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Analysis of Klf4 factor stoichiometry effects during iPS cell derivation from mouse embryonic fibroblasts

ABSTRACT: Oct3/4, Sox2, Klf4, and c-Myc re-wire somatic cells to achieve induced pluripotency (iPS cells). However, subtle differences in reprogramming methodology may confound comparative studies of reprogramming-induced gene expression changes. We specifically focused on the design of polycistronic reprogramming constructs, which encode all four factors linked with 2A peptides. Notably, publically available cassettes were found to employ one of two Klf4 variants (Klf4S and Klf4L; GenBank Accession No’s: AAC52939.1 and AAC04892.1), differing only by nine N-terminal amino acids. In a polycistronic context, these two variants generated dissimilar protein stoichiometry, where Klf4L vectors produced more Klf4 protein than those encoding Klf4S. Employing piggyBac transposons, we systematically assessed gene expression changes during mouse embryonic fibroblast (MEF) reprogramming using a panel of novel and publically available polycistronic cassettes (Klf4S: OKMS, STEMCCA, EB-C5, WTSI; Klf4L: OSKM, MKOS, OK+9MS, OKN-HAMS). Strikingly, global gene expression patterns elicited by the panel of polycistronic cassettes at an intermediate stage of reprogramming (day6) diverged according to the Klf4 variant they encoded. Klf4L expressing vectors induced gene expression changes in MEFs similar to over-expression of Klf4 alone. Moreover, we found that these gene expression clusters were associated with differences in reprogramming hallmarks, including colony formation frequencies and the stabilization of transgene-independent pluripotency. Our data exposes a Klf4 reference cDNA variation that alters polycistronic factor stoichiometry, implicating a threshold in determining reprogramming outcomes. We hope this study will help to guide the comparison of compatible public gene expression data sets.

ORGANISM(S): Mus musculus

PROVIDER: GSE65468 | GEO | 2015/03/13

SECONDARY ACCESSION(S): PRJNA274124

REPOSITORIES: GEO

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Project description:Since the first generation of induced Pluripotent Stem cells (iPSCs), several reprogramming systems have been used to study its molecular mechanisms. However, the system of choice largely affects the reprogramming efficiency, influencing our view on the mechanisms. Here we demonstrate that reprogramming triggered by less efficient polycistronic reprogramming cassettes not only highlights Mesenchymal-Epithelial Transition (MET) as a roadblock, but also faces more severe difficulties to attain a pluripotent state even post-MET. Also, in contrast to previous findings, more efficient cassettes can reprogram both wild type and Nanog-/- fibroblasts with comparable efficiencies, routes and kinetics, rebutting previous studies that Nanog is critical for iPSC generation. We revealed that the 9 amino acids in the N-terminus of Klf4 in polycistronic reprogramming cassettes are the dominant factor causing these critical differences. Our data establishes that some reprogramming roadblocks are system-dependent, highlighting the need to pursue mechanistic studies with close attention to the systems to better understand reprogramming. The aim of the experiment is to compare the reprogramming pathways driven by two different polycistronic cassettes (MKOS and OKMS). We have isolated cells at intermediate stages of both MKOS and OKMS reprogramming and analysed their gene expression profiles. 2N- are CD44- ICAM1-, Nanog-GFP-, 3N- are CD44- ICAM1+, Nanog-GFP-, 3N+ are CD44- ICAM1+, Nanog-GFP+, all from day 10 of reprogramming. MKOS/OKMS iPSCs are established iPSC clones, TNG an Embryonic Stem Cell line carrying a Nanog-GFP reporter published in Chambers et al. Cell, 113, 643-655, from this line TNG MKOS and OKMS Embryonic Stem Cells were generated after targeting the Sp3 locus with the MKOS or the OKMS cassette respectively,E14 a reference Embryonic Stem Cell line and MEF are Mouse Embryonic Fibroblasts either wild type or generaterd from TNG MKOS or OKMS ESCs. D6 is the D6s4B5 iPSC line published in O'Malley et al. Nature, 499, 88-91.

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Analysis of Klf4 factor stoichiometry effects during iPS cell derivation from mouse embryonic fibroblasts

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