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Netrin-1 regulates somatic cell reprogramming and pluripotency maintenance.

Ozmadenci D, Féraud O, Markossian S, Kress E, Ducarouge B, Gibert B, Ge J, Durand I, Gadot N, Plateroti M, Bennaceur-Griscelli A, Scoazec JY, Gil J, Deng H, Bernet A, Mehlen P, Lavial F - Nat Commun (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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, 69008 Lyon, France.

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.

No MeSH data available.


Related in: MedlinePlus

Recombinant Netrin-1 treatment is not detrimental to iPS cells quality.(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.
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f2: Recombinant Netrin-1 treatment is not detrimental to iPS cells quality.(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.

Mentions: 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.


Netrin-1 regulates somatic cell reprogramming and pluripotency maintenance.

Ozmadenci D, Féraud O, Markossian S, Kress E, Ducarouge B, Gibert B, Ge J, Durand I, Gadot N, Plateroti M, Bennaceur-Griscelli A, Scoazec JY, Gil J, Deng H, Bernet A, Mehlen P, Lavial F - Nat Commun (2015)

Recombinant Netrin-1 treatment is not detrimental to iPS cells quality.(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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4510695&req=5

f2: Recombinant Netrin-1 treatment is not detrimental to iPS cells quality.(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.
Mentions: 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.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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, 69008 Lyon, France.

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.

No MeSH data available.


Related in: MedlinePlus