LETTER doi:10.1038/nature12885 C/EBPa poises B cells for rapid reprogramming into induced pluripotent stem cells Bruno Di Stefano 1,2 , Jose Luis Sardina 1,2 *, Chris van Oevelen 1,2 *, Samuel Collombet 3,4,5 , Eric M. Kallin 1,2 {, Guillermo P. Vicent 1,2 , Jun Lu 6 , Denis Thieffry 3,4,5 , Miguel Beato 1,2 & Thomas Graf 1,2,7 CCAAT/enhancer binding protein-a (C/EBPa) induces transdif- ferentiation of B cells into macrophages at high efficiencies and enhances reprogramming into induced pluripotent stem (iPS) cells when co-expressed with the transcription factors Oct4 (Pou5f1), Sox2, Klf4 and Myc (hereafter called OSKM) 1,2 . However, how C/EBPa accomplishes these effects is unclear. Here we find that in mouse primary B cells transient C/EBPa expression followed by OSKM activation induces a 100-fold increase in iPS cell reprogramming efficiency, involving 95% of the population. During this conversion, pluripotency and epithelial–mesenchymal transition genes become markedly upregulated, and 60% of the cells express Oct4 within 2 days. C/EBPa acts as a ‘path-breaker’ as it transiently makes the chromatin of pluripotency genes more accessible to DNase I. C/EBPa also induces the expression of the dioxygenase Tet2 and promotes its transloca- tion to the nucleus where it binds to regulatory regions of pluripo- tency genes that become demethylated after OSKM induction. In line with these findings, overexpression of Tet2 enhances OSKM- induced B-cell reprogramming. Because the enzyme is also required for efficient C/EBPa-induced immune cell conversion 3 , our data indi- cate that Tet2 provides a mechanistic link between iPS cell reprogram- ming and B-cell transdifferentiation. The rapid iPS reprogramming approach described here should help to fully elucidate the process and has potential clinical applications. Reprogramming of somatic cells into induced pluripotent stem (iPS) cells by the OSKM transcription factors (also called Yamanaka factors) can be divided into a stochastic and a deterministic phase 4,5 . During this process cells undergo a mesenchymal–epithelial transition (MET) and activate endogenous pluripotency genes, paralleled by changes in histone marks, nucleosome positioning and chromatin accessibility 6 . In addition, their promoters become de-methylated, preceded by oxida- tion of methylated CpGs through the dioxygenases Tet2 and Tet1, both of which have been implicated in the establishment of pluripotency 7,8 . Earlier work showed that co-expression of C/EBPa with OSKM increases the reprogramming efficiency of B cells ,15-fold, reaching ,3% of the population 1 . Here we describe that a pulse of C/EBPa followed by OSKM overexpression permits the rapid reprogramming of B cells into iPS cells by activating Tet2 and facilitating accessibility of pluripotency gene promoters to Oct4 binding. Highly efficient reprogramming of somatic cells has recently also been reported with a loss-of-function approach 9 . Committed B-cell precursors (hereafter referred to as B cells) can be induced to transdifferentiate into macrophages at 100% efficiency by forced C/EBPa expression, deregulating ,7,500 genes 10 . Reasoning that chromatin of cells in transition might be more ‘open’ than that of end stages, we tested the effect of transiently exposing B cells to C/EBPa, followed by OSKM expression. B cells were isolated from the bone marrow of reprogrammable mice (containing a tetracycline-controlled transactivator (rtTA) and a doxycycline-responsive OSKM cassette 11 ), infected them with C/EBPa-ER-hCD4 retrovirus, sorted human CD4 1 cells 4 days later and incubated them for different times with b-estradiol (E2) followed by a wash-out (Fig. 1a). Subsequently, OSKM was induced by doxycycline treatment and Nanog 1 colonies scored 12 days post induction (d.p.i.). B cells continuously co-expressing C/EBPa with OSKM showed an 11-fold enhancement in reprogramming efficiency com- pared to cells induced with OSKM alone (B1OSKM cells), confirming earlier reports 1,12 . In contrast, cells treated for 18h with E2 and then treated with doxycycline (Ba91OSKM cells) exhibited a 103-fold colony increase, with cells pulsed for 6h already showing a 74-fold increase (Fig. 1b–d). Clonal assays showed that 92–94% of viable colonies were Nanog 1 after 12 days (Extended Data Fig. 1a–c). OSKM induction of B cells pre-treated with E2 did not increase Nanog 1 colony numbers nor Oct4 expression levels (Extended Data Fig. 2a). A mutant of C/EBPa (BRM-2 (ref. 13)) defective for DNA binding failed to enhance iPS cell generation (Extended Data Fig. 2b). Induced Ba91OSKM cells remained .50% viable (Extended Data Fig. 2c). C/EBPa pulses after OSKM induction had no effect on reprogramming efficiency (Extended Data Fig. 2d), indicating that C/EBPa acts as a path-breaker for OSKM- induced reprogramming. Stable iPS cell lines derived from Ba91OSKM cells (aiPS cells) displayed similar gene expression profiles as embry- onic stem (ES) cells, differentiated into all three germ layers in vitro and in vivo, and efficiently contributed to coat colour chimaerism (Fig. 1e and Extended Data Fig. 3a–e). Ba91OSKM-derived iPS cell colonies could be identified as early as 4 d.p.i. and their numbers increased modestly after 8days (Fig. 1f, g), whereas B1OSKM colonies continued to increase 8–10 d.p.i. (Fig. 1f, g). Retroviral hCD4 expression was found to be silenced within 2–4 days in Ba91OSKM cells compared to ,8 days in B1OSKM cells (Fig. 1h). To test the effect of C/EBPa on OSKM-induced transgene independence, Ba9 and B cells were doxycycline-treated for different times (Fig. 1i). The first transgene-independent iPS cell colonies were observed after 4 days for Ba91OSKM cells, compared to 9–10 days for B1OSKM controls (Fig. 1j). Together these results show that the C/EBPa pulse accelerates iPS cell reprogramming by 4–6 days. Gene expression analyses of Ba91OSKM cells 8 d.p.i. showed the upregulation of 764 out of 1,668 genes expressed more highly in ES cells than in B cells, including all well described pluripotency genes (Fig. 2a). Unsupervised hierarchical clustering analysis revealed that 8 d.p.i. Ba1OSKM cells clustered with ES/iPS cells (Extended Data Fig. 4a). A large part of known pluripotency genes were activated 2–6 d.p.i. (Fig. 2b, c and Extended Data Fig. 4b), reaching levels com- parable to aiPS cells and ES cells within ,1 week (Fig. 2c and Extended Data Fig. 4c). In contrast, they remained essentially silent in B1OSKM cells (Fig. 2b, c). In addition, five genes described to be activated very late (21 d.p.i.) during fibroblast reprogramming 14 became upregulated within 2–4 days in Ba91OSKM cells (Extended Data Fig. 4d). Using Oct4– GFP reporter mice 12 crossed with reprogrammable mice 11 ,Ba91OSKM *These authors contributed equally to this work. 1 Gene Regulation, Stem Cells and Cancer Programme, Centre for Genomic Regulation (CRG), Dr Aiguader 88, 08003 Barcelona, Spain. 2 Universitat Pompeu Fabra (UPF), Dr Aiguader 88, 08003 Barcelona, Spain. 3 Ecole Normale Supe ´ rieure, Institut de Biologie de l’ENS, 45 Rue d’Ulm, Paris F-75005, France. 4 Inserm, U1024, Paris F-75005, France. 5 CNRS, UMR 8197, Paris F-75005, France. 6 Yale Cancer Center, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, USA. 7 Institucio ´ Catalana de Recerca i Estudis Avançats (ICREA), Pg Lluis Companys 23, 08010 Barcelona, Spain. {Present address: Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York 10065, USA. 13 FEBRUARY 2014 | VOL 506 | NATURE | 235 Macmillan Publishers Limited. All rights reserved ©2014