Isscr07poster_sessions[1].pdf

Poster Session IV
Wednesday, June 20
Presenter’s name is in bold and is subject to change.
electric field. In particular, cell displacement rate was higher for cells culturedonto hydrogel substrate and myotubes contraction rate increased as a conse- THE ROLE OF EPHB/EPHRIN-B INTERACTIONS IN CELL
quence of the frequency increasing. This frequency-dependent response of ATTACHMENT AND SPREADING OF MESENCHYMAL STEMS
satellite cells cultured on micropatterned substrates was confirmed by gene DERIVED FROM HUMAN DENTAL PULP TISSUE
Stokowski, Agneiska1, Koblar, Simon A.2, Gronthos, Stan1
1Div of Haematology, Institute of Medical & Veterinary Sciences, Adelaide, Australia, 2School of Molecular and Biomedical Science (Genetics), University GENE EXPRESSION RESPONSES OF TWO MOUSE EMBRYONIC
STEM CELL LINES TO NOCODAZOLE TREATMENT
We have recently identified a novel mesenchymal stem cell-like population Mamo, Solomon1, Kobolak, Julianna1, Becker, Sonja2, Horsch, Marion2, derived from adult third molars called dental pulp stem cells (DPSC). DPSC Beckers, Johannes2, Dinnyes, Andras1
are thought to arise from a neural crest origin and demonstrate the capacity 1Genetic Reprogramming Group, Agricultural Biotechnology Center, to develop into pulp fibroblasts, odontoblasts, perivascular cells, adipocytes Godollo, Hungary, 2Institute of Experimental Genetics, GSF-National and neural-like cells. The Eph family of receptor tyrosine kinases and their Research Center for Environment and Health, Neuherberg, Germany ligands, the ephrin molecules, play an essential role in the migration of neu-ral crest cells during development. The present study examined the expres- Treatment of embryonic stem (ES) cells with microtubule disrupting agents sion pattern and function of EphB/ephrin-B molecules on DPSC mobilization.
like nocodazol is a common practice to arrest the cell cycle at G2/M phase Multiple receptors and ligands were identified on DPSC by real time PCR and for synchronization. The goals of our study were to treat the contrasting ES immunohistochemistry. The function of EphB/ephrin-B molecules during cells with nocodazole, analyze, and compare the gene expression profiles, DPSC attachment and spreading was assessed in the presence of different and finally functionally characterize the differentially regulated genes. The Eph/ephrin-Fc fusion proteins using an established in vitro cell-spreading mouse R1-9 ES cells established from 129X1/SvJ X 129/Sv-CP of 3.5-day assay. In response to either EphB2-Fc or ephrin-B1-Fc, DPSC formed rounder blastocyst and HM-1 ES cells established from inbred (129/Ola X 129/Ola) and smaller cell bodies demonstrated by F-actin distribution, and restricted mouse strain were used for the study. ES cells were cultured on mitomycin C their ability to spread. Additionally, immature focal adhesions were identified treated mouse embryonic fibroblast feeder cell layer. Cells were grown in at the edge of the cell membrane, reminiscent of spreading initiation centres standard ES cell medium changed daily [high glucose DMEM (Invitrogen, (SIC), which have only been observed at early stages of cell spreading. In the Carlsbad, CA) supplemented with Na-pyruvate (0.11% w/w; Invitrogen), presence of Inhibitors, Eph forward signaling through the mitogen activated 0.1mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), fetal bovine protein kinase (MAPK) pathway restricted DPSC spreading, while reverse sig- serum (15% v/v; HyClone; Logan, UT), 1000 U/ml murine-LIF (CHEMICON naling through the ephrin ligand was mediated via the phosphorylation of International, Temecula, CA) and antibiotics (penicillin: 50 U/ml, strepto- the Src homology (SH2) domain activating downstream signaling cascade.
mycin: 50 µg/ml (Sigma-Aldrich)]. Nocodazole (Sigma-Aldrich) was added to Collectively, these studies indicated that EphB/ephrin-B molecules provide an the medium at the concentration of 3 µg/ml for 13 hours and cells were col- inhibitory environment for DPSC attachment, consequently resulting in the lected thereafter. Total RNA was isolated from aliquots of R1-9 ES cells at lack of spreading and detachment. These results may have implications for passage 13 and HM-1 ES cells at passage 23 using RNeasy Midi kit (Qiagen, dental pulp development and regeneration.
Düsseldorf, Germany) procedures. For hybridization, 15 µg of total RNA eachfrom the contrasting samples were used for reverse transcription and label- ing either with Cy3 or Cy5 dyes (Amersham, Buckinghamshire, UK). Thelabeled samples were dissolved in hybridization buffer and added to the FREQUENCY-DEPENDENT GENE EXPRESSION OF ELECTRICALLY
cDNA arrays (custom produced at GSF) containing about 21,000 sequences STIMULATED MICROPATTERNED MOUSE SATELLITE STEM CELLS
and hybridized for 17 hours at 42°C. The microarray results of four inde- ednesday
Carnio, Silvia, Carnio, Silvia
pendent hybridizations were analyzed and differentially regulated genes Principi e Impianti di Ingegneria Chimica, University of Padova, Italy, Padova, were identified. Finally, the results of five randomly selected genes were veri- fied by real-time PCR analysis. The microarray analysis revealed 116 tran-scripts that showed significant variation (p < 0.01) between the two nocoda- The comprehension of spatio-temporal events that affect gene expression is zole treated ES cell lines. Of these, only 8 transcripts were up regulated and one of the most important and intriguing goals of molecular biology. The the rest down regulated in the HM-1 ES cells. Most of these genes were possibility of tuning gene expression by changing the characteristics of the overrepresented in important biological processes like development (17%), external environment has an enormous potential in all fields of modern biol- cell organization and biogenesis (10%), regulatory roles (17%), organogene- ogy. In this work, we have investigated the influence of pulsed exogenous sis (13%), metabolism (16%), signaling cascade (8%) and vesicle mediated electrical field at different frequencies on gene expression profile of muscular transport (4%). Moreover, the verification analysis using real-time PCR has satellite stem cells cultured onto micro-patterned templates. Stem cells were confirmed the results of microarray. Thus based on the detailed analysis and derived from single muscle fibers and seeded onto soft hydrogel surfaces, confirmation of the results with independent method, it is plausible to con- coated with micro-contact printing (µCP) technique. Pulse square waves of clude that the expression profile reflected the true biological responses of different frequencies (ranging between 1 and 0.01 Hz) were applied at dif- these ES cells to nocodazole treatment, and provide valuable information for ferent time points. Transcriptional analysis of electrically stimulated aligned comparative analysis of the two cell lines. Supported by Wellcome Trust satellite stem cell cultures was performed by cDNA microarrays. Total RNA (Grant No.070246), EU FP6 (MEXT-CT-2003-509582, LSHG-CT-2006-518240 from oriented electrically stimulated cultured cells was extracted and used and MRTN-CT-2006-035468), and Hungarian National Science Fund (OTKA for cDNA microarrays; RNA from single muscle fibers was used as control.
Satellite cells on the hydrogel surface are viable and myogenic as they arepositive to desmin and MyoD. They are able to proliferate, fuse and differen-tiate into aligned myotubes that follow the topology template of themicropatterned parallel lanes. Aligned myotubes, positive to both desminand myosin, show synchronous spontaneous contraction. Electrical stimula-tion affects morphology and spatial organization of satellite cells. This spatialarrangement is driven by chemotaxis phenomena. Different cell motilitieswere observed for different applied frequencies of the pulsed exogenous w w w . i s s c r . o r g
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Source: http://medrat.abc.hu/ISSCR07Poster_Sessions_page327.pdf