Unknown,Transcriptomics,Genomics,Proteomics

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Characterization of the gene expression profile of porcine iPS-like cells derived by two reprogramming techniques from translocated t(Y;14) fibroblasts

ABSTRACT: In the present study we compare the effect of integrative and non-integrative strategies on pluripotency during pig somatic cells reprogramming to pluripotency. Microarray analysis was used to compare the gene expression profile of non reprogrammed porcine embryonic fibroblasts (PEF) and of iPS-like cells obtained by cell reprogramming of control amniocytes (PB20) or fibroblasts from animals carrying a t(Y;14) translocation (I, NI). We investigate the impact of different reprogramming techniques by using a first integrative method based on viral infection (PB20, I) and a second non-integrative method based on the use of SendaÃ¯ viruses (NI). Moreover, we analyzed the changes in gene expression profile upon embryoÃ¯d formation in the NI-iPS-like cells (EB). We cultured one cell line of porcine embryonic fibroblast, one cell line of iPS-like cells derived from normal amniocytes (PB20), 2 cell lines derived from t(Y;14) fibroblast by the integrative technique (I3 and I4) both at early (ep) and late passages (lp), and three cell lines derived from t(Y;14) fibroblasts by the non-integrative method (NI12, NI13 and NI20). EmbryoÃ¯d bodies from NI cell lines were obtained by 10 days of floating culture in a specific medium under low agitation (EB12, EB13 and EB20). Total RNA from cell pellets of each sample was extracted in triplicate using the Nucleospin RNA kit (Macherey-Nagel) and checked for quantity and quality. After the microarrays experimentation, quality control and normalization, 34 over 36 samples were conserved (represented here).

ORGANISM(S): Sus scrofa

SUBMITTER: Herve Acloque

PROVIDER: E-GEOD-71792 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of nonproductive and staggered productive intermediates arise at different reprogramming time points1-11. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells12,13 prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that during reprogramming the cells progressively lose donor cell identity and gradually acquire iPS cell properties1,2,7,8,10. Here, we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200 that are absent in fibroblasts and iPS cells. Single cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic reprogramming phase2. Expression profiling revealed that the transcriptional regulators Nr0b1 and Etv5 are specifically expressed in this early reprogramming state, preceding activation of key pluripotency regulators such as Rex1, Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus mark some of the earliest known regulators of iPS cell induction. Our study shows an ordered sequence of transitions during the earliest steps of iPS cell reprogramming that deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition. Samples for poised (CD73+ or CD49d+) and non-poised (CD73-) reprogramming samples were FACS sorted 6 and 9 days after induction of Klf4, Oct4, Sox2 and cMyc in Rosa-rtTA +/- mouse embryonic fibroblasts (MEFs). 'Total' populations are expression analyses for unsorted populations analyzed at the same time points. Control populations were also sampled: mouse embryonic fibroblasts (MEFs), partially reprogrammed cells (SC4) and mouse embryonic stem cell (ESC).

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Characterization of the gene expression profile of porcine iPS-like cells derived by two reprogramming techniques from translocated t(Y;14) fibroblasts

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