Netrin-1 regulates somatic cell reprogramming and pluripotency maintenance

Ozmadenci, Duygu; Féraud, Olivier; Markossian, Suzy; Kress, Elsa; Ducarouge, Benjamin; Gibert, Benjamin; Ge, Jian; Durand, Isabelle; Gadot, Nicolas; Plateroti, Michela; Bennaceur-Griscelli, Annelise; Scoazec, Jean-Yves; Gil, Jesus; Deng, Hongkui; Bernet, Agnes; Mehlen, Patrick; Lavial, Fabrice

doi:10.1038/ncomms8398

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Published: 08 July 2015

Netrin-1 regulates somatic cell reprogramming and pluripotency maintenance

Duygu Ozmadenci¹,
Olivier Féraud²,
Suzy Markossian³,
Elsa Kress⁴,
Benjamin Ducarouge¹,
Benjamin Gibert¹,
Jian Ge⁵,
Isabelle Durand⁶,
Nicolas Gadot⁷,
Michela Plateroti⁴,
Annelise Bennaceur-Griscelli²,
Jean-Yves Scoazec⁷,
Jesus Gil⁸,
Hongkui Deng⁵,
Agnes Bernet¹,
Patrick Mehlen¹^na1 &
…
Fabrice Lavial^1,9^na1

Nature Communications volume 6, Article number: 7398 (2015) Cite this article

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Abstract

The generation of induced pluripotent stem (iPS) cells holds great promise in regenerative medicine. The use of the transcription factors Oct4, Sox2, Klf4 and c-Myc for reprogramming is extensively documented, but comparatively little is known about soluble molecules promoting reprogramming. Here we identify the secreted cue Netrin-1 and its receptor DCC, described for their respective survival/death functions in normal and oncogenic contexts, as reprogramming modulators. In various somatic cells, we found that reprogramming is accompanied by a transient transcriptional repression of Netrin-1 mediated by an Mbd3/Mta1/Chd4-containing NuRD complex. Mechanistically, Netrin-1 imbalance induces apoptosis mediated by the receptor DCC in a p53-independent manner. Correction of the Netrin-1/DCC equilibrium constrains apoptosis and improves reprogramming efficiency. Our work also sheds light on Netrin-1’s function in protecting embryonic stem cells from apoptosis mediated by its receptor UNC5b, and shows that the treatment with recombinant Netrin-1 improves the generation of mouse and human iPS cells.

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Introduction

Somatic cell reprogramming to pluripotency involves epigenetic modifications, changes in gene expression, protein degradation and protein synthesis. Reprogramming resets differentiated cells to a pluripotent state and can be achieved by nuclear transfer, cell fusion or transduction of certain transcription factors. The original approach relies on the ectopic expression of the factors Oct4, Sox2, Klf4 and c-myc—collectively known as OSKM^1,2,3. The relative flaws in the understanding of the molecular mechanisms governing induced pluripotent stem (iPS) cells generation hinder the efficient derivation of reprogrammed cells without genetic manipulation. Whereas the modulation of the initial OSKM cocktail with other factors has been extensively documented, comparatively little is known about soluble molecules promoting the process, even if such recombinant factors could be highly valuable for therapeutic applications. Several processes acting as reprogramming roadblocks harbour tumour-suppressive activity such as programmed cell death (PCD) and senescence^4,5,6. As an example, the p53/Puma axis limits iPS cell generation at the late stage of reprogramming and has been related to the dual pro-oncogenic and pro-PCD effect of c-Myc⁷. However, the mechanisms governing cell death independently of p53/c-Myc in the early days of reprogramming remain unclear^4,5,6.

Here we identify the Netrin-1 and its dependence receptor DCC (Deleted in Colorectal Carcinoma) as regulators of somatic cell reprogramming to pluripotency. Netrin-1 is a secreted laminin-related molecule initially identified as an axon guidance cue and more recently proposed as a multifunctional protein implicated both during nervous system development and adult pathologies^8,9. Of interest in the scope of this study, Netrin-1 was shown to act as an oncogene by limiting apoptosis induced by its main dependence receptors DCC and UNC5s (UNC5a, UNC5b, UNC5c and UNC5d)^8,9,10,11. Using a strategy centred on the genomic regions differentially bound by OSKM in somatic and pluripotent cells, we identify members of the Netrin-1/DCC signalling pathway as putative reprogramming roadblocks. We therefore examine Netrin-1 function during iPS cell generation and reveal that the early stage of the process is associated with a transient Netrin-1 transcriptional repression mediated by Oct4 and Klf4 repressive action on the Netrin-1 promoter. We show that such Netrin-1 deficiency limits reprogramming by engaging DCC-induced apoptosis. In parallel, we demonstrate that Netrin-1 protects established pluripotent cells from apoptosis induced by its receptor UNC5b. Importantly, we demonstrate that the Netrin-1/DCC imbalance is corrected by the non-invasive treatment with recombinant Netrin-1 that improves reprogramming efficiency of human and mouse somatic cells.

Results

Netrin-1 level controls iPS cell generation

To identify novel pluripotent reprogramming impediments, we developed a method centred on the OSKM ‘differentially bound regions’ (DBRs; Fig. 1a)¹². DBR were identified by comparing the chromatin immunoprecipitation (ChIP)-Sequencing data for OSKM binding in fibroblasts 48 h after OSKM induction and in established pluripotent cells. This approach led to the identification of 264 ‘DBR genomic regions’ harbouring a differential OSKM binding in both cell types¹². We selected the 705 genes located within DBR as candidate genes potentially hindering pluripotent reprogramming (Fig. 1a). We hypothesized that the partial OSKM binding will delay or abrogate their regulation upon reprogramming and might therefore alter iPS cell generation. By performing gene ontology analysis centred on ‘PCD’, a major reprogramming impediment, we restricted the list to 51 candidates (Step1). Next, 29 candidates were selected (Step2) due to their dynamic expression during mouse embryonic fibroblasts (MEFs) reprogramming (Supplementary Table 1)¹³. The candidates were subjected to a protein interaction network prediction that revealed an unexpected over-representation of members of the Netrin-1 (Ntn1) signalling pathway with genes corresponding to the Netrin-1 ligand and its receptors DCC, UNC5c and UNC5d (Fig. 1b and Supplementary Table 1).

**Figure 1: Netrin-1 level governs reprogramming.**

Ntn1 expression was assessed during MEF reprogramming induced by OSKM retroviral infection and in the resulting iPS cells grown in 2i/LIF (leukemia inhibitory factor) culture conditions. Such approach revealed a biphasic Ntn1 expression profile with a marked downregulation during the first 6 days of pluripotent reprogramming followed by a strong reactivation in iPS cells (Fig. 1c). The same pattern, even though with a faster kinetic, is observed in OSKM dox-inducible reprogrammable MEF, ruling out possible side effects of retroviral infection on gene expression (Supplementary Fig. 1a)¹⁴.

The dynamic expression of Netrin-1 during reprogramming prompted us to investigate whether this factor regulates iPS cell generation. Oct4/GFP knock-in MEF were infected with lentiviral particles encoding a scrambled short hairpin RNA (shRNA) or three different shRNA-targeting Netrin-1 2 days before reprogramming induction by OSKM retroviral infection and 2i/LIF culture conditions (Fig. 1d and Supplementary Fig. 1b). Netrin-1 depletion led to a significant decrease in iPS cell generation efficiency, as denoted by counting GFP-positive colonies activating endogenous Oct4 promoter (Fig. 1e,f). Similar results were obtained by alkaline phosphatase (AP)-positive colony counting, further confirming Netrin-1 effect on iPS cells generation (Supplementary Fig. 1c,d).

Recombinant Netrin-1 improves reprogramming efficiency

Conversely, Netrin-1 downregulation at the early stage of reprogramming prompted us to ask whether the non-invasive treatment with soluble recombinant Netrin-1 (rNetrin-1) could compensate for such endogenous deficiency and improve reprogramming features. We induced Oct4/GFP MEF to reprogramme using non-integrative RNA Sendai virus and 2i/LIF culture conditions. In such settings, the treatment with rNetrin-1 led to a eightfold increase in the number of Oct4/GFP-positive colonies (Fig. 1g,h). A similar trend was observed with other reprogramming strategies such as OSKM retroviral infection (Supplementary Fig. 1e–g). Conversely, and as expected, a Netrin-1-blocking antibody reduces AP+ colony formation (Supplementary Fig. 1h,i).

We then asked whether Netrin-1 effect on iPS cell generation was restricted to MEFs or could be broadened to other adult somatic cells. We derived various primary cells and detected comparable Netrin-1 expression levels in adult fibroblasts from ear (AEF) or tail tip (ATF) and in intestinal epithelium where Netrin-1 is involved in cell survival and homeostasis (Fig. 1i)¹⁰. Interestingly, OSKM expression induced by doxycycline (dox) treatment in cultured intestinal epithelium led to the same transient Ntn1 downregulation than in MEF (Fig. 1j), whereas such phenomenon was not observed in AEF (Supplementary Fig. 1j). Next, we plated intestinal epithelium on irradiated feeder and iPS cells formation by dox treatment. As reported, AP-positive iPS colonies reactivating the naive pluripotency marker Nanog emerged around day 15 (Fig. 1k and Supplementary Fig. 1k)¹⁴. In such context, we demonstrated that Netrin-1 knockdown, before reprogramming induction, reduced by 75% the number of Nanog-expressing iPS colonies, whereas recombinant Netrin-1 (rNetrin-1) treatment increased iPS cells generation by a fourfold factor (Fig. 1l,m). Netrin-1 depletion also reduced iPS cell generation efficiency from AEF (Fig. 1n).

We next assessed whether recombinant Netrin-1 treatment improves human somatic cells reprogramming. Treatment of human foreskin fibroblasts (HFFs) with rNetrin-1 induced to reprogramme by non-integrative RNA Sendai virus led to a 15-fold increase in reprogramming efficiency, as evaluated by counting the number of SSEA4-positive colonies (Fig. 1o). These findings identify (i) Netrin-1 as a novel factor governing iPS cell formation and (ii) recombinant Netrin-1 as a novel soluble factor improving reprogramming efficiency of various human and mouse somatic cells.

In a therapeutic perspective of improving reprogramming efficiency, we addressed whether recombinant Netrin-1 (rNetrin-1) treatment is detrimental to the iPS cell quality. We characterized pluripotency features and genomic stability of mouse (miPs) and human iPS (hiPS) cells derived with rNetrin-1. We expanded mouse Oct4/GFP iPS (miPS) clones obtained with integrative and non-integrative methods in standard conditions (‘control’) or with rNetrin-1 (0.6 μg ml⁻¹; ‘rNetrin-1 derived’). The 9 ‘rNetrin-1 derived’ miPS clones generated using RNA sendai virus harboured similar levels for Nanog, Rex1 and Esrrb RNA than the three ‘control’ miPS clones and mouse embryonic stem (ES) cells, showing that rNetrin-1 is not detrimental to the transcriptional reactivation of such naive pluripotent circuitry (Fig. 2a). In parallel, we performed RNA-sequencing analysis to compare Oct4/GFP MEF, partially reprogrammed (pre-) miPS cells, ‘control’ miPS cells and two different ‘rNetrin-1 derived’ miPS clones generated by OSKM retroviral infection (Fig. 2b and Supplementary Fig. 2a). The mouse pre-iPS cells do not reactivate the Oct4/GFP endogenous locus and do not silence the retroviral transgenes¹⁵. The calculation of the euclidian distances revealed a strong correlation between the ‘control’ and the two ‘rNetrin-1-derived’ miPS clones, confirming similar transcriptome resetting in both conditions (Fig. 2b). In particular, ‘rNetrin-1-derived’ miPS cells harbour similar Nanog, Esrrb and Dppa3 levels than ‘control’ miPS cells, whereas the pre-iPS cells failed at reactivating these genes (Supplementary Fig. 2b). After 40 passages in culture, the quantification of major chromosomal alterations did not reveal any significant difference between miPS cell lines, suggesting that rNetrin-1 treatment does not impact massively chromosomal stability (Supplementary Fig. 2c). In terms of differentiation potential, we showed that ‘rNetrin-1-derived’ miPS cells turn off the endogenous Oct4/GFP promoter activity following LIF withdrawal for 5 days (Supplementary Fig. 2d). Embryoid body formation assays demonstrated their ability to give rise to derivatives of the three germ layers (Fig. 2c). The histological analysis of teratoma formed by ‘rNetrin-1 derived’ miPS cells confirmed the presence of tissues representing all three germ layers (Fig. 2d). In parallel, two hiPS cell lines derived from foreskin fibroblasts with rNetrin-1 were characterized at the molecular and functional levels. As shown in Fig. 2e,f, ‘rNetrin-1 derived’ hiPS cells express the pluripotency-associated markers OCT4, TRA1-60 and SSEA4. Karyotype analysis revealed no abnormalities and teratoma formation confirmed the presence of derivatives of the three germ layers (Fig. 2g,h). Altogether, these results suggest that the treatment with recombinant Netrin-1 is not detrimental to mouse and hiPS cell features.

**Figure 2: Recombinant Netrin-1 treatment is not detrimental to iPS cells quality.**

Netrin-1 limits DCC-induced apoptosis during reprogramming

To decipher the mechanisms downstream of Netrin-1, the expression of its receptors was assessed during MEF reprogramming. In the first 6 days, DCC expression remains steady, whereas UNC5b and UNC5c are rapidly downregulated (Fig. 3a and Supplementary Fig. 2e). DCC belongs to the functional family of dependence receptors sharing the ability of triggering apoptosis in the absence of their ligand (Fig. 3b). The expression of such receptor thus renders the cell dependent for its survival on ligand availability^8,16,17. In Netrin-1-limiting conditions, unbound DCC triggers apoptosis via a cleavage of its intracellular domain¹⁸. We asked whether Netrin-1 transient downregulation limits reprogramming by engaging DCC-induced apoptosis. To test this hypothesis, DCC expression was silenced with shRNA lentiviral particles before MEF reprogramming induced by OSKM retroviral infection. DCC knockdown using three different shRNA significantly improves reprogramming efficiency, as denoted by counting the number of Oct4/GFP colonies (Fig. 3c,d and Supplementary Fig. 2f). More importantly, the Netrin-1 depletion effect is rescued by DCC knockdown, identifying the pair Netrin-1/DCC as a novel mediator of reprogramming (Fig. 3e). In contrast, silencing of the Netrin-1 receptor UNC5b did not modify iPS cell generation efficiency (Supplementary Fig. 2g,h). We generated a mice model in which a DCC point mutation at the critical amino acid required for intracellular cleavage (aspartic acid 1290) is sufficient to block DCC-induced apoptosis while leaving unaffected the positive DCC signalling observed upon Netrin-1 binding (Fig. 3f)¹⁹. MEFs derived from DCC^wt/wt and DCC^{D1290N/D1290N} littermate embryos were reprogrammed by retroviral OSKM infection and 2i/LIF culture conditions. DCC^{D1290N/D1290N} MEFs gave rise to iPS cell colonies with a slightly higher efficiency than DCC^wt/wt MEFs, indicating that DCC effect on reprogramming efficiency is at least partly due to its pro-apoptotic activity (Fig. 3g). Recombinant Netrin-1 (rNetrin-1) effect was compared during DCC^wt/wt and DCC^{D1290N/D1290N} MEF reprogramming. The rNetrin-1 and Netrin-1 blocking antibody effects are nearly abrogated in DCC^{D1290N/D1290N} MEF, confirming that Netrin-1 effect is partly mediated by the inhibition of DCC pro-apoptotic activity (Supplementary Fig. 2i,j).

**Figure 3: DCC pro-apoptotic activity limits reprogramming occurrence.**

Reprogramming factors, especially c-Myc, activate the p53 pathway whose alleviation reduces apoptosis and improves iPS cells generation^4,5,6. We investigated whether Netrin-1 effect was dependent on c-Myc/p53. First, reprogramming experiments were performed without c-Myc (OSK) using integrative and non-integrative methods⁴. We demonstrated that, in the absence of c-Myc, the modulation of Netrin-1 level still impacts iPS cell generation, even if at a lesser extent (Supplementary Fig. 2k,l). To assess whether Netrin-1 effect was p53 mediated, p53^−/− MEFs were infected with shRNA against Ntn1 before OSKM reprogramming. Such approach still led to a significant decrease in the number of AP-positive colonies (Fig. 3h,i). The treatments with rNetrin-1 or Netrin-1 blocking antibody are also still effective in the absence of p53 (Supplementary Fig. 2m,n). Moreover, p53 protein levels remained unchanged by rNetrin-1 treatment at reprogramming day 4 (Fig. 3j). Taken together, these data support the view that DCC pro-apoptotic activity limits pluripotent reprogramming mainly in a c-Myc/p53-independent manner.

We next analysed apoptosis occurrence at the early stage of reprogramming by FACS combining the cell surface markers thy1 and SSEA1 with 4,6-diamidino-2-phenylindole (DAPI) and Annexin V^15,20,21. The starting MEF population express thy1, whereas SSEA1 is repressed (thy1^Positive/SSEA1^Negative). FACS analysis performed at days 3 and 5 post OSKM infection confirmed the emergence of a thy1^Negative/SSEA1^Negative subpopulation (thy1^Neg) enriched in reprogramming cells, as shown by morphology and AP staining (Fig. 4a and Supplementary Fig. 3a). We showed that, in the early days of reprogramming, Netrin-1/DCC ratio modulation directly impacts apoptosis occurrence with thy1^Neg cells being particularly sensitive to such phenomenon (Fig. 4b,c and Supplementary Fig. 3b–e). Following Ntn1 silencing, the percentage of thy1^Neg apoptotic cells is increased by 30% at reprogramming day 3 (Fig. 4b,c and Supplementary Fig. 3e). Conversely, following DCC knockdown, the percentage of thy1^Neg apoptotic cells is decreased by 20% and 40% at reprogramming day3 and 5, respectively. As expected, Ntn1 and DCC double knockdown led to a reduction in apoptosis occurrence (Fig. 4b,c and Supplementary Fig. 3e). Taken together, these data support the view that the early Netrin-1/DCC imbalance limits iPS generation by inducing DCC-mediated apoptosis predominantly in thy1^Neg reprogramming cells. Ntn1 and DCC expression levels were next assessed at this stage of reprogramming. At day 3, Ntn1 expression is strongly downregulated in thy1^Neg cells, whereas DCC remains steady—situation favouring DCC-mediated cell death (Fig. 4d). However, 3 days later, the pattern is inverted with Ntn1 reactivation and DCC silencing, possibly reflecting the elimination of the DCC-expressing cells (Fig. 4d). The Ntn1 activation is maintained at day 9 in the thy1^Negative/SSEA1^Positive cells co-expressing Nanog and in established iPS cells (Supplementary Fig. 3f)^15,21.

**Figure 4: Ntn1/DCC balance modulates apoptosis at the early stage of reprogramming.**

Oct4, Klf4 and the NuRD complex repress Netrin-1 expression

We then asked which epigenetic mechanism triggers Netrin-1 transient transcriptional repression. In agreement with its location within DBR, the Ntn1 locus is occupied by Oct4, Sox2 and Klf4 in mouse ES cells (mm8 genome browser: Oct4-binding chr11: 68210308-68210332, Sox2-binding chr11: 68210051-68210500, Klf4-binding chr11: 68210297-68210303; Supplementary Fig. 4a)^12,22. We evaluated OSKM effect on the activity of Netrin-1 promoter²³. Luciferase assays performed on OSKM dox-inducible MEF transfected with different promoter constructs demonstrated the ability of the reprogramming cocktail to repress specifically Netrin-1 promoter activity, whereas control SV40 promoter activity remains steady (Fig. 4e,f). A similar approach was employed in HEK293T cells with single reprogramming factors to reveal that Oct4 and Klf4 are responsible for Netrin-1 promoter repression (Fig. 4g).

Different epigenetic complexes, such as Polycomb, WDR5 and NuRD, are associated with early development and connected to the reprogramming factors activity^24,25,26,27. Among them, the nucleosome remodelling and deacetylase (NuRD) complex member Mbd3 has been identified as a strong reprogramming roadblock, with OSKM inducing a global redistribution of Mbd3 on target genes in MEF (1,177 binding regions in MEF compared with 8,657 after OSKM induction)²⁶. ChIP-Sequencing data revealed that, upon dox induction in MEF, Mbd3 gets specifically recruited on the Netrin-1 promoter, at a genomic location that corresponds exactly to Oct4-, Sox2- and Klf4-binding site in mouse ES cells (mm8 genome browser: Mbd3-binding chr11: 68209776-68210746; Fig. 4h)¹². As depicted in Fig. 4i, Mbd3 knockdown before reprogramming induction led to a rescue of Ntn1 downregulation. In agreement with this finding, comparison of Netrin-1 expression profile during MEF^wt/wt and Mbd3^fl/- reprogramming shed light on a significantly higher induction of Ntn1 in day 8 Mbd3^fl/- MEF reprogramming than in day 11 MEF^wt/wt, whereas Ntn3 expression profile remains unaffected (Supplementary Fig. 4b). Luciferase assays confirmed that Oct4 and Klf4 repressive effect on Netrin-1 promoter activity is mainly mediated by Mbd3 (Fig. 4j). Moreover, depletion of the other NuRD components Mta1 and Chd4 before reprogramming also rescued Ntn1 downregulation, whereas Mta2 knockdown had no effect (Fig. 4k). Taken together, these data demonstrate that a Mbd3/Mta1/Chd4-containing NuRD complex is responsible for the transient Netrin-1 transcriptional repression observed upon reprogramming.

Netrin-1 limits apoptosis in pluripotent cells

Finally, owing to the high Netrin-1 expression level in iPS cells, we asked whether Netrin-1 contributes to pluripotency maintenance. Taking advantage of blastocyst-derived stem cells in vitro, we showed that Ntn1 expression is confined to ES cells and absent from the extra-embryonic-derived TS and XEN cells, whereas UNC5b is detected in the three in vitro cellular models, even if at a reduced level compared with MEF (Fig. 5a). We next showed that the Netrin-1 promoter activity is strong in pluripotent ES cells compared with MEF, because of genomic elements located in the promoter part B (Fig. 5b). We asked whether Netrin-1 is expressed during pre-implantation development but Ntn1 mRNA could not be detected in blastocysts by in situ hybridization. This observation was confirmed by exploiting single-cell PCR resource performed in mouse blastomeres and in ES cells²⁸. Ntn1 and its receptors (DCC, UNC5a-d) are not detected at the blastocyst or epiblast stages in vivo but Ntn1 and UNC5b are activated during the ES cell derivation process (Supplementary Fig. 4c). To evaluate Netrin-1 function, Netrin-1 knockdown ES cells were established by lentiviral infection with two different shRNA (Fig. 5c–g and Supplementary Fig. 4d). In a colony formation assay, we showed that Netrin-1 depletion led to a drastic reduction in the number of AP-positive colonies obtained in the presence or absence of LIF, demonstrating that Netrin-1 contributes to maintain ES cell self-renewal properties (Fig. 5c–e). Moreover, when the morphology of the mouse ES colonies was compared, a 30% decrease in the percentage of ‘undifferentiated’ colonies (round, strongly and homogeneously positive for AP) and a concomitant 30% increase in the percentage of ‘mixed’ colonies (weak and heterogeneous AP staining) was detected with the Netrin-1-depleted population, suggesting a defect in the stability of the pluripotent state (Fig. 5f). This phenotype could be due to a defect in cell cycle and/or survival in such challenging conditions. No significant change on cell cycle could be detected upon Netrin-1 knockdown (Supplementary Fig. 4e). However, Netrin-1 depletion led to a significant increase of the percentage of apoptotic cells, whereas necrotic occurrence remains unaffected, demonstrating a protective effect of Netrin-1 in pluripotent cells (Fig. 5g and Supplementary Fig. 4f). Conversely, stable mouse ES clones expressing exogenously Netrin-1 present an improved self-renewal ability in colony formation assay (Fig. 5h–j). The effect on the ES cell morphology was particularly striking with colonies that remained undifferentiated even after 5 days in differentiation conditions in the absence of LIF (Fig. 5h). Interestingly, such Netrin-1 exogenous expression reduced significantly apoptosis occurrence in ES cells (Fig. 5k,l and Supplementary Fig. 4g). Owing to UNC5b expression in ES cells, we asked whether Netrin-1 function was mediated by this dependence receptor. UNC5b knockdown rescues Netrin-1 depletion effect on colony formation assay and apoptosis occurrence, identifying the pair Netrin-1/UNC5b as novel regulators of apoptosis in pluripotent ES cells in vitro (Fig. 5m–p and Supplementary Fig. 4h).

**Figure 5: Netrin-1 promotes pluripotency features *in vitro*.**

Discussion

Together, the data presented here described the importance of the dependence receptor paradigm both in the regulation of reprogramming and of pluripotency, the balance between Netrin-1 and its receptors DCC or UNC5b being key to regulate both processes. Dependence receptors form a family of more than 20 membrane receptors that are not linked by their structure, but by the common functional trait of triggering active apoptosis in the absence of their respective ligand. Thus, cells expressing these types of receptor are dependent on the presence of ligands in the extracellular environment to survive.

Along this line, it was recently formerly demonstrated that the Netrin-1/DCC pair plays a major role in cancer with DCC constraining tumour progression^19,29 and with Netrin-1-promoting tumour progression^10,30 via their respective pro-apoptotic/survival activity. This dual activity may also be implicated in neuronal navigation and developmental angiogenesis^11,31

In this study, we identified unexpected functions for the secreted cue Netrin-1 in regulating iPS cell generation and pluripotency maintenance (Fig. 5q)²⁶. On the one hand, we propose an integrated model deciphering how the early dichotomous function of OSKM leads to the recruitment of a repressive Mbd3/Mta1/Chd4-containing NuRD complex to the Ntn1 promoter, leading to Netrin-1 deficiency. This finding reinforces and broadens the view that early OSKM impaired binding to the somatic genome, combined with the recruitment of antagonistic epigenetic complexes, result in aberrant gene expression that is detrimental for iPS cell generation^12,26. We show that the destabilization of the balance between Netrin-1 and its receptor DCC leads to apoptosis induction at the early stage of reprogramming and reduces iPS cell generation. Importantly, we established Netrin-1 as a recombinant protein able to improve the generation of miPS and hiPS cells under specific culture conditions. On the other hand, our work shed light on another Netrin-1 function in ES cell self-renewal, notably by constraining UNC5b-mediated apoptosis. Our work, by providing novel insights into the early stage of reprogramming and pluripotency maintenance, should shorten the road to develop clinically useful iPS cells.

Methods

Cell culture and RNAi experiments

MEFs were derived from E13.5 embryos from different backgrounds. Mouse pre-iPS, iPS and Cgr8 ES cells were cultured on gelatin or on irradiated MEF in KSR+LIF or KSR 2i+LIF³² as previously described³³. Mouse XEN and TS cells were grown as previously reported³³. Intestinal epithelium was grown and induced to form iPS cells, as previously reported¹⁴. 293T and plat-E cells were grown in DMEM supplemented with 10% FCS and penicillin/streptomycin. For reprogramming, when stipulated, 2i components were added 5–6 days post OKM induction (infection or dox treatment). PD0325901 and chiron were purchased from Merck Millipore. shRNA experiments were performed using pLKO.1 vectors and short interfering RNA (siRNA) using esirna from Sigma (SHCLNG-NM_008744 for Ntn1, SHCLNG-NM_007831 for DCC, SHCLNG-NM_029770 for UNC5b and EMU022741 for Mbd3, SHCLNG-NM_054081 for Mta1, SHCLNG-NM_11842 for Mta2 and SHCLNG-NM_145979 for Chd4). TRC clone numbers are available upon request. Luciferase assays were performed using dual luciferase reporter assay system (Promega, E1910). MEF and HEK293 cells were transfected using Lipofectamine 2000 (Life Technologies). Human Mbd3 stealth siRNA were employed (Life Technologies, hss147580 and hss147581). The different constructs of human Netrin-1 promoter were already described²³. HA-Flagged Netrin-1 cDNA was cloned into the PacI and XhoI restriction sites of pcagg-ires-puro plasmid giving rise to pcagg-Netrin-1-ires-puro vector (pcagg-ires-puro is a kind gift from Ian Chambers). ES cells were stably transfected using FugeneHD reagent (Promega, E2691).

Antibodies

Primary antibodies used in this study for immunofluorescence and FACS are as follows: anti-Oct4 (Santa Cruz, C10), anti-Nanog (Cosmobio, RCA B000 2P-F), anti-thy1 (Ebiosciences, 53-2.1), anti-SSEA1 (Stem Cell Technologies, 60060PE) and NL493-conjugated anti-SSEA4 antibody (R&D Systems, SC023), anti-Tra1-60 (Stemgent, 09-0009).

Retroviral and lentiviral production

Plat-E cells were used to produce pMXs-based retroviruses containing the cDNA for Oct4, Sox2, Klf4 or cMyc, as previously described³². PlatE-generated mCherry retrovirus was used to monitor MEF infection efficiency in each experiment. 293T cells were used to produce lentiviral pLKO-derived particles.

Mouse iPS cells generation

For iPS induction, MEFs were seeded per well of a six-well plate and 12 h later overnight infections were performed using equal amounts of each indicated retrovirus in the presence of 8 μg ml⁻¹ polybrene (Sigma, 107689). The day after, wells were rinsed twice and cells were cultivated in medium supplemented or not with 150 or 750 ng ml⁻¹ recombinant Netrin-1 (rNetrin-1). Forty-eight hours after infection, MEFs were reseeded onto irradiated feeders in iPS media. Media were replaced every day with medium alone or freshly supplemented with rNetrin-1. Emerging iPS colonies were monitored until days 12–14 when cells were harvested or individual colonies picked for further analysis. Similar protocol was employed with RNA sendai virus (cytotune 2.0, Life Technologies). The dox-inducible mice were purchased from the Jackson Laboratories (stock number 011001). MEFs, adult fibroblasts from ear and tail tip and intestinal epithelium were plated on irradiated feeders and treated with doxycycline at 2 μg ml⁻¹. Recombinant Netrin-1 was purchased from Adipogen (AB40B-0075). Because of the poor stability of the recombinant Netrin-1 molecule and variability between batches, batch activity was systematically tested in cell death assays using UNC5b-transfected cells. The blocking Netrin-1 antibody was obtained from the Netris Pharma company.

Human iPS cells generation

Reprogramming experiments have been performed on 5 × 10⁵ HFFs (Millipore) seeded in a well of six-well plates and grown in FibroGRO-LS medium (Millipore) until replating on MEFs. Cells were infected overnight with Sendaï viruses (Cytotune, Life Technologies) each of them at multiplicity of infection (MOI)=3. The day after, wells were rinsed twice and cells were cultivated in medium supplemented or not with 150 or 750 ng ml⁻¹ rNetrin-1. Media were replaced every day with medium alone or freshly supplemented with rNetrin-1. Seven days post infection, cells were seeded onto freshly mitomycin-C-treated MEF at a density of 1 × 10⁵ or 2 × 10⁵ HFF cells per plate. The next day medium was switched to DMEM/F12 culture medium supplemented with 20% PluriQ Serum Replacement (GlobalStem), 0.1 mmol l⁻¹ non-essential amino acids, 1 mmol l⁻¹ L-glutamine, 0.1 mmol l⁻¹ 2-mercaptoethanol, penicillin/streptomycin (all of them from Life Technologies), 12.5 ng ml⁻¹ recombinant human basic fibroblast growth factor (Milytenyi Biotec). At day 26, fully reprogrammed colonies were manually picked and transferred to freshly mitomycin-C-treated MEF for amplification.

Apoptosis detection

Cells were stained with anti-thy1 and apoptosis quantified with the Dead Cell Apoptosis Kit with Annexin V FITC and DAPI (Life technologies, V13242).

Colony formation assay was performed as previously described³².

FACS sorting

Analysis was performed on a BD LSRFortessa. Sorting was performed on a BD FACSDiVa. Cells were sorted, washed immediately and centrifuged before being plated directly in fresh medium or frozen for RNA extraction and gene expression analysis.

RNA-Sequencing

RNA quality was analysed using a Bioanalyser (Agilent). Libraries were constructed and sequenced on an illumina Hiseq 2000 by the MGX company. GEO record series GSE54107.

Teratoma formation assay

5 × 10⁶ iPS cells were injected under the kidney capsules of 7-week-old severe combined immunodeficient (SCID) mice (CB17/SCID, Charles River). After 3 weeks, the mice were euthanized and lesions were surgically removed and fixed in formol or in 4% paraformaldehyde for cryosections. Same procedure was employed with 5 × 10⁵ iPS cells injection in testicles. Human teratoma formation was conducted as previously described³⁴. All animal procedures were performed in accordance with the institutional guidelines (French ceccapp project 01369.01).

Quantifications and Statistics

Comparison of the reprogramming efficiencies was performed on littermate embryos. At least two different batches of MEFs and reagents were used. Statistical analyses of mean and variance were performed with Prism 6 (GraphPad Software) and Student’s t-test. The littermates belong to an experimental group determined by the genotyping.

Additional information

How to cite this article: Ozmadenci, D. et al. Netrin-1 regulates somatic cell reprogramming and pluripotency maintenance. Nat. Commun. 6:7398 doi: 10.1038/ncomms8398 (2015).

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Acknowledgements

We are grateful to Hervé Acloque for the STN cell line, and to Michelle Percharde for reagents and protocols. We thank Jose Silva and Rodrigo Santos for Oct4/GFP MEF. We thank the contribution of AniRA-PBES platform (SFR Biosciences-UMS3444/US8), especially Sophie Blanc, Marie Teixeira and Denise Aubert for technical advice and support regarding characterisation of stem cells. We also thank Frederic Flamant. We are grateful to Veronique Azuara for discussions and critical reading of the manuscript. This work was supported by institutional grants from CNRS, INSERM, University of Lyon, Centre Léon Bérard, from the Fondation pour la Recherche Médicale, ATIP/AVENIR (F.L.) and from the Ligue Contre le Cancer, INCA, ANR, ERC, FRM, EU FP7 Hermione-2man and Fondation Bettencourt (P.M.).

Author information

Patrick Mehlen and Fabrice Lavial: These authors contributed equally to this work

Authors and Affiliations

Apoptosis, Cancer and Development Laboratory - Equipe labellisée ‘La Ligue’, LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon, INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, 69008, France
Duygu Ozmadenci, Benjamin Ducarouge, Benjamin Gibert, Agnes Bernet, Patrick Mehlen & Fabrice Lavial
INSERM U935, ESTeam Paris-Sud, Université Paris-Sud, Villejuif, France
Olivier Féraud & Annelise Bennaceur-Griscelli
Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique, École Normale Supérieure de Lyon, Lyon, 69364, France
Suzy Markossian
CGϕMC UMR 5534 - Université Lyon 1, Campus de la Doua, Bâtiment Gregor Mendel 16, Rue Dubois, 69622, Villeurbanne, France
Elsa Kress & Michela Plateroti
Center for Life Sciences, Peking University, Yiheyuan Road Haidian District, Beijing 100871, P.R. China
Jian Ge & Hongkui Deng
Cytometry Facility, Centre de Cancérologie de Lyon, INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, 69008, France
Isabelle Durand
Anipath, Université de Lyon, Hospices Civils de Lyon, Hôpital Edouard Herriot, Anatomie Pathologique, Lyon, 69437, France
Nicolas Gadot & Jean-Yves Scoazec
Cell Proliferation Group, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, W12 0NN, London, UK
Jesus Gil
Cellular Reprogramming and Oncogenesis Laboratory, ATIP/Avenir Laboratory, Centre de Recherche en Cancérologie de Lyon, INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, 69008, France
Fabrice Lavial

Authors

Duygu Ozmadenci
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Olivier Féraud
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Suzy Markossian
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Jian Ge
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Annelise Bennaceur-Griscelli
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Agnes Bernet
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Patrick Mehlen
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Fabrice Lavial
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Contributions

D.O. performed the experiments from Figs 3, 4 and 5. O.F. and L.B.-G. performed the human reprogramming experiments and the characterization of human iPS cells from Figs 1 and 2. E.K. and M.P. prepared intestinal epithelium for reprogramming experiments. S.M. performed teratoma experiments with mouse iPS cells for Fig. 2. B.D. and B.G. provided mice for MEF derivation. J.G. and H.D. performed reprogramming experiments. ID performed flow cytometry analyses from Figs 4 and 5. N.G. and J.-Y.S. performed histological analysis of mouse teratoma from Fig. 2. J.G. performed iPS cells characterization assays and provided F.L. with reagents. F.L. performed experiments from Fig. 1–5. F.L., A.B. and P.M. designed the study and F.L. and P.M. wrote the manuscript.

Corresponding authors

Correspondence to Patrick Mehlen or Fabrice Lavial.

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Ozmadenci, D., Féraud, O., Markossian, S. et al. Netrin-1 regulates somatic cell reprogramming and pluripotency maintenance. Nat Commun 6, 7398 (2015). https://doi.org/10.1038/ncomms8398

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Received: 08 March 2015
Accepted: 05 May 2015
Published: 08 July 2015
DOI: https://doi.org/10.1038/ncomms8398

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