KLF4 N-terminal variance modulates induced reprogramming to pluripotency.
Bottom Line: Yet, subtle differences in methodology confound comparative studies of reprogramming mechanisms.Strikingly, global gene expression patterns elicited by published polycistronic cassettes diverged according to each KLF4 variant.Our data expose a Klf4 reference cDNA variation that alters polycistronic factor stoichiometry, predicts reprogramming hallmarks, and guides comparison of compatible public data sets.
Affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8507, Japan.Show MeSH
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Mentions: Global gene expression has been used to define reprogramming paths. In order to link gene expression to initial reprogramming behaviors, we subjected d6 mCherry+ cells from PB-TAC-OSKM, -OKMS, -OK+9MS, and -MKOS to microarray analysis (Figures 3A and 3B). Based on OSKM and OKMS GO term enrichment (data not shown) and results from previous reports, we established sample indexing criteria (Figure 3A, left). Krt14 and Krt17 (encoding structural proteins), as well as Sfn (a regulator of epithelial cell growth), are claimed to activate in reprogramming populations with preferred iPSC fate (O’Malley et al., 2013). Expression of Ocln and Cdh1 indicates MET responses (Samavarchi-Tehrani et al., 2010), while additional cell-surface changes are reflected by upregulation of Cadm1 and Fut9 (the fucosyltransferase responsible for producing SSEA-1). The developmental regulators Tbx21 and Bcl11a (transiently upregulated non-ESC markers; Polo et al., 2012) and the pre-iPSC marker Nr0b1 (Mikkelsen et al., 2008) show the extent of reprogramming. As early as d6, these ten marker genes were >2-fold differentially expressed between OK+9MS (green series) and OKMS (red series; Figure 3A, left), and <2-fold differentially expressed in a pairwise correlation of OSKM to OK+9MS (R = 0.993; Figure 3A, top right), certifying our indexing for further pairwise comparisons. Correlation and indexing of OSKM to OKMS verified the influence of KLF4 on early gene expression responses (Figure 3A, bottom right).
Affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8507, Japan.