(a) Identification of pluripotent reprogramming impediments. DBR genes were selected using UCSC genome browser ( http://genome.ucsc.edu), gene ontology performed with DAVID ( http://david.abcc.ncifcrf.gov/home.jsp; gene ontology:0008624, 0006917, 0012502, 0043065). Candidate list was refined to 29 genes using RNA-Seq, microarray data and literature. (b) Predicted protein interaction network ( http://www.genemania.org). Blue and red dots correspond to candidates, grey dots to potential partners. (c) Ntn1 expression is biphasic during reprogramming. Quantitative reverse transcription–PCR (Q-RT–PCR) depicts Ntn1 expression levels during reprogramming induced by OSKM retroviral expression. Data are expressed relative to MEF as the mean±s.d. (n=3). (d) Schematic of the time schedule of reprogramming experiment. (e) Typical mouse Oct4/GFP iPS colony 14 days following OSKM infection. Scale bars, 80 μm. (f) Netrin-1 depletion reduces reprogramming efficiency. The number of Oct4/GFP-positive colonies produced from sh-scrambled MEFs is set at 100% for each individual experiment. Data are the mean±s.d. (n=3). Student's t-test, P<0.05. (g) Schematic of the time schedule of reprogramming experiment. (h) Recombinant Netrin-1 (rNetrin-1) effect on mouse Oct4/GFP iPS cells generation induced by OSKM RNA sendai virus. rNetrin-1 (0.15or 0.75 μg ml⁻¹) was added daily to the media. Data are the mean±s.d. (n=2). Student's t-test, P<0.01. (i) Netrin-1 expression in various somatic cells. Q-RT–PCR depicts Ntn1 level in mouse embryonic fibroblast (MEF), adult ear (AEF) and tail tip (ATF) fibroblasts and intestinal epithelium. Data are expressed relative to MEF as the mean±s.d. (n=3). (j) Netrin-1 expression is biphasic during intestinal epithelium reprogramming. Q-RTPCR depicts Ntn1 expression at day 2 and day 5 post OSKM induction (dox treatment). Data are expressed relative to untreated intestinal epithelium at day 2 and day 5 as the mean±s.d. (n=2). (k) Oct4/Nanog immunostaining of a miPS colony derived from intestinal epithelium. Scale bars, 100 μm. (l) Netrin-1 depletion reduces reprogramming efficiency of intestinal epithelium. The number of Nanog-positive colonies produced from sh-scrambled sample is set at 100%. Data are the mean±s.d. (n=2). Student's t-test, **P<0.01. (m) Recombinant Netrin-1 (rNetrin-1) effect on iPS cells generation from mouse intestinal epithelium. Data are the mean±s.d. (n=2). Student's t-test, *P<0.05. (n) Netrin-1 depeltion reduces reprogramming efficiency of adult ear fibroblasts (AEFs). Nanog immunostaining was performed after 12–14 days of reprogramming. Data are the mean±s.d. (n=2). Student's t-test, ***P<0.001. (o) Recombinant Netrin-1 effect on human iPS colonies emergence from human foreskin fibroblasts. SSEA4-positive hiPS colonies were counted day 26 post infection.

(a) Naive pluripotency markers expression in ‘control' and ‘rNetrin-1 derived' miPS cells from RNA Sendai virus. Mouse ES cells, three ‘control' iPS clones, nine ‘rNetrin-1-derived' iPS clones and MEF were analysed for Nanog, Esrrb, Rex1/Zfp42 and Oct4 expression by quantitative reverse transcription–PCR. (b) Heatmap of the euclidean distances between the samples as calculated from the variance stabilizing transformation of the count data (DESeq). Samples consist of Oct4/GFP MEF, mouse pre-iPS, ‘control' and ‘rNetrin-1-derived' iPS clones derived by OSKM retroviral infection. (c) ‘Control' and ‘rNetrin-1-derived' mouse iPS cells form embryoid bodies containing derivatives of the three germ layers. Data are normalized to housekeeping genes and expressed relative to t0 as the mean±s.d. (n=3). (d) Teratoma histological analysis from ‘ rNetrin-1-derived' miPS cells showing contribution to the three germ layers. Scale bars, 100 μm. (e) Pluripotency markers expression in ‘rNetrin-1-derived' human iPS cells. Immunostainings for OCT4 and Tra1-60 were performed on a hiPS colony. Scale bars, 50 μm. (f) SSEA4 and TRA1-60 FACS analysis of ‘rNetrin-1-derived' hiPS cells. (g) Karyotype of ‘rNetrin-1-derived' hiPS cells. (h) Histological analysis of a ‘rNetrin-1-derived' hiPS cells teratoma. Scale bars, 200 μm.

(a) DCC expression is maintained at the early stage of reprogramming. Quantitative reverse transcription–PCR depicts DCC and Ntn1 expression profile during reprogramming. Data are expressed relative to MEF as the mean±s.d. (n=3). (b) Schematic depicting the dependence receptor paradigm. In presence of its ligand Netrin-1, DCC transduces a positive signalling leading to differentiation and guidance cues. In the absence of or in Netrin-1-limiting conditions, DCC actively induces caspase-dependent apoptosis. (c) Schematic of the time schedule of reprogramming experiment. (d) DCC depletion improves reprogramming efficiency. The number of Oct4/GFP colonies produced from sh-scrambled MEFs is set at 100% for each individual experiment. Data are the mean±s.d. (n=3). Student's t-test, *P<0.05 or **P<0.01. (e) Netrin-1 depletion effect is rescued by DCC knockdown. Data are the mean±s.d. (n=3). Student's t-test, *P<0.05 or **P<0.01. (f) Scheme depicting DCC^D1290N point mutant model. In the presence of Netrin-1, DCC-positive signalling remains unaffected. In settings of ligand absence, DCC pro-apoptotic activity is abrogated. (g) DCC pro-apoptotic activity abrogation improves reprogramming efficiency. MEFs were derived from embryos of DCC^wt/D1290N mice intercrosses. DCC^wt/wt and DCC^{D1290N/D1290N} were subjected to reprogramming and AP-positive colonies scored at days 12–14. The number of colonies from MEF^wt/wt is set at 100% for each individual experiment. Data are the mean±s.d. (n=3). Comparable transduction efficiencies were achieved with DCC^wt/wt and DCC^{D1290N/D1290N} MEF. Student's t-test, *P<0.05. (h–j) Netrin-1 effect is mainly p53 independent. (h–i) Netrin-1 depletion reduces reprogramming efficiency of p53^−/− MEF. Data are the mean±s.d. (n=3). Student's t-test, *P<0.05 or **P<0.01 or ***P<0.001. (j) p53 protein levels are not modulated by rNetrin-1 treatment. Western blot for p53 and ACTIN was performed on MEF treated with dox for 4 days in the presence or absence of recombinant Netrin-1 (0.75 μg ml⁻¹).

(a) Representative FACS profile of thy1^High and thy1^Neg cells 3–5 days post OSKM infection. Cells were sorted and plated back in reprogramming media for 6–9 additional days before AP activity staining. (b) Representative FACS profile of apoptosis in thy1^Neg cells following Ntn1 and/or DCC depletion. Apoptotic cells percentages are indicated. (c) Quantification of thy1^Neg apoptotic cells following Ntn1 and/or DCC depletion at days 3 and 5. The apoptotic cell percentage in sh-scrambled thy1^Neg MEFs is set at 100%. Data are the mean±s.d. (n=3). Student's t-test, *P<0.05 or **P<0.01 or ***P<0.001; NS, non-significant. (d) Ntn1 and DCC expression levels in thy1^Neg cells. Cells were sorted at days 3 and 6 post OSKM retroviral infection. Quantitative reverse transcription–PCR (Q-RT–PCR) data are expressed relative to MEF as the mean±s.d. (n=3). (e) Scheme depicting Netrin-1 promoter constructs: part A, B and A+B. pGL3-control vector with SV40 promoter and enhancer driving luciferase expression is used as ‘control vector'. (f) Dox-inducible MEFs were transfected with Netrin-1 promoter luciferase constructs and treated with or without dox for 48 h. Data are normalized to Renilla activity and expressed as the mean±s.d. (n=2). Student's t-test, P<0.05. (g) Regulation of Netrin-1 promoter activity by single reprogramming factors. HEK293T cells were transfected with Netrin-1 promoter part A+B and reprogramming factors. Data are the mean±s.d. (n=3). Student's t-test, P<0.05. (h) Mbd3 is recruited to Ntn1 promoter upon reprogramming. ChIP-seq resource was employed to assess Ntn1 promoter occupancy in mouse ES cells and in MEF following OSKM induction. Data are extracted from refs , . Mbd3 is recruited on chromosome 11 (position 68209776-68210746 NCBI36/mm8) in the vicinity of Oct4- (chr11 68210308-68210332), Sox2- (chr11 68210051-68210500) and Klf4- (chr11 68210297-68210303) binding sites. (i) Netrin-1 transcriptional silencing is rescued by Mbd3 depletion. MEFs were transfected with Mbd3 siRNA 24 h before OSKM infection and cells harvested 36 h post OSKM infection. Data are the mean±s.d. (n=3). Student's t-test, *P<0.05 or **P<0.01. (j) OSKM repressive effect on Ntn1 promoter activity is mediated by Mbd3. HEK293T cells were transfected with Mbd3 siRNA before Ntn1 promoter luciferase construct and reprogramming factors. Luciferase activity was assessed 48 h post transfection and normalized to Renilla activity. Data are the mean±s.d. (n=2). Student's t-test, *P<0.05. (k) Netrin-1 silencing is mediated by Mta1 and Chd4. MEFs were infected with lentiviral particles targeting the NuRD complex members Mta1, Mta2 and Chd4 and reprogramming induced 48 h later. Q-RTPCR depicts Ntn1, Mta1, Mta2 and Chd4 expression levels at reprogramming day 2. Data are the mean±s.d. (n=3). Student's t-test, *P<0.05 or **P<0.01.

(a) Netrin-1 and UNC5b expression in blastocyst-derived stem cells. Semi-quantitative RTPCR depicts Ntn1, UNC5b and housekeeping rs17 expression levels. (b) Netrin-1 promoter activity in pluripotent versus somatic contexts. Netrin-1 promoter constructs were transfected in mouse embryonic stem (mES) and mouse embryonic fibroblasts (MEF) cells. Data are normalized to Renilla activity and expressed as the mean±s.d. (n=3). Student's t-test, P<0.05. (c–f) Netrin-1 knockdown decreases colony formation ability of mES cells. sh scrambled, shNtn1#1 and shNtn1#2 mES cells were plated at low density and grown for 6 days in the presence (proliferating condition) or absence (differentiation condition) of LIF before AP staining. (c) Colony formation assay results. (d,e) Counting of AP-positive colonies obtained in the presence (d) or in the absence (e) of LIF. Data are expressed as the mean±s.d. (n=3). Student's t-test, P<0.05. (f) Netrin-1 depletion affects mES cells pluripotency. ‘Unidifferentiated' and ‘mixed' colonies were scored after colony formation assay. The n indicates the number of colonies counted. Data are representative of three independent experiments. (g) Representative FACS profile of apoptosis in sh scrambled and shNtn1#1 mES cells. Cells were plated at low density for 3–5 days and stained with Annexin V/DAPI. (h–j) Netrin-1 exogenous expression improves mES cells self-renewal properties. (h) AP pictures of ‘control' and ‘Netrin-1-expressing' mES cells. Scale bars, 100 μm. (i) Counting of AP-positive colonies. Same protocol as c. Data are expressed as the mean±s.d. (n=3). (j) ‘Undifferentiated' and ‘mixed' colonies were scored after colony formation assay. Data are representative of three independent experiments. (k) Representative FACS profile of apoptosis in ‘control' and ‘Netrin-1-expressing' mES cells. (l) Quantification of apoptosis in ‘control' and ‘Netrin-1-expressing' ES cells. Data are the mean±s.d. (n=3). Student's t-test, *P<0.05. (m–p) Netrin-1 depletion effect is rescued by UNC5b knockdown. (m) Bright-field pictures of colony formation assay results. (n) AP-positive colony counting. Data are expressed as the mean±s.d. (n=3). (o,p) Netrin-1 depletion effect on apoptosis is rescued by UNC5b knockdown. (o) Representative FACS profile of apoptosis in sh scrambled, shNtn1, shUNC5b and shNtn1+shUNC5b mES cells. (p) Quantification of apoptosis following Netrin-1 and/or UNC5b depletion. The apoptotic cells percentage in sh-scrambled mES cells is set at 100%. Data are the mean±s.d. (n=2). Student's t-test, *P<0.05 or **P<0.01. (q) Model recapitulating Netrin-1 function in reprogramming and pluripotency maintenance.