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Embryoid body formation from embryonic and induced pluripotent stem cells:Benefits of bioreactors

World Journal of Stem Cells 2009年第1期1卷 11-21页

作者： Sasitorn Rungarunlert Mongkol Techakumphu Melinda K Pirity andras dinnyes Agricultural Biotechnology Center H-2100 GdllHungary andras dinnyes Molecular Animal Biotechnology LaboratorySzent Istvan UniversityH-2100 GdllHungary Department of Obstetrics Gynaecology and ReproductionFaculty of Veterinary ScienceChulalongkorn UniversityBangkok 10330Thailand BioTalentum Ltd. Aulich Lajos u.26.H-2100GdllHungary

Embryonic stem(ES)cells have the ability to differ-entiate into all germ layers,holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapies and for drug *** body (EB)formation from ES cells is a common method for producing different cell lineages for further applications. However,conventional techniques such as hanging drop or static suspension culture are either inherently incapable of large scale production or exhibit limited control over cell aggregation during EB formation and subsequent EB *** standardized mass EB production,a well defined scale-up platform is ***,novel scenario methods of EB formation in hydrodynamic conditions created by bioreactor culture systems using stirred suspension systems(spinner flasks),rotating cell culture system and rotary orbital culture have allowed large-scale EB *** use allows for continuous monitoring and control of the physical and chemical environment which is difficult to achieve by traditional *** review summarizes the current state of production of EBs derived from pluripotent cells in various culture ***,an overview of high quality EB formation strategies coupled with systems for in vitro differentiation into various cell types to be applied in cell replacement therapy is provided in this review. Recently,new insights in induced pluripotent stem(iPS) cell technology showed that differentiation and lineage commitment are not irreversible processes and this has opened new avenues in stem cell *** cells are equivalent to ES cells in terms of both self-renewal and differentiation ***,culture systems for expansion and differentiation of iPS cells can also apply methodologies developed with ES cells,although direct evidence of their use for iPS cells is still limited.

关键词： Embryoid body Embryonic stem cells Induced pluripotent stem cells Bioreactors Different- iation

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Germline competence of mouse ES and iPS cell lines: Chimera technologies and genetic background

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World Journal of Stem Cells 2009年第1期1卷 22-29页

作者： Ana Claudia Carstea Melinda K Pirity andras dinnyes Genetic Reprogramming Group Agricultural Biotechnology CenterSzent Gyrgyi A u.4 H-2100 GdllHungary Biotalentum Ltd Aulich L. u 26H-2100 GdllHungary Molecular Animal Biotechnology Laboratory Szent Istvan UniversityPater K u 1H-2103GdllHungary

In mice,gene targeting by homologous recombination continues to play an essential role in the understanding of functional *** strategy allows precise location of the site of transgene integration and is most commonly used to ablate gene expression("knock-out"),or to introduce mutant or modified alleles at the locus of interest("knock-in").The efficacy of producing live,transgenic mice challenges our understanding of this complex process,and of the factors which influence germline competence of embryonic stem cell ***,evidence indicates that culture conditions and in vitro manipulation can affect the germline-competence of Embryonic Stem cell(ES cell) lines by accumulation of chromosome abnormalities and/or epigenetic alterations of the ES cell genome. The effectiveness of ES cell derivation is greatly strain-dependent and it may also influence the germline transmission *** technical improvements in the production of germline chimeras have been focused on means of generating ES cells lines with a higher germline *** are a number of options for generating chimeras from ES cells (ES chimera mice);however,each method has its advantages and *** developments in induced pluripotent stem(iPS)cell technology have opened new avenues for generation of animals from genetically modified somatic cells by means of chimera *** aim of this review is to give a brief account of how the factors mentioned above are influencing the germline transmission capacity and the developmental potential of mouse pluripotent stem cell *** most recent methods for generating specifically ES and iPS chimera mice,including the advantages and disadvantages of each method are also discussed.

关键词： Chimeras Transgenic Embryonic stem cells Epigenetic changes Germline competence Induced pluripotent stem cells

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Gene targeting and Calcium handling efficiencies in mouse embryonic stem cell lines

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World Journal of Stem Cells 2010年第6期2卷 127-140页

作者： Solomon Mamo Julianna Kobolak Istvan Borbíró Tamás Bíró Istvan Bock andras dinnyes Genetic Reprogramming Group Agricultural Biotechnology CenterSzent-Gyorgyi Albert ut.4H-2100 GdllHungary Solomon MamoLyons Research FarmUniversity College DublinNewcastleCo DublinIreland Department of Physiology Research Center for Molecular MedicineMedical and Health Science CenterUniversity of DebrecenNagyerdei krt.98H-4032 DebrecenHungary Abiol Ltd. Nagyerdei krt.98H-4032 DebrecenHungary BioTalentum Ltd. Aulich l.26GdllHungary Andras DinnyesMolecular Animal Biotechnology LaboratorySzent Istvan UniversityPáter K.u.1H-2103 GdllHungary

AIM:To compare gene targeting efficiencies,expression profiles,and Ca2+ handling potentials in two widely used mouse embryonic stem cell ***:The two widely used mouse embryonic stem cell lines,R1 and HM-1,were cultured and maintained on Mitomycin C treated mouse embryonic fibroblast feeder cell layers,following standard culture *** were incubated with primary and secondary antibodies before fluorescence activated cell sorting analysis to compare known pluripotency ***,cells were harvested by trypsinization and transfected with a kinase-inactive murine Tyk2 targeting construct,following the BioRad and Amaxa transfection ***,the cells were cultured and neomycin-resistant cells were picked after 13 d of *** clones were screened twice by polymerase chain reaction(PCR) and finally confirmed by Southern blot analysis before *** gene expression profiles of more than 20 400 probes were also compared and significantly regulated genes were conf irmed by real time PCR *** handling potentials of these cell lines were also compared using various ***:We found signif icant differences in transfection eff iciencies of the two cell lines(91% ± 6.1% vs 75% ± 4.2%,P = 0.01).Differences in the targeting efficiencies were also significant whether the Amaxa or BioRad platforms were used for *** did not observe signif icant differences in the levels of many known pluripotency ***,our genomewide expression analysis using more than 20 400 spotted cDNA arrays identified 55 differentially regulated transcripts(P < 0.05) implicated in various important biological processes,including binding molecular functions(particularly Ca2+ binding roles).Subsequently,we measured Ca2+ signals in these cell lines in response to various calcium agonists,both in high and low Ca2+ solutions,and found signif icant differences(P < 0.05) in the regulation of Ca2+ homeostasis betwee

关键词： Embryonic stem cells Microarray Calcium Agonists Transfection Cell culture Pluripotency Gene targeting

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