Poster Abstracts

No. Author Title 1 Cinzia Calzarossa AMNIOTIC FLUID CELLS: A PRELIMINARY STUDY ON THEIR NEURAL

POTENTIAL

2 Zhenzhi Chng Function of Ectodermin in Human Embryonic Stem Cells

3 Claudia Colussi Global Changes in Chromatin Landscape Characterize Duchenne Muscle Dystrophy Response to Histone Deacetylase Inhibitors.

4 Rod Dashwood Dietary HDAC inhibitors: from cell to mice to man

5 Ulrich Hoffmueller Cell Characterization in Regenerative Medicine Applying DNA Methylation Markers

6 Kamran Hussain Goosecoid Function in hESC Differentiation

7 Emma S. Lucas EXAMINATION OF GLOBAL DNA METHYLATION AND DNA METHYLTRANSFERASE EXPRESSION REVEALS DISTINGUISHING FEATURES OF EMBRYONIC STEM CELLS AND FIBROBLASTS

8 Helle Joergensen Chromatin modifiers preferentially regulate replication timing of satellite repeats in ES cells

9 Andriani Margariti Smooth Muscle Cells Differentiated from Embryonic Stem Cells are Regulated by HDAC7

10 Alejandro Marín-Menéndez

New protocols to characterize and separate stem cells and late progenitors

11 Aziz Mustafa Automated Selection and Harvest of mES Colonies on Feeder Cell Monolayers and Gelatine-coated Dishes

12 Stefan Nonchev Putative stem cell niche disruption in the mouse mutants ‘Bald Mill Hill’ (bmh)

13 Carmen Ramirez-Castillejo

New protocols to characterize and separate stem cells and late progenitors

14 H.I. (Trudy) Roach The abnormal expression of proteases by chondrocytes in osteoarthritis follows “unsilencing” due to DNA de-methylation of specific CpG sites in the relevant promoters

15 Nicole MR Schmitz Limited redundancy in phosphorylation of retinoblastoma tumor suppressor protein by cyclin-dependent kinases in acute lymphoblastic leukaemia

16 Andrew Shore DNA methylation controls tissue specific expression of uncoupling protein 1 in adipose tissue.

17 Stefania Mattiussi Epigenetic Reprogramming during SS-dependent Differentiation of Mouse Embryonic Stem Cells:Role of Nitric Oxide and Histone Deacetylases

18 Sanjay Thakrar Somatic cell nuclear transfer and epigenetic reprogramming in zebrafish

19 Sanna Timoskainen Epigenetic reprogramming of OCT4 and NANOG in epithelial cells treated with carcinoma cell extract

20 Thomas Touboul Signalling pathways controlling the differentiation of human Embryonic Stem Cells into definitive endoderm

21 Dirk Winnemoeller Modulating cellular functions by Nanog protein transduction

22 Michelle Wood Investigating the epigenetic events leading to heritable silencing of Cdkn1c (p57

KIP2)

POSTER 1 Cinzia Calzarossa AMNIOTIC FLUID CELLS: A PRELIMINARY STUDY ON THEIR NEURAL POTENTIAL 1) Cinzia Calzarossa 2) Laura Coloca 3) Manuela Mellone 4) Lidia Cova 5) Daniela Giardino 6) Vincenzo Silani 1)Dipartimento di Scienze Neurologiche, Centro ”Dino Ferrari”, IRCCS Istituto Auxologico Italiano, Università degli Studi di Milano, Milano. 2)Laboratorio di Ricerche di Citogenetica Medica e Genetica Molecolare, IRCCS Istituto Auxologico Italiano, Milano. 3)Dipartimento di Scienze Neurologiche, Centro ”Dino Ferrari”, IRCCS Istituto Auxologico Italiano, Università degli Studi di Milano, Milano 4)Dipartimento di Scienze Neurologiche, Centro ”Dino Ferrari”, IRCCS Istituto Auxologico Italiano, Università degli Studi di Milano, Milano 5)Laboratorio di Ricerche di Citogenetica Medica e Genetica Molecolare, IRCCS Istituto Auxologico Italiano, Milano 6)Dipartimento di Scienze Neurologiche, Centro ”Dino Ferrari”, IRCCS Istituto Auxologico Italiano, Università degli Studi di Milano, Milano Human amniotic derived Fluid Cells (AFCs) represent a new resource of stem cells which potential has not been extensively investigated and characterised. The aim of this study was to determinate the AFCs neuro-glia plasticity and potentiality “in vitro” for their future use in the treatment of incurable neurodegenerative diseases. Naïve AFCs were analyzed for the expression of transcripts and proteins of neural markers, neurotophins and cytochines. Furthermore, epigenetic induction towards neural phenotypes were performed by exposure to astrocytes conditionated medium. Briefly, by RT-PCR, AFCs expressed all the neural markers tested, such as Nestin, ?tubulin III, glial fibrillary acid protein (GFAP), brain derived nerve growth factor (BDNF), glial derived neurotrophic factor (GDNF), neurotrophin 3 (NT-3). We confirmed also the presence of other cytokine transcripts, such as tumor necrosis factor ?/? and interleukin 6 and 8. Western blot reveled the presence of Nestin, immature ?tubulin III (Tuj1) and dopamine transporter (DAT), whereas GFAP and dopamine precursor Tyrosine Hydroxilase (TH) were not detected. Moreover, immunocytochemistry pointed out that only few AFCs were positive for the proliferation marker Ki67, while, on the contrary, almost all expressed Nestin, Tuj1 and mature ?tubulin III. In addition, the neuro-glial potential of these cells was proved by their capability to synthesize and release the neurotrophic factor BDNF. Preliminary data on AFCs plasticity were derived after treatment with astrocytes conditionated medium with the detected induction towards neuronal phenotype, proved by the increase of Tuj1 positive cells together with the reduction of Ki67+ cell number. In conclusion, these preliminary data demonstrate the neural and immunomodulatory potentiality of AFCs and sustain their possible use in the cell therapy of neurodegenerative diseases, after in vivo solid pre-clinical demonstration in animal models.

POSTER 2 Zhenzhi Chng Function of Ectodermin in Human Embryonic Stem Cells Zhenzhi CHNG, Roger PEDERSEN, Ludovic VALLIER CIMR, Department of Surgery, Wellcome Trust/MRC Building, Addenbrookes Hospital, Hills Road, Cambridge CB2 2XY Human embryonic stem cells (hESCs) are pluripotent cells derived from embryos at the blastocyst stage. Their embryonic origin confers upon them the ability to proliferate indefinitely in vitro while maintaining the capacity to differentiate into a large variety of cells types. Our group has recently shown that the addition of Activin or Nodal in a chemically defined medium is sufficient to maintain pluripotency of hESCs. On the other hand, inhibition of Activin/Nodal signalling drives differentiation of hESCs towards neuroectoderm, (Vallier et al., 2004). Together, these results suggest that Activin/Nodal signalling pathway maintains the pluripotent status of hESCs by blocking neuroectoderm differentiation. To further understand the molecular mechanisms involving Activin/Nodal signalling in pluripotency, we are studying in hESCs the function of key Smad-binding partners which are known to play a role in neuroectoderm specification. One of these is Ectodermin, a member of the Transcriptional Intermediary Factor 1 (TIF1) family which inhibits mesendoderm differentiation during Xenopus development. Ectodermin is reported to promote neuroectoderm differentiation by enhancing proteasomal degradation of Smad4, a common Smad mediator of the TGFß/Activin/Nodal and BMP4 signalling cascade (Dupont et al., 2005). The function of Ectodermin appears to be conserved in mammalian cells since its overexpression is capable of blocking the anti-proliferative effect of TGFß on cancerous cell lines. Interestingly, Ectodermin has also a function in cell fate commitment of human hematopoietic stem cells which is to mediate erythrocyte differentiation by binding phosphorylated Smad2/3 (He et al., 2006). Our preliminary studies show that Ectodermin is highly expressed in hESCs, with the protein localized predominantly in the nucleus, and that Ectodermin binds Smad4 as well as phosphorylated Smad2/3. By overexpressing Ectodermin in hESCs, we found that it does not block TGFß/Activin/Nodal or BMP4 signalling. Indeed, luciferase reporter assays show that raising the levels of Ectodermin in hESCs increases the transcriptional response to Activin signaling without affecting pluripotency. Finally, differentiation of Ectodermin-overexpressing hESCs shows that Ectodermin is a key mediator of TGFß gene responses during cell fate determination. These findings underscore the importance of Activin/Nodal signalling in hESCs and provide additional insights into its mechanisms of action in pluripotency and differentiation.

POSTER 3 COLUSSI CLAUDIA Global Changes in Chromatin Landscape Characterize Duchenne Muscle Dystrophy Response to Histone Deacetylase Inhibitors. Claudia Colussi1, Barbara Illi2, Stefania Straino1, Aymone Gurtner3, Mario Pescatori4, Enzo Ricci4, Pierlorenzo Puri5, Giulia Piaggio3, Maurizio C. Capogrossi1, Carlo Gaetano1. 1Laboratorio di Patologia Vascolare, Istituto Dermopatico dell' Immacolata, Roma, Italy. 2Laboratorio di Biologia Vascolare e Terapia Genica, Istituto Cardiologico Monzino, Milano, Italy. 3Laboratorio di Oncogenesi Molecolare, Istituto Regina Elena, Roma, Italy. 4 Università Cattolica del Sacro Cuore, Policlinico A.Gemelli, Roma Italy. 5 Istituto Dulbecco, Fondazione Telethon, Parco Scientifico di Castel Romano, Castel Romano, Italy The Duchenne (DMD) and Becker (BMD) type of muscular dystrophies are inherited genetic diseases characterized by progressive muscle degeneration in the presence of mutations and/or deletions of the dystrophin gene product. This study provides the evidence that DMD is characterized by an increased expression and function of the class-I histone deacetylases (HDAC) 2 and 3 which coincides with the presence of an abnormal global and local pattern of histone modifications in the skeletal muscle of human patients and MDX mice. In fact, a variety of specific histone H3 modifications, including Serine 10 phosphorylation, acetylation of Lysines 9 and 14 and methylation of Lysine 79, were abundant in MDX, BMD or DMD patients compared to normal controls. These observations, suggesting for the presence of a transcription-competent open chromatin configuration, were further confirmed by chromatin immunoprecipitation experiments from different MDX DNA promoter regions. Remarkably, histone deacetylase inhibitors (HDACi) reduced HDAC expression restoring global histone modification, chromatin structure and gene expression control at normal level thus establishing a functional link between chromatin remodelling and muscle regeneration processes.

POSTER 4 Rod Dashwood Dietary HDAC inhibitors: from cell to mice to man Melinda C Myzak, Emily Ho and Roderick H Dashwood Linus Pauling Institute, Oregon State University, Corvallis OR 97331, USA Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables, such as broccoli. This anticarcinogen was first identified as a potent inducer of Phase 2 enzymes, but evidence is mounting that SFN acts through epigenetic mechanisms (1-6). It has been shown to inhibit histone deacetylase (HDAC) activity in human colon and prostate cancer lines, with a concomitant increase in global and local histone acetylation status, such as on the promoter region of the P21 gene. SFN also inhibited the growth of prostate cancer xenografts and spontaneous intestinal polyps in mouse models, with evidence for altered histone acetylation and HDAC activities. In human subjects, a single dose of 68 g BroccoSprouts inhibited HDAC activity in peripheral blood mononuclear cells 3-6 h after consumption. These findings provide evidence that one mechanism of cancer chemoprevention by SFN is via epigenetic modulation of HDACs. 1. MC Myzak, PA Karplus, F-L Chung, RH Dashwood (2004) A novel mechanism of chemoprotection by sulforaphane: Inhibition of histone deacetylase. Cancer Res 64:5767-5774. 2. RH Dashwood, MC Myzak, E Ho (2006) Dietary HDAC inhibitors: time to rethink weak ligands in cancer chemoprevention? Carcinogenesis 27: 344-349. 3. MC Myzak, K Hardin, R Wang, RH Dashwood, E Ho (2006) Sulforaphane inhibits HDAC activity in BPH-1, LnCaP and PC-3 prostate epithelial cells. Carcinogenesis 27: 811-819. 4. MC Myzak, WM Dashwood, GA Orner, E Ho, RH Dashwood (2006) Sulforaphane inhibits HDAC in vivo and suppresses tumorigenesis in Apcmin mice. FASEB J 20: 506-508. 5. MC Myzak, RH Dashwood (2006) HDACs as targets for dietary cancer preventive agents: lessons learned with butyrate, diallyl disulfide and sulforaphane. Curr Drug Targets 7(4): 443-52. 6. MC Myzak, K Hardin, P Tong, RH Dashwood, E Ho (2007) Sulforaphane retards the growth of human PC-3 prostate cancer xenografts and inhibits HDAC activity in human subjects. Exp Biol Med, in press.

POSTER 5 Ulrich Hoffmueller Cell Characterization in Regenerative Medicine Applying DNA Methylation Markers Ulrich Hoffmueller1, Stephen Rapko2, Udo Baron1, Georg Wieczorek1, Leslie Wolfe2, Sven Olek1 1) Epiontis GmbH, Berlin, Germany, 2) Genzyme Corp., Cambridge, MA Reliable cell characterization plays a pivotal role in tissue engineering and regenerative medicine. During the research and development process, it provides insights and orientation in respect to changes and differentiation steps that occur in the cells. With a perspective on clinical application of a cell product, thorough quality control is essential to ensure product efficacy, patient safety and to fulfill regulatory requirements. Previously applied techniques for quality control are either based on protein markers, mRNA expression levels or specific enzymatic activity. To a large extend, these markers describe the current short term status of a cell, whereas the key property of a cellular therapeutic is its cell type identity and long term specialization. In the need of better quality control means, new techniques are sought after. The epigenetic phenomenon of DNA methylation possesses a large potential for cell characterization. Specific methylation patterns correlate with differentiation states, cell type and long-term cell functions. In a first step, using genome wide discovery methods and knowledge based approaches, DNA methylation candidate markers are identified. Upon marker validation they are applicable to determine identity, purity and potency of cell samples. Several high performance methylation assay methods permit sensitive and quantitative characterization of cellular therapeutics. The application of the DNA methylation technology is presented on two examples: (1) The results of the collaboration between Genzyme and Epiontis, demonstrating how DNA methylation analysis can be applied for testing the chondrocyte product Carticel®. (2) Marker discovery for a panel of primary human cells comprising several cell types from the mesenchymal lineage. These results indicate that DNA methylation analysis qualifies as a suitable technique for cell characterization and routine release quality control tests for products in tissue engineering and regenerative medicine.

POSTER 6 Kamran Hussain Goosecoid Function in hESC Differentiation Kamran Hussain, Joseph Smith and Roger Pedersen Department of Surgery CIMR Wellcome Trust/MRC Building Addenbrookes Hospital Hills Road, Cambridge, CB2 2XY In Xenopus, zebrafish and mouse, Goosecoid (GSC), has been shown to regulate pluripotent stem cell fate during gastrulation, where mesoderm, endoderm and ectoderm germ layers are formed and the dorsal-ventral and anterior-posterior axes become evident in mammalian embryos. We examined the function of GSC in the differentiation of human Embryonic Stem Cells (hESCs) via two methods, using either a Cre-inducible Z/GSC construct to express a GSC transgene or using the novel TAT-NLS system to deliver His-TAT-GSC protein. For Z/GSC the full length cDNA clone of GSC was cloned into the Z/EG vector (Novak et al., 2000), replacing the original eGFP. Stable Z/GSC expression clones were generated by lipofecting this construct into H9 hESCs to produce neomycin-resistant colonies, which grew into established hESC lines characterised by Cre-inducible over-expression of GSC. The His-TAT-GSC plasmid vector was generated by cloning the full length cDNA clone of GSC into the pTRIEx-1 vector, already containing the His-TAT- sequence. Column purified protein was produced by amplification of the construct in Rosetta competent cells (Novagen), using IPTG to induce over-expression of the His-TAT-GSC protein. When GSC levels were increased in undifferentiated hESCs, cell colony survival was adversely affected. However in hESCs induced to differentiate towards trophoblast by exposure to BMP4, GSC expression caused down-regulation of trophectoderm markers HAND-1, H19 and FOXF1, and up-regulation of extraembryonic endoderm markers alpha-fetoprotein (AFP) and Hepatocyte Nuclear Factor -4 (HNF4). In addition, in hESCs induced to differentiate towards definitive endoderm (Vallier et al., unpublished), GSC expression caused up-regulation of the chemokine receptor CXCR4 and the hepatocyte differentiation markers HEX and HNF4, as well as the extraembryonic endoderm marker AFP. Taken together these results suggest that GSC may be playing a role during differentiative events in early mammalian development, when the extraembryonic tissues as well as the three primary germs layers are established.

POSTER 7 Emma S. Lucas EXAMINATION OF GLOBAL DNA METHYLATION AND DNA METHYLTRANSFERASE EXPRESSION REVEALS DISTINGUISHING FEATURES OF EMBRYONIC STEM CELLS AND FIBROBLASTS Emma S. Lucas1, Nigel M. Smith2, Chris Denning1 and Lorraine E. Young1 1) School of Human Development, The University of Nottingham, Queen’s Medical Centre, Nottingham, NG7 2UH. 2) Department of Clinical Cytogenetics, Nottingham City Hospital, Nottingham, NG5 1PB This study aimed to examine the relative level and organisation of cytosine methylation and DNA methyltransferases in embryonic stem (ES) cells and fibroblasts, using immunocytochemistry. Cytosine methylation is associated with transcriptional silencing, and is stably maintained during cell division, thus mapping cytosine methylation changes during differentiation will begin to unravel a key cell specification mechanism. Image quantification of 5-methylcytosine (5-mC) immunofluorescence in interphase nuclei revealed that HUES-7 and NOTT1 human ES cells were 20% less methylated than BJ fibroblasts, and murine HM-1 ES cells were 30% less methylated than E13.5 embryonic fibroblasts (MEFs). Surprisingly, macroscopic examination of metaphase chromosomes from ES cells and fibroblasts of both species revealed no obvious changes in the chromosomal distribution of 5-mC between cell types. Mouse chromosomes exhibited intense centromeric staining, whereas human chromosome methylation was distributed in a banded pattern that varied considerably between chromosomes. In contrast to the metaphase distribution, the interphase nuclear distribution of 5-mC differed between ES cells and fibroblasts in both species, with more intensely staining foci in fibroblasts relative to ES cells. In general, however, mouse cells exhibited larger 5-mC foci than human cells, consistent with species differences in general chromatin organisation. Examination of DNA methyltransferase expression revealed that the maintenance methyltransferase, DNMT1, was readily detected by immunofluorescence in nuclei of BJ fibroblasts, MEFs and HM-1 ES cells, but not in either human ES cell line. In both species, the de novo methyltransferase DNMT3B was only detected in ES cells, and not in fibroblasts. This study demonstrates relatively undermethylation of ES cells in comparison to fibroblasts, and indicates that this is due to differential expression of methyltransferases. Relative undermethylation in ES cells may reflect a state of transcriptional readiness to allow differentiation down any developmental pathway. The regulation of DNA methylation may also vary between mouse and human ES cells.

POSTER 8 Helle Joergensen Chromatin modifiers preferentially regulate replication timing of satellite repeats in ES cells Helle F Jørgensen1,+, Véronique Azuara1,‡, Shannon Amoils1, Anna Terry1, Mikhail Spivakov1, Tatyana Nesterova2 , Bradley S Cobb1, Bernard Ramsahoye3, Matthias Merkenschlager1, Amanda G Fisher1,+ 1 Lymphocyte Development and 2 Developmental Epigenetics Groups, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN, UK. 3 John Hughes Bennett Laboratory, Division of Oncology, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK. Embryonic stem (ES) cells have an unusual chromatin profile where many developmental regulator genes that are not yet expressed are marked by both active and repressive histone modifications. This poised or bivalent state is also characterised by locus replication in early S-phase in ES cells, while replication timing is delayed in cells with restricted developmental options. Here we used a panel of mutant ES cells lacking important repressive chromatin modifiers to dissect the relationship between chromatin and replication timing. We show a profound difference in the behavior of single copy and repeat sequences. Temporal replication of most single copy loci, including a 5 Mb contiguous region surrounding Rex1, was unaffected in mutant ES cells. In contrast, replication timing of satellite sequences was sensitive to loss of repressive chromatin modifiers including Eed, Dnmt1, Suv39h1/h2 and Dicer, highlighting the importance of these factors for maintaining correct pericentric replication in pluripotent ES cells.

POSTER 9 Andriani Margariti Smooth Muscle Cells Differentiated from Embryonic Stem Cells are Regulated by HDAC7 Andriani Margariti, Qingzhong Xiao, Yanhua Hu, Lingfang Zeng and Qingbo Xu Cardiovascular Division, King’s College London, SE5 9PJ, UK Embryonic stem (ES) cells have the unique capability to transform and replenish the different tissue types in vivo, while they are able to differentiate into specific cell lineages in response to different stimuli in vitro. Vascular progenitor cells that can give rise into either endothelial cells or smooth muscle cells (SMCs) could be important cell types for both vessel development in embryos and vascular disease in adults. On the other hand, the homeostasis of histone acetylation (HAT) and deacetylation (HDAC) possesses a central role in the regulation of gene expression. This study aimed to determine whether HDAC7 had an impact on embryonic stem (ES) cell differentiation toward smooth muscle cells (SMCs). We demonstrate that HDAC7 expression correlates to SMC marker gene induction during ES cell differentiation. Upregulation of HDAC7 expression leads to increase in SMC marker expression, while downregulation of HDAC7 by laminar flow or siRNA caused decrease of SMC marker expression. In ex vivo culture of embryonic cells from SM22-LacZ transgenic mice, over-expression of HDAC7 significantly increased beta-gal positive cell numbers and activity, indicating a crucial role of HDAC7 in SMC differentiation during embryonic development. We also provide evidence that PDGF induces differentiation toward SMCs through the upregulation of HDAC7 transcription and splicing. Our experiments show that PDGF treatment results in an alternative splicing of HDAC7, which correlates to SMC differentiation. HDAC7 siRNA ablates PDGF-induced SMC marker gene expression. These results suggest that PDGF exerts its function through HDAC7. Therefore, these findings provide novel information on the mechanism involved in SMC differentiation, and identify HDAC7 as a new target in therapeutic intervention on vascular disease, where inhibition of vascular progenitor cell differentiation into SMC would be beneficial.

POSTER 10 Alejandro Marín-Menéndez New protocols to characterize and separate stem cells and late progenitors Alejandro Marin-Menendez, Paola Castro-Garcia, Carmen Ramirez-Castillejo Regional Center of Biomedical Research(CRIB). Faculty of Medicine. University of Castilla La-Mancha. Almansa nº14 02006 Albacete Spain Stem cell is as a quiescent cell population with self-renewal capacity in permanent equilibrium with intermediate progenitors, one step more committed to their final phenotype. In contrast to stem cells, progenitors have selft-renewal capacity limited and faster cell cycles which lead them to accumulate chromosomal aberrations due to the telomeric shortening and become tumour cells. Stem cells have been identified in different adult tissues and also in the majority of cancer processes. Despite of these former facts, the differentiation between stem cells and progenitors is not easy because they share the majority of cellular markers. Research people working in stem cells, specially in neural stem cells use different assays to characterize the population of stem cells and to differentiate this populations from the progenitors cells with faster proliferation. These assays are growing curve, neurospheres formed units, size of the neurospheres and Bromodeoxyuridine incorporation. But the majority of these parameters are counted by hand with Neubauer camera or directly by total counting at the inverted microscope. These types of measurement are very tough and the results are very variables. We are establishing a methodology to count the number of neurospheres, size of the neurospheres in culture, and growing curve by flow citometry, with a fast protocol with more reliable results than the previous manually counting.

POSTER 11 Aziz Mustafa Automated Selection and Harvest of mES Colonies on Feeder Cell Monolayers and Gelatine-coated Dishes Dr. Ute Schäfer (1), Dr. Rafael Backhaus (2),Dipl. Ing. Sonja Asche (2) 1) IFOM, University Witten-Herdecke, Cologne, Germany; 2) AVISO Trade GmbH, Talstraße 44, D-07545 Gera, Germany Embryonic mouse stem cells are grown on feeder cell monolayers or on gelatine-coated plates. Selection of single cells or clones can be laborious and challenging. The CellCelectorTM allows for precise harvest of cells and clones due to high resolution imaging, objective software analysis and high precision robotic harvest. The newly developed Scrape-Module permits now uncontaminated harvest of stem cells and clones from feeder cell monolayers or gelatine-coated dishes.

POSTER 12 Stefan Nonchev Putative stem cell niche disruption in the mouse mutants ‘Bald Mill Hill’ (bmh) Dimitri Salameire and Stefan Nonchev University Joseph Fourier, Grenoble 1, Institute Albert Bonniot, INSERM U823, La Tronche, 38 706, France The mouse hairless gene encodes a nuclear receptor co-repressor implicated in the control of in the epidermal differentiation and hair follicle (HF) cycling. The hairless protein (HR) integrity is required for proliferation, migration and differentiation of keratinocyte precursors in the stratum basale and the outer root sheath (ORS) of the HF. This protein interacts with HDACs and seems to harbour motifs required for chromatin remodelling activities. HR likely affects fate and identity of precursor cells by interfering in the WNT and VDR signalling pathways. Spontaneous and targeted mutations at the Hr locus are instrumental in deciphering interaction networks underlying the correct HF development and skin morphogenesis. We have used the recently identified mutants “bald Mill Hill” (bmh) to address gene expression and morphology in the putative stem cell reservoirs of the epidermis and HF. Our data show that disintegration of the lower part of the HF in early catagen is associated to defects in their adhesion properties and in particular to a specific loss of E-cadherin expression. In these mutants the HF is gradually transformed in an abnormal skin structure termed utricle. The utricle’s epithelium appears to be gradually converted to epidermal identity. Indeed, gene gun transfection of bmh skin explants indicates that specific markers for the granular layer (Filaggrin reporter constructs) of the epidermis are expressed in epidermal pattern in upper and lower utricles in bmh mice. Using a panel of stem cell markers to delineate the HF bulge area, we observed a marked disruption of this putative stem cell niche in first catagen of the hair cycle. These results suggest that Hr might control key events in the first steps of precursors proliferation and differentiation, rather than be responsible for the maintenance of stem cell identity.

POSTER 13 Carmen Ramirez-Castillejo New protocols to characterize and separate stem cells and late progenitors. Alejandro Marin-Menendez, Paola Castro-Garcia, Carmen Ramirez-Castillejo Regional Center of Biomedical Research (CRIB) Faculty of Medicine University of Castilla La-Mancha Almansa 14 02006 Albacete Spain Stem cell is as a quiescent cell population with self-renewal capacity in permanent equilibrium with intermediate progenitors, one step more committed to their final phenotype. In contrast to stem cells, progenitors have selft-renewal capacity limited and faster cell cycles which lead them to accumulate chromosomal aberrations due to the telomeric shortening and become tumour cells. Stem cells have been identified in different adult tissues and also in the majority of cancer processes. Despite of these former facts, the differentiation between stem cells and progenitors is not easy because they share the majority of cellular markers. Research people working in stem cells, specially in neural stem cells use different assays to characterize the population of stem cells and to differentiate this populations from the progenitors cells with faster proliferation. These assays are growing curve, neurospheres formed units, size of the neurospheres and Bromodeoxyuridine incorporation. But the majority of these parameters are counted by hand with Neubauer camera or directly by total counting at the inverted microscope. These types of measurement are very tough and the results are very variables. We are establishing a methodology to count the number of neurospheres, size of the neurospheres in culture, and growing curve by flow citometry, with a fast protocol with more reliable results than the previous manually counting.

POSTER 14 H.I. (Trudy) Roach The abnormal expression of proteases by chondrocytes in osteoarthritis follows “unsilencing” due to DNA de-methylation of specific CpG sites in the relevant promoters H.I. Roach (1), N. Yamada (2), K. Hashimoto (2), F. Bronner (1) Bone & Joint Research Group, University of Southampton, Southampton, UK; (2) Tohoku University School of Medicine, Sendai, Japan; (3) University of Connecticut Health Center, Farmington, USA Osteoarthritis (OA) is due to progressive erosion of the articular cartilage so that it can no longer fulfil its role as shock absorber. Once started, the disease cannot be halted. The cartilage degradation is mediated by matrix metalloproteases, mostly MMP-3, -9, -13 and aggrecanases, especially ADAMTS-4 and -5. While the synovium may be a source of the enzymes early in the disease, it is aberrant expression by the articular chondrocytes themselves that underlies the progressive, uncurable nature of the disease. Initially, only a few cells near the surface show aberrant expression. These cells proliferate and abnormal expression is transmitted to daughter cells, suggesting that epigenetic mechanisms might be involved in switching on the aberrant protease expression. With time, all chondrocytes abnormally express proteases and contribute to cartilage erosion. To determine whether the induction of proteases was associated with an “unsilencing”, we investigated whether normally methylated DNA had become demethylated in OA. We extracted DNA from the articular cartilage of controls and OA patients, treated with methylation-sensitive restriction enzymes followed by PCR to assess methylation status of specific CpGs in the promoter regions of MMP-3, -9, 13 and ADAMTS-4. In OA, the fraction of non-methylated CpG sites effectively doubled for each of the four enzymes examined. Notwithstanding considerable variation between different patients, there was at least one CpG site in the promoter of each enzyme that exhibited a statistically significant loss of DNA methylation in samples from OA patients compared with control samples. The results suggest that the abnormal expression of matrix-degrading enzymes in OA follows DNA demethylation. Prevention of demethylation may therefore reduce enzyme-induced cartilage damage and ameliorate the devastating effects of cartilage erosion.

POSTER 15 Nicole MR Schmitz Limited redundancy in phosphorylation of retinoblastoma tumor suppressor protein by cyclin-dependent kinases in acute lymphoblastic leukaemia Nicole M R Schmitz * †, Andreas Hirt * †, Markus Aebi ‡, and Kurt Leibundgut * † From the Department of Clinical Research*, University of Bern, Bern, Switzerland; the Department of Paediatrics †, University Children’s Hospital Inselspital, Bern, Switzerland; and the Swiss Federal Institute of Technology, Institute of Microbiology ‡, Zürich, Switzerland. Cyclin-dependent kinases (CDKs) successively phosphorylate the retinoblastoma protein (RB) at the restriction point in G1 phase. Hyperphosphorylation results in functional inactivation of RB, activation of the E2F transcriptional program, and entry of cells into S phase. RB, unphosphorylated at serine 608, has growth suppressive activity. Phosphorylation of serines 608/612 inhibits binding of E2F-1 to RB. In Nalm-6 acute lymphoblastic leukemia extracts serine 608 is phosphorylated by CDK4,6 complexes, but not by CDK2. We reasoned that phosphorylation of serines 608/612 by redundant CDKs could accelerate phospho group formation, and determined which G1 CDK contributes to serine 612 phosphorylation. Here, we report that CDK4 complexes from Nalm-6 extracts phosphorylated in vitro the CDK2-preferred serine 612, which was inhibited by p16INK4a, and fascaplysin. In contrast, serine 780 and serine 795 were efficiently phosphorylated by CDK4, but not by CDK2. The data suggest that the redundancy in phosphorylation of RB by CDK2 and CDK4 in Nalm-6 extracts is limited. Serine 612 phosphorylation by CDK4 also occurred in extracts of childhood acute lymphoblastic leukemia cells, but not in extracts of mobilized CD34+ hemopoietic progenitor cells. This phenomenon could contribute to the commitment of childhood ALL cells to proliferate, and explain their refractoriness to differentiation-inducing agents.

POSTER 16 Andrew Shore DNA methylation controls tissue specific expression of uncoupling protein 1 in adipose tissue. Andrew Shore-1 John Speakman-3 Paul Kemp-1 Michael Lomax-2 1-National Heart and Lung Institute, Imperial College London. 2-Institute of Medical Sciences, University of Aberdeen. 3-School of Biological Sciences, University of Aberdeen. Background: Regulation of UCP1 expression, the functional marker gene for brown adipose tissue (BAT), in response to cold stress is controlled by a distal enhancer element in the promoter, which contains two cAMP response elements (CREs). We have investigated whether gene-silencing mechanisms could regulate these CREs by examining DNA methylation during alterations in UCP1 gene expression in vivo and in vitro. Methods: Bisulphite mapping was used to determine the methylation state of the UCP1 enhancer in both expressing brown adipose tissue (BAT), non expressing white adipose tissue (WAT) and liver in mice. UCP1 expression in BAT was stimulated by cold stress. Control and deacclimatised groups were also included. Model cell lines for BAT (HIB1B) and WAT (3T3-L1) were used to determine the effect methylation on reporter vectors containing fragments of a -3.1KB region of DNA upstream of the start of transcription with and without forskolin stimulation. Results: The UCP1 enhancer exhibits significantly lower levels of CpG methylation in expressing BAT than in non-expressing WAT and liver (p<0.01). There are significantly lower levels of methylation in CpG’s within the CREs compared to regions flanking the enhancer (P<0.01).Methylation levels do not change when UCP1 expression is stimulated in vivo during cold stress. Comparing methylated and unmethylated reporter constructs in white (3T3-L1) and brown (HIB1B) adipocyte cell lines we present evidence to determine the functional significance of these changes and propose additional sites of interest on the UCP1 promoter. Conclusions: Our results show that methylation of the UCP1 enhancer is lower in BAT but that cold stress does not change the methylation state. We propose that this imprint is of developmental significance in the determination of mesenchymal stem cells to the BAT lineage. Funding: Research relating to this abstract was funded by the BBSRC

POSTER 17 MATTIUSSI STEFANIA Epigenetic Reprogramming during SS-dependent Differentiation of Mouse Embryonic Stem Cells:Role of Nitric Oxide and Histone Deacetylases Barbara Illi°, Francesco Spallotta, Stefania Mattiussi, Maurizio C. Capogrossi, Carlo Gaetano. ° Laboratorio di Biologia Vascolare e Terapia Genica, Centro Cardiologico Fondazione “I. Monzino”, IRCCS, Via Parea 4, Milan, ITALY Laboratorio di Patologia Vascolare, Istituto Dermopatico dell' Immacolata, Via Monti di Creta 104, Rome, ITALY. Introduction: Nitric oxide (NO) is involved in SS signalling in vascular cells and facilitates cardiomyogenesis in ES. In this report, we show the results of experiments performed to assess the molecular mechanism activated by NO in the SS-dependent cardiovascular differentiation of ES. Methods and Results: The expression of developmentally regulated genes requires the fine tuning of the chromatin condensation/decondensation process in a temporally and spatially-regulated manner, a phenomenon strictly dependent on the activity of chromatin modifying enzymes such as Histone Acetyltransferases (HATs) and Histone Deacetylases (HDACs). Further experiments revealed that, after LIF removal, class II HDACs, specifically HDAC4 and 7, shuttled from the cytoplasm to the nucleus of ES cells, confirming the role of these chromatin modifiers in the epigenetic reprogramming of ES cells’ fate. Indeed, treatment of ES with the histone deacetylase inhibitor Trichosatin A (TSA) blocked the spontaneous ES differentiation towards the endothelial lineage. Consistently, the inhibition of HAT activity with Anacardic Acid (AA) anticipated the expression of cardiovascular markers in ES and this result was in parallel with a marked decrease in Oct 3/4 expression in AA-treated cells. The nuclear shuttling of class II HDACs occurring as early as 1 hour after LIF deprivation, was transient as these molecules returned to the cytoplasm in 6 hours. Intriguingly, the direct exposure of ES to NO donors allowed the nuclear retention of these enzymes beyond the 6 hours timepoint. This observation was paralleled by a significant reduction of histone H3 acetylation, indicating the presence of a prolonged histone deacetylase activity in the ES nuclei upon NO treatment. Conclusions: These data suggest a direct role of NO in the regulation of class II HDACs function and in the chromatin remodelling of ES cells and provide new insights about the role of NO during the cardiovascular commitment of embryonic stem cells.

POSTER 18 Sanjay Thakrar Somatic cell nuclear transfer and epigenetic reprogramming in zebrafish Sanjay Thakrar (1), Ian Wilmut (2), John Mullins (1) and Sari Pennings (1) Centre for Cardiovascular Science (1) and Centre for Regenerative Medicine (2), Queen’s Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ. In normal embryonic development, cells lose their pluripotent status and become restricted to a particular differentiation pathway. The activated and repressed states of gene expression, as mediated by DNA methylation and histone modifications, are remarkably stable and not normally reversed or redirected. Such changes however can take place reproducibly in somatic cell nuclear transfer (SCNT) experiments. This shows that the epigenetic program of a differentiated cell nucleus can be reversed by appropriate reprogramming in the recipient egg cytoplasm. Nevertheless, cloning by SCNT is extremely inefficient, and this is thought to be due to insufficient epigenetic reprogramming of the donor cell nucleus. Thus, the main aim of this project is to gain insight into the general epigenetic mechanisms that are essential for reprogramming during SCNT using zebrafish. Accordingly, this poster contains details of the preparatory work conducted for the SCNT process, such as the acquisition of unfertilised recipient oocytes, the visualisation of their polar bodies and maternal pro-nuclei, the derivation of differentiated and undifferentiated donor cells from tissue culture, and finally, preliminary immunocytochemistry studies of epigenetic marks in wild-type embryos.

POSTER 19 Sanna Timoskainen Epigenetic reprogramming of OCT4 and NANOG in epithelial cells treated with carcinoma cell extract Sanna Timoskainen, Christel T. Freberg, John Arne Dahl and Philippe Collas Institute of Basic Medical Sciences, Department of Biochemistry, University of Oslo, PO Box 1112 Blindern, 0317 Oslo, Norway Reprogramming of differentiated cells to pluripotency has been shown to occur by nuclear transplantation into an enucleated egg, fusion with embryonic stem cells or by retroviral transduction of pluripotency factors. We recently reported a reprogramming of epithelial 293T cells treated with an extract of human embryonal carcinoma NCCIT cells (Taranger et al., 2005, Mol. Biol. Cell 16, 5719-5735). Reprogramming is associated with induction of expression of pluripotency genes such as NANOG, OCT4 and enhancement of differentiation potential, suggestive of a reprogramming to a more pluripotent state. We now present evidence of reprogramming of DNA methylation and histone modifications in the OCT4 and NANOG regulatory regions. Bisulfite sequencing analysis throughout the OCT4 regulatory locus shows that OCT4 is methylated in 293T cells but unmethylated in NCCIT cells. Treatment of 293T cells with NCCIT extract promotes demethylation of OCT4. Demethylation is most pronounced in the OCT4 proximal promoter and the distal enhancer. Demethylation occurs as early as week 1 and persists for at least 3 weeks. Likewise, the NANOG promoter is demethylated. Chromatin immunoprecipitation (ChIP) analysis reveals enhanced histone H3 lysine (K) 9 acetylation and H3K9 demethylation on the OCT4 and NANOG loci, in agreement with transcriptional induction of the genes. Minimal changes in di- and trimethylated H3K4 and trimethylated H3K27 levels occur. Cells treated with their own extract may also display transient OCT4 DNA methylation and histone modification alterations that parallel a transient OCT4 upregulation. Therefore, cells exposed to carcinoma extract undergo chromatin modifications indicative of epigenetic reprogramming of pluripotency genes. Nevertheless, extract treatment per se may cause transient epigenetic perturbations manifested by a transcriptional ‘noise’. Current attempts at manipulating the epigenome prior to and after extract treatment aim at inducing more consistent and profound DNA methylation and histone modification changes upon nuclear reprogramming in vitro.

POSTER 20 Thomas Touboul Signalling pathways controlling the differentiation of human Embryonic Stem Cells into definitive endoderm Thomas Touboul (1), Ludovic Vallier (2), Anne Weber (1), Roger Pedersen (2) 1) Inserm U804;Universite Paris-Sud. 2) University of Cambridge, Pedersen Lab, Wellcome Tust/MRC building Addenbrookes hospital,Hills Road CB2 0XY Cambridge The pluripotent status of human embryonic stem cells (hESCs) confers upon them unique value for regenerative medicine, as they are capable of generating a large variety of cell types. However, generating fully functional cells from hESCs and achieving this goal using clinically-compatible conditions remain major challenges. The presence of undefined components in standard culture media and protocols (including animal-derived serum, feeder cells and extracellular matrices) has significantly impeded the achievement of these objectives. Modelling the early steps of embryonic development in vitro may provide the best approach for generating differentiated cells with native properties. The first cell fate decision during vertebrate embryonic development specifies the ectoderm and mesendoderm, the two primary germ layers from which all the body tissues arise. The mesendoderm subdivides in definitive mesoderm and endoderm during the process of gastrulation from which are derivated organs such as pancreas, liver or lungs. The efficiency of cell therapy for the treatment of liver degenerative diseases and diabetes has increased the interest in producing a large quantity of mature liver cells or pancreatic ß-cells. Here, we describe the development and validation of chemically defined culture conditions for achieving specification of hESCs into mesendoderm and particularly into definitive endoderm. Importantly, these defined culture conditions are devoid of animal products, thereby eliminating factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Using a chemically defined medium (CDM) devoid of serum, we studied the efficiency of different growth factors including Activin, FGF2 and BMP4 for driving the differentiation of hESCs into mesendoderm. Based on this analysis, we developed an approach to generate populations of cells expressing the endoderm marker Sox17, GSC and Mixl1 without expressing the extra-embryonic marker Sox7. FACS analyses showed that 80% of the cells express the definitive endoderm CXCR4 demonstrating the near homogeneity of the population generated under these culture conditions. We also investigated the individual effect of FGF, BMP and TGFb signalling pathways on the differentiation of these endoderm progenitors into more definitive cell types including liver cells. Our preliminary results suggest that the endoderm cells progenitors generated in CDM are capable of differentiating further into cells expressing early liver markers including HNF4 and Hex. In sum, we produced definitive endoderm progenitors in chemically defined medium, by using a protocol of differentiation that mimics development. These cells went through a gene expression profile similar to the cells that migrate through the primitive streak during gastrulation. The main goal remaining is to generate mature definitive cell, and we are now attempting to drive these cells to a hepatic fate.

POSTER 21 Dirk Winnemoeller Modulating cellular functions by Nanog protein transduction Dirk Winnemoeller, Michael Peitz, Bernhard Muenst & Frank Edenhofer Stem Cell Engineering Group, Institute of Reconstructive Neurobiology, LIFE & BRAIN Center, University of Bonn and Hertie Foundation Sigmund-Freud-Straße 25, 53105 Bonn, Germany. Embryonic stem (ES) cells differ from somatic cells by their unique capacity to renew themselves and to give rise to all differentiated cell types. The transcription factors Oct4, Nanog and Sox2 build a core transcriptional network that constitutes ES cell identity. These three factors co-occupy the promoters of a large population of genes encoding developmentally important homeodomain transcription factors. Moreover, Nanog alone promotes transfer of pluripotency to somatic cells fused to ES cells. We present a novel experimental strategy to induce Nanog activity into cells in a protein-based manner thereby reversibly modulating stem cell properties without genetic modification. Based on our previously reported Cre protein transduction system we engineered a cell permeable version of the stem cell factor Nanog (TAT-Nanog fusion protein). Treatment with TAT-Nanog induces self-renewal properties in ES cells even in the absence of leukemia inhibitory factor (LIF) in a reversible manner. Moreover, we show the potential of TAT-Nanog to modulate properties of mature cells. For that we used NIH 3T3 cells and murine embryonic fibroblasts (MEFs) as model systems to study the effect of the Nanog stemness factor in differentiated cells. Introduction of Nanog activity into somatic NIH 3T3 cells resulted in an increased growth rate and a transformed phenotype as demonstrated by foci formation and colony growth in soft agar. TAT-Nanog transduction into primary MEFs induced an increased proliferation and bypassed senescence resulting in an ongoing cell expansion. In conclusion our system of cellular manipulation provides a powerful model to introduce stemness factors into somatic cells without altering the genome and overcoming gene silencing. We expect this approach to greatly facilitate the analysis of the molecular link between stemness, epigenetic modifications and transformation.

POSTER 22 Michelle Wood Investigating the epigenetic events leading to heritable silencing of Cdkn1c (p57

KIP2)

Michelle Wood, Dr Rosalind John Genomic imprinting is a process of allele specific gene regulation. Heritable modifications such as DNA methylation and chromatin modifications establish and maintain differences between the parental genomes, resulting in expression from only one allele. Cdkn1c is a maternally expressed gene found within a 1-Mb imprinting domain containing ~16 imprinted genes. Expression of Cdkn1c is controlled by an imprint control region (KvDMR1) containing a differentially methylated CpG island that is only methylated on the maternal allele. Methylation of the KvDMR1 results in silencing of a non-coding RNA transcript Kcnq1ot1. On the paternal allele Kcnq1ot1 is expressed and the surrounding imprinted genes are silent. The aim of this work was to set up an in vitro system using imprint erased embryonic germ cells to look at the epigenetic modifications associated with non-imprinted chromatin and to determine if there is a default process of silencing that occurs as these cells differentiate in culture. Bisulphite sequencing and MSRE-PCR was used to look at the change in methylation within the Cdkn1c promoter region (a somatic differentially methylated mark) and the KvDMR1. Changes in imprinted gene expression during differentiation of the EG cells were examined by RT-PCR. Chromatin immunoprecipitation will be used to look at the changes in histone modifications. Initial results indicate that de novo methylation of Cdkn1c promoter does not occur on differentiation of the EG cells. An examination of the expression levels of the imprinted genes suggests that there may be a default process of silencing in the EG cells that does not require DNA methylation.