cells, such as skin cells, transforming the skin cells into induced pluripotent stem cells (iPS), While a core set of factors necessary for reprogramming has been identified the process is extremely inefficient (less than .1% of cells exposed to factors are transformed). The impetus to improve the efficiency of reprogramming is high. Approaches in regenerative medicine, such as replacing organs and tissues lost to injury and disease, face the obstacle of donor cell rejection by the host. Nuclear reprogramming can create iPS cells that are genetically identical to that of the donor, thus allowing a patient to be the donor for an iPS cell line that may serve as starting material for replacement tissue and organs which will not be rejected by the patient. A key component to increasing the efficiency of reprogramming is a more comprehensive understanding of pluripotency. This would help to define more extensive genetic networks and additional players that could be introduced or engineered to increase reprogramming efficiency. Much of the information gained from studying pluripotency has been done so using genomic approaches; such as DNA microarrays or "chips"", and variations on chip experiments such as chIP-chip, chromatin immunoprecipitation followed by chip analysis, and more recently bio-chIP and bio-chIP-chip which is in vivo biotinylation mediated chIP coupled with target mapping (chip). The significant findings achieved using these approaches reflect the shifting landscape of how biological qustions are being asked-on a global rather than gene by gene level. Innovative and improved methods are enabling us to understand the extensive gene and protein networks involved in pluripotency which will, in turn, inform reprogramming approaches. Recently, much attention has been focused on a small suite of transcription factors including the "reprogramming factors" (Oct4, Sox2, Klf4, and c-Myc) and others, such as Nanog, found to be required to maintain cells in a pluripotent state (Stadtfeld et al., 2008). In a recent paper by Stu Orkin's lab at the Harvard Stem Cell Institute and Dana Farber Cancer Center (Kim et al., 2008), they explored the molecular basis of these requirements using a powerful bio ChIP (in vivo-biotinylated chIP) and bio chIP-chIP approach, which gets around some of the limitations of many antibodies in traditional chIP experiments. Orkin's group identified target promoters of nine of the reprogramming factors on a global scale in embryonic stem cells. They were able to examine their promoters and distinguish two classes of target genes based on the requirements to activate or repress them suggesting differential regulation of these targets related to the factors occupying the promoter. While other groups have used chip based approaches to probe the downstream targets of Oct4, Sox2,and Nanog in mouse and human embryonic stem cells, the studies by Orkin and colleagues expand the set analyzed to nine transcription factors, examined on a global scale and provide a background with which to ask more detailed questions about the reprogramming activities of these factors. Another recent study, also taking a whole genome approach toward understanding the genetic networks involved in pluripotency, Bing Lim and colleagues were interested in defining the mechanism by which the Wnt pathway, ubiquitous in many developmental processes, plays a role in maintaining pluripotency. The researchers coupled chromatin immunpprecipitation and DNA chip analysis (ChIP-on-chip) in order to identify previously unknown targets of Tcf3, a downstream effector of the Wnt pathway known to be involved in pluripotency. Their experiments revealed that, along with a rang of other targets previously implicated in developmental processes, Tcf3. Interestingly, they found that Tcf3 binds and represses Oct4 one of the core reprogramming factors which affects Oct4 as well Nanog levels, another factor involved in pluripotency. The genomic approaches taken allowed researchers to examine multiple genes at once, revealing a complex genetic network that would not have so clearly emerged using a more piecemeal approach. Whole genome studies have come into their own at an ideal time to serve as a tool to better our understanding of pluripotency, as well as differentiation. A greater knowledge of the two states will ultimately help us understand the means for more efficient and targeted flux between the two. References - Kim, J., Chu, J., Shen, X., Wang, J., Orkin, S.H. (2008) An extended transcriptional network for pluripotency of embryonic stem cells. Cell 132, 1049-61.
- Stadtfeld, M., Maherali, N., Breault, D.T., Hochedlinger, K. (2008) Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell. 2, 230-40.
- Tam, W.L., Lim, C.Y., Han, J., Zhang, J., Ang, Y.S., Ng, H.H., Yang H, Lim, B. (2008) Tcf3 Regulates Embryonic Stem Cell Pluripotency and Self-Renewal by the Transcriptional Control of Multiple Lineage Pathways. Stem Cells. May 8. [Epub ahead of print]
| | | Spotlight Article Dissecting direct reprogramming through integrative genomic analysis This month's spotlighted article, published online in the journal Nature, is by new HSCI Principal Faculty member Alexander Meissner, PhD, Assistant Professor in Harvard's new inter-school Department of Stem Cell and Regenerative Biology and Associate Member of the Broad Institute. Abstract: Somatic cells can be reprogrammed to a pluripotent state through the ectopic expression of defined transcription factors. Understanding the mechanism and kinetics of this transformation may shed light on the nature of developmental potency and suggest strategies with improved efficiency or safety. Here we report an integrative genomic analysis of reprogramming of mouse fibroblasts and B lymphocytes. Lineage-committed cells show a complex response to the ectopic expression involving induction of genes downstream of individual reprogramming factors. Fully reprogrammed cells show gene expression and epigenetic states that are highly similar to embryonic stem cells. In contrast, stable partially reprogrammed cell lines show reactivation of a distinctive subset of stem-cell-related genes, incomplete repression of lineage-specifying transcription factors, and DNA hypermethylation at pluripotency-related loci. These observations suggest that some cells may become trapped in partially reprogrammed states owing to incomplete repression of transcription factors, and that DNA de-methylation is an inefficient step in the transition to pluripotency. We demonstrate that RNA inhibition of transcription factors can facilitate reprogramming, and that treatment with DNA methyltransferase inhibitors can improve the overall efficiency of the reprogramming process. Mikkelsen TS, Hanna J, Zhang X, Ku M, Wernig M, Schorderet P, Bernstein BE, Jaenisch R, Lander ES, Meissner A. Dissecting direct reprogramming through integrative genomic analysis. Nature. 2008 May 28. | Review and Commentary Articles - Weissleder R, Pittet MJ. Imaging in the era of molecular oncology. Nature. 2008 Apr 3;452(7187):580-9. Review. Read Abstract.
- Martin-Puig S, Wang Z, Chien KR. Lives of a heart cell: tracing the origins of cardiac progenitors. Cell Stem Cell. 2008 Apr 10;2(4):320-31. Read Abstract.
- Wang Y, Armstrong SA. Cancer: inappropriate expression of stem cell programs? Cell Stem Cell. 2008 Apr 10;2(4):297-9. Read Abstract.
- Koh I, Hong R, Weissleder R, Josephson L. Sensitive NMR Sensors Detect Antibodies to Influenza. Angew Chem Int Ed Eng. 2008 Apr 21;47(22):4119-4121. No abstract available. Read Abstract.
- Punzo C, Cepko CL. Ultrasound-guided in utero injections allow studies of the development and function of the eye. Dev Dyn. 2008 Apr;237(4):1034-42. Read Abstract.
- Papayannopoulou T, Scadden DT. Stem-cell ecology and stem cells in motion. Blood. 2008 Apr 15;111(8):3923-30. Read Abstract.
- Williams DA. Foamy virus vectors come of age. Mol Ther. 2008 Apr;16(4):635-6. No abstract available. Read Abstract.
| Blood - Min IM, Pietramaggiori G, Kim FS, Passegue E, Stevenson KE, Wagers AJ. The transcription factor EGR1 controls both the proliferation and localization of hematopoietic stem cells. Cell Stem Cell. 2008 Apr 10;2(4):380-91. Read Abstract.
- Muller LU, Milsom MD, Kim MO, Schambach A, Schuesler T, Williams DA. Rapid Lentiviral Transduction Preserves the Engraftment Potential of Fanca(-/-) Hematopoietic Stem Cells. Mol Ther. 2008 Apr 8. Read Abstract.
| Cancer - Kirstetter P, Schuster MB, Bereshchenko O, Moore S, Dvinge H, Kurz E, Theilgaard-Monch K, Mansson R, Pedersen TA, Pabst T, Schrock E, Porse BT, Jacobsen SE, Bertone P, Tenen DG, Nerlov C. Modeling of C/EBPalpha mutant acute myeloid leukemia reveals a common expression signature of committed myeloid leukemia-initiating cells. Cancer Cell. 2008 Apr;13(4):299-310. Read Abstract.
- Wernig G, Kharas MG, Okabe R, Moore SA, Leeman DS, Cullen DE, Gozo M, McDowell EP, Levine RL, Doukas J, Mak CC, Noronha G, Martin M, Ko YD, Lee BH, Soll RM, Tefferi A, Hood JD, Gilliland DG. Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of a murine model of JAK2V617F-induced polycythemia vera. Cancer Cell. 2008 Apr;13(4):311-20. Read Abstract.
- Austin KM, Gupta ML, Coats SA, Tulpule A, Mostoslavsky G, Balazs AB, Mulligan RC, Daley G, Pellman D, Shimamura A. Mitotic spindle destabilization and genomic instability in Shwachman-Diamond syndrome. J Clin Invest. 2008 Apr;118(4):1511-8. Read Abstract.
- Quintas-Cardama A, Tong W, Manshouri T, Vega F, Lennon PA, Cools J, Gilliland DG, Lee F, Cortes J, Kantarjian H, Garcia-Manero G. Activity of tyrosine kinase inhibitors against human NUP214-ABL1-positive T cell malignancies. Leukemia. 2008 Apr 10. Read Abstract.
| Cell Biology - Saharinen P, Eklund L, Miettinen J, Wirkkala R, Anisimov A, Winderlich M, Nottebaum A, Vestweber D, Deutsch U, Koh GY, Olsen BR, Alitalo K. Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-cell and cell-matrix contacts. Nat Cell Biol. 2008 May;10(5):527-37. Epub 2008 Apr 20. Read Abstract.
| Developmental Biology - Wu X, Tu X, Joeng KS, Hilton MJ, Williams DA, Long F. Rac1 activation controls nuclear localization of beta-catenin during canonical Wnt signaling. Cell. 2008 Apr 18;133(2):340-53. Read Abstract.
- Wang J, Levasseur DN, Orkin SH. Requirement of Nanog dimerization for stem cell self-renewal and pluripotency. Proc Natl Acad Sci USA. 2008 Apr 29;105(17):6326-31. Epub 2008 Apr 24. Read Abstract.
- Sue N, Jack BH, Eaton SA, Pearson RC, Funnell AP, Turner J, Czolij R, Denyer G, Bao S, Molero-Navajas JC, Perkins A, Fujiwara Y, Orkin SH, Bell-Anderson K, Crossley M. Targeted disruption of the Basic Kruppel-like Factor (Klf3) gene reveals a role in adipogenesis. Mol Cell Biol. 2008 Apr 7. Read Abstract.
- Kim DS, Ross SE, Trimarchi JM, Aach J, Greenberg ME, Cepko CL. Identification of molecular markers of bipolar cells in the murine retina. J Comp Neurol. 2008 Apr 10;507(5):1795-810. Read Abstract.
| Diabetes - Cha HC, Oak NR, Kang S, Tran TA, Kobayashi S, Chiang SH, Tenen DG, Macdougald OA. Phosphorylation of C/EBPalpha regulates GLUT4 expression and glucose transport in adipocytes. J Biol Chem. 2008 Apr 11. Read Abstract.
| Imaging - Shah K, Hingtgen S, Kasmieh R, Figueiredo JL, Garcia-Garcia E, Martinez-Serrano A, Breakefield X, Weissleder R. Bimodal viral vectors and in vivo imaging reveal the fate of human neural stem cells in experimental glioma model. J Neurosci. 2008 Apr 23;28(17):4406-13. Read Abstract.
- Kelly KA, Bardeesy N, Anbazhagan R, Gurumurthy S, Berger J, Alencar H, Depinho RA, Mahmood U, Weissleder R. Targeted nanoparticles for imaging incipient pancreatic ductal adenocarcinoma. PLoS Med. 2008 Apr 15;5(4):e85. Read Abstract.
- Kelly KA, Setlur SR, Ross R, Anbazhagan R, Waterman P, Rubin MA, Weissleder R. Detection of early prostate cancer using a hepsin-targeted imaging agent. Cancer Res. 2008 Apr 1;68(7):2286-91. Read Abstract.
| Muscular System - Luth ES, Jun SJ, Wessen MK, Liadaki K, Gussoni E, Kunkel LM. Bone marrow side population cells are enriched for progenitors capable of myogenic differentiation. J Cell Sci. 2008 May 1;121(Pt 9):1426-34. Epub 2008 Apr 8. Read Abstract.
| Nervous System - Wang X, Liu J, Zhu H, Tejima E, Tsuji K, Murata Y, Atochin DN, Huang PL, Zhang C, Lo EH. Effects of Neuroglobin Overexpression on Acute Brain Injury and Long-Term Outcomes After Focal Cerebral Ischemia. Stroke. 2008 Apr 10. Read Abstract.
- Kohno T, Wang H, Amaya F, Brenner GJ, Cheng JK, Ji RR, Woolf CJ. Bradykinin enhances AMPA and NMDA receptor activity in spinal cord dorsal horn neurons by activating multiple kinases to produce pain hypersensitivity. J Neurosci. 2008 Apr 23;28(17):4533-40. Read Abstract.
- Lagali PS, Balya D, Awatramani GB, Munch TA, Kim DS, Busskamp V, Cepko CL, Roska B. Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration. Nat Neurosci. 2008 Apr 27. Read Abstract.
- Hedlund EM, Pruszak J, Lardaro T, Ludwig W, Viñuela A, Kim KS, Isacson O. Embryonic Stem (ES) Cell-derived Pitx3-eGFP Midbrain Dopamine Neurons Survive Enrichment by FACS and Function in an Animal Model of Parkinson's Disease. Stem Cells. 2008 Apr 3. Read Abstract.
- Wernig M, Zhao JP, Pruszak J, Hedlund E, Fu D, Soldner F, Broccoli V, Constantine-Paton M, Isacson O, Jaenisch R. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Proc Natl Acad Sci USA. 2008 Apr 15;105(15):5856-61. Epub 2008 Apr 7. Read Abstract.
- Mendez I, Vinuela A, Astradsson A, Mukhida K, Hallett P, Robertson H, Tierney T, Holness R, Dagher A, Trojanowski JQ, Isacson O. Dopamine neurons implanted into people with Parkinson's disease survive without pathology for 14 years. Nat Med. 2008 May;14(5):507-9. Epub 2008 Apr 6. Read Abstract.
| Renal System - Zhang J, Brown RP, Shaw M, Vaidya VS, Zhou Y, Espandiari P, Sadrieh N, Stratmeyer M, Keenan J, Kilty CG, Bonventre JV, Goering PL. Immunolocalization of Kim-1, RPA-1, and RPA-2 in Kidney of Gentamicin-, Mercury-, or Chromium-Treated Rats: Relationship to Renal Distributions of iNOS and Nitrotyrosine. Toxicol Pathol. 2008 Apr 25. Read Abstract.
| Tissue Engineering - Rocha FG, Sundback CA, Krebs NJ, Leach JK, Mooney DJ, Ashley SW, Vacanti JP, Whang EE. The effect of sustained delivery of vascular endothelial growth factor on angiogenesis in tissue-engineered intestine. Biomaterials. 2008 Jul;29(19):2884-90. Epub 2008 Apr 8. Read Abstract.
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