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

In mice,gene targeting by homologous recombination continues to play an essential role in the understanding of functional genomics.This 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 lines.Increasingly,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 capability.Recent technical improvements in the production of germline chimeras have been focused on means of generating ES cells lines with a higher germline potential.There are a number of options for generating chimeras from ES cells (ES chimera mice);however,each method has its advantages and disadvantages.Recent 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 technologies.The 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 lines.The most recent methods for generating specifically ES and iPS chimera mice,including the advantages and disadvantages of each method are also discussed.

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

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 screening.Embryoid 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 aggregation.For standardized mass EB production,a well defined scale-up platform is necessary.Recently,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 formation.Their use allows for continuous monitoring and control of the physical and chemical environment which is difficult to achieve by traditional methods.This review summarizes the current state of production of EBs derived from pluripotent cells in various culture systems.Furthermore,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 research.These cells are equivalent to ES cells in terms of both self-renewal and differentiation capacity.Hence,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.

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

AIM:To compare gene targeting efficiencies,expression profiles,and Ca2+ handling potentials in two widely used mouse embryonic stem cell lines.METHODS: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 procedures.Cells were incubated with primary and secondary antibodies before fluorescence activated cell sorting analysis to compare known pluripotency markers.Moreover,cells were harvested by trypsinization and transfected with a kinase-inactive murine Tyk2 targeting construct,following the BioRad and Amaxa transfection procedures.Subsequently,the cells were cultured and neomycin-resistant cells were picked after 13 d of selection.Surviving clones were screened twice by polymerase chain reaction(PCR) and finally confirmed by Southern blot analysis before comparison.Global gene expression profiles of more than 20 400 probes were also compared and significantly regulated genes were conf irmed by real time PCR analysis.Calcium handling potentials of these cell lines were also compared using various agonists.RESULTS: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 comparison.We did not observe signif icant differences in the levels of many known pluripotency markers.However,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 between the investigated cell lines.Then we further compared the detection and expression of various membrane and in-tracellular Ca2+ receptors and similarly found signif icant(P < 0.05) variations in a number of calcium receptors between these cell lines.CONCLUSION:Results of this study emphasize the importance of considering intrinsic cellular variations,during selection of cell lines for experiments and interpretations of experimental results.