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Automated Identification and Location Analysis of Marked Stem Cells Colonies in Optical Microscopy Images.doc

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Automated Identification and Location Analysis of Marked Stem Cells Colonies in Optical Microscopy Images

AutomatedIdentificationandLocationAnalysisof MarkedStemCellsColoniesinOpticalMicroscopy Images VincenzoPaduano1,3,DanielaTagliaferri2,GeppinoFalco2,3*,MicheleCeccarelli1,3 * 1BioinformaticsLab,GeneticResearchInstitute‘‘G.Salvatore’’(IRGS)c/oBioGeMs.c.ar.l.,ArianoIrpino,Avellino,Italy,2StemCellResearchLab,GeneticResearchInstitute ‘‘G.Salvatore’’(IRGS)c/oBioGeMs.c.ar.l.,c.daCamporeale,ArianoIrpino,Avellino,Italy,3DepartmentofScienceandTechnologies,UniversityofSannio,viaPort’Arsa, Benevento,Benevento,Italy Abstract Embryonicstemcells(ESCs)arecharacterizedbytworemarkablepeculiarities:thecapacitytopropagateasundifferentiated cells (self-renewal) and the ability to differentiate in ectoderm, endoderm, and mesoderm derivatives (pluripotency). AlthoughthemajorityofESCsdividewithoutlosingthepluripotency,ithasbecomeevidentthatESCculturesconsistsof multiplecellpopulationshighlightedbytheexpressionofearlygermlineagemarkersduringspontaneousdifferentiation. Hence, the identification and characterization of ESCs subpopulations represents an efficient approach to improve the comprehension of correlation between gene expression and cell specification status. To study markers of ESCs heterogeneity,wedevelopedananalysispipelinewhichcanautomaticallyprocessimagesofstemcellcoloniesinoptical microscopy.Thequestionwetrytoaddressistofindoutthestatisticallysignificantpreferredlocationsofthemarkedcells. Wetestedouralgorithmonasetofimagesofstemcellcolonies toanalyzetheexpressionpatternofthe Zscan4 gene, whichwasanelitecandidategenetobestudiedbecauseitisspecificallyexpressedinsubpopulationofESCs.Tovalidate theproposedmethodweanalyzedthebehaviorofcontrolgeneswhosepatternhadbeenassociatedtobiologicalstatus suchasdifferentiation(EndoA),pluripotency(Pou5f1),andpluripotencyfluctuation(Nanog).WefoundthatZscan4isnot uniformlyexpressedinsideastemcellcolony,andthatittendstobeexpressedtowardsthecenterofthecolony,moreover cellsexpressingZscan4clustereachother.Thisisofsignificantimportancebec