Cell surface marker mediated purification of iPS cell intermediates from a reprogrammable mouse model

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dc.contributor.authorNefzger, C.M.
dc.contributor.authorAlaei, S.
dc.contributor.authorKnaupp, A.S.
dc.contributor.authorHolmes, M.L.
dc.contributor.authorPolo, J.M.
dc.date.issued2014
dc.description.abstractMature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).
dc.description.statementofresponsibilityChristian M. Nefzger, Sara Alaei, Anja S. Knaupp, Melissa L. Holmes, Jose M. Polo
dc.identifier.citationJournal of Visualized Experiments, 2014; 91(91):1-8
dc.identifier.doi10.3791/51728
dc.identifier.issn1940-087X
dc.identifier.issn1940-087X
dc.identifier.orcidPolo, J.M. [0000-0002-2531-778X]
dc.identifier.urihttps://hdl.handle.net/2440/133534
dc.language.isoen
dc.publisherJOURNAL OF VISUALIZED EXPERIMENTS
dc.rights© 2014 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License
dc.source.urihttps://doi.org/10.3791/51728
dc.subjectStem Cell Biology, Issue 91, Induced pluripotent stem cells; reprogramming; intermediates; fluorescent activated cells sorting; cell surface marker; reprogrammable mouse model; derivation of mouse embryonic fibroblasts
dc.subject.meshFibroblasts
dc.subject.meshAnimals
dc.subject.meshMice, Transgenic
dc.subject.meshMice
dc.subject.meshCell Adhesion Molecules
dc.subject.meshTranscription Factors
dc.subject.meshAntigens, Neoplasm
dc.subject.meshAntigens, Surface
dc.subject.meshFlow Cytometry
dc.subject.meshPregnancy
dc.subject.meshFemale
dc.subject.meshMale
dc.subject.meshEmbryo, Mammalian
dc.subject.meshInduced Pluripotent Stem Cells
dc.subject.meshEpithelial Cell Adhesion Molecule
dc.subject.meshLewis X Antigen
dc.subject.meshThy-1 Antigens
dc.titleCell surface marker mediated purification of iPS cell intermediates from a reprogrammable mouse model
dc.typeJournal article
pubs.publication-statusPublished

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