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Autophagy and mTORC1 regulate the stochastic phase of somatic cell reprogramming

Nature Cell Biology volume 17, pages 715–725 (2015)Cite this article

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Abstract

We describe robust induction of autophagy during the reprogramming of mouse fibroblasts to induced pluripotent stem cells by four reprogramming factors (Sox2, Oct4, Klf4 and c-Myc), henceforth 4F. This process occurs independently of p53 activation, and is mediated by the synergistic downregulation of mechanistic target of rapamycin complex 1 (mTORC1) and the induction of autophagy-related genes. The 4F coordinately repress mTORC1, but bifurcate in their regulation of autophagy-related genes, with Klf4 and c-Myc inducing them but Sox2 and Oct4 inhibiting them. On one hand, inhibition of mTORC1 facilitates reprogramming by promoting cell reshaping (mitochondrial remodelling and cell size reduction). On the other hand, mTORC1 paradoxically impairs reprogramming by triggering autophagy. Autophagy does not participate in cell reshaping in reprogramming but instead degrades p62, whose accumulation in autophagy-deficient cells facilitates reprogramming. Our results thus reveal a complex signalling network involving mTORC1 inhibition and autophagy induction in the early phase of reprogramming, whose delicate balance ultimately determines reprogramming efficiency.

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Figure 1: Induction of autophagy in early reprogramming.
Figure 2: The 4F regulate autophagy differently in a p53-independent manner.
Figure 3: Autophagy impairs reprogramming.
Figure 4: Autophagy is not responsible for mitochondrial remodelling in reprogramming.
Figure 5: Inhibition of mTORC1 mediates mitochondrial remodelling.
Figure 6: p62 is an autophagy target in reprogramming.

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Acknowledgements

We thank W. Lee and J. CY. Ho from the University of Hong Kong for their technical support with the electron microscopy. We thank all members of the Pei and Esteban laboratory for their support, and J. Chen, Y. Chen, S. Chu, F. Cui, S. Huang, D. P. Ibáñez, K. Lai, R. Luo, X. Qing and F. Zhang for technical assistance. We also thank A. Schambach from the Department of Experimental Hematology, Hannover Medical School for providing the polycistronic 4F-dT lentiviral vector, Y. Chen from Tsinghua University for providing the Atg5 KO mice (with permission from N. Mizushima and RIKEN BioResource Center) and Q. Chen from the Institute of Zoology, Chinese Academy of Sciences. This work was financially supported by the National Basic Research Program of China (973 Program of China, 2011CBA01106, 2011CBA01004, 2011CB965201 and 2012CB966802), the ‘Strategic Priority Research Program’ of the Chinese Academy of Sciences (XDA01020106, XDA01020202 and XDA01020401), the National Natural Science Foundation of China (31301057, 31421004, 91213304 and 31371513), the Ministry of Science and Technology International Technology Cooperation Program (2012DFH30050) and a joint German–Chinese grant from the German Academic Exchange Service (DAAD), the German Ministry of Research and the Ministry of Science and Technology of China to D.P. and A. Schambach. H-F.T. is financially supported by the Theme Based Research Scheme (T12-705/11) and the Innovation and Technology Support Programme (Tier 3) (ITS/303/12).

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Author notes

  1. Yasong Wu and Yuan Li: These authors contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Regenerative Biology of the Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China

    Yasong Wu, Yuan Li, Hui Zhang, Yinghua Huang, Ping Zhao, Yujia Tang, Xiaohui Qiu, Yue Ying, Su Ni, Meng Zhang, Longqi Liu, Yan Xu, Qiang Zhuang, Zhiwei Luo, Christina Benda, Hong Song, Liangxue Lai, Xingguo Liu, Xichen Bao, Miguel A. Esteban, Baoming Qin & Duanqing Pei

  2. School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China

    Yuan Li

  3. Cardiology Division, Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong SAR, China

    Wen Li & Hung-Fat Tse

  4. Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China

    Wen Li

  5. School of Life Sciences, Anhui University, Hefei 230601, China

    Meng Zhang

  6. Health Science Center, Shenzhen University, Shenzhen 518060, China

    Baohua Liu

  7. Shenzhen Institutes of Research and Innovation, the University of Hong Kong, Hong Kong SAR, China

    Hung-Fat Tse

  8. Hong Kong-Guangdong Joint Laboratory on Stem Cell and Regenerative Medicine, the University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, China

    Hung-Fat Tse, Miguel A. Esteban, Baoming Qin & Duanqing Pei

  9. CUHK-GIBH Joint Research Laboratory of Stem Cells and Regenerative Medicine, School of Biomedical Sciences, Faculty of Medicine, the Chinese University of Hong Kong, Hong Kong, China

    Wai-Yee Chan

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  1. Yasong Wu
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Contributions

B.Q., M.A.E. and D.P. conceived the idea. B.Q. supervised the study with help from D.P. and M.A.E. Y.W. and Y.L. conducted most of the experiments. Y.W., Y.L. and B.Q. analysed the data. H.Z., Y.H., P.Z., Y.T., X.Q., Y.Y., S.N., M.Z., L.L., Y.X., Q.Z., Z.L., C.B. and H.S. contributed to the experiments. W.L. contributed to electron microscopy. L.L., H-F.T., X.B. and M.A.E. provided infrastructural support. X.L., B.L. and W-Y.C. provided relevant advice. B.Q., M.A.E. and D.P. wrote the manuscript. D.P. approved the final version.

Corresponding authors

Correspondence to Baoming Qin or Duanqing Pei.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 4 mRNA expression in different reprogramming systems and autophagy induction using the polycistronic lentivirus and the 2° system.

(a) qPCR analysis of the 4F mRNA level in retroviral reprogramming relative to Ctrl day 6. Means ± s.e.m. from n = 3 independent experiments with 3 technical replicates are shown. (b) Phase contrast and fluorescence of Oct4-GFP (OG2) ESCs in 2i+LIF on gelatin (ESCs 2i), serum+LIF on feeder (ESCs serum) and differentiation (withdrawal of 2i and LIF for 3 days, Diff. D3). Scale bars: 100 μm. (c) Western blotting for LC3B, p62 and ACTIN in MEFs, mouse ESCs in 2i+LIF and serum+LIF medium. Low (L) and high (H) cell density indicate 2- or 3-day culture respectively. (d) Quantification of LC3B-II relative to ACTIN in c. (e) qPCR analysis of Oct4 and Nanog in cells after 3-day differentiation (right panel in b) relative to ESCs in 2i+LIF. Data shown are the mean of 3 replicates and are from 1 experiment. (f) Western blotting for LC3B, p62 and ACTIN in MEFs, mouse ESCs in 2i+LIF and 3-day differentiation. (g) Quantification of LC3B-II relative to ACTIN in (f). (h) qPCR analysis for the 4F in the 4F-dT system on day 6 (Ctrl, 1XLenti-Flag). Data shown are the mean of 3 replicates and are from 1 of 2 representative experiments. (i) Western blotting for LC3B and ACTIN in MEFs transduced with Ctrl or 4F-dT on day 3, 5 and 7 post infection. (j) qPCR analysis for 4F in the 2° system relative to Ctrl (-DOX). Data shown are the mean of 3 replicates and are from 1 of 2 representative experiments. (k) Western blotting for LC3B and ACTIN in the 2° system, −/+Baf. Uncropped images of blots are shown in Supplementary Fig. 6. Source data are provided in Supplementary Table 4.

Supplementary Figure 5 The Yamanaka factors regulate autophagy differently independent of p53.

(a) qPCR for a large panel of Atg genes in MEFs transduced with 4F retroviruses at different time points and ESCs. (b) Western blotting for Atg genes in MEFs and ESCs. (c) qPCR analysis of the knockdown efficiency of combined shRNAs for Atg5, Beclin1 and Vps34 on 4F day 5. Only 1 control of shLuc is shown. (d) Western blotting for LC3B in MEFs transduced with combined factors on day 1, 3 and 5, treated with Baf. Ctrl, 2XFlag Numbers indicate quantification of LC3B-II relative to ACTIN. (e) qPCR for indicated genes in p53 KO TTFs transduced with 4F. Means ± s.e.m. from n = 3 independent experiments with 3 technical replicates are shown. (f) qPCR for the indicated genes in p53 KO TTFs transduced with 2 factors. Ctrl, 2XFlag. (g) Western blotting for LC3B and ACTIN in MEFs transduced with Ctrl (4XFlag) or 4F in iCD1+BMP4 medium under −/+Baf conditions. The samples were from one experiment and run on the same gel. The additional bands were sliced out. A solid line was put to separate the two slice parts. (h) Western blotting for LC3B and ACTIN in MEFs transduced with Ctrl (1XLenti-Flag) or 3F-dT, −/+Baf. Data shown are the mean of 3 replicates and are from 1 of 2 representative experiments (a,f). Source data are provided in Supplementary Table 4. Uncropped images of blots are shown in Supplementary Fig. 6.

Supplementary Figure 6 Autophagy impairs 4F reprogramming.

(a) Apoptosis analysis of 4F and Atg shRNA transduced cells. (b) Western blotting for the indicated genes to confirm the expression of Atg gene WT and dominant-negative (DN) mutants in transduced MEFs. (c) Western blotting for LC3B in MEFs transduced with 4F and DN mutants on day 5 (−/+Baf). (d) Effect of the above mutants on reprogramming. (e) Western blotting for LC3B in MEFs transduced with 4F and the indicated WT genes on day 5 (−/+Baf). (f) Effect of these WT genes on reprogramming. (g) FACS analysis of dTomato-positive cells in 4F-dT transduced MEFs from Atg5 WT or KO embryos. Data are plotted from 1 experiment with 3 technical replicates. (h) Western blotting for exogenous SOX2 in 4F-dT transduced MEFs from Atg5 WT and KO embryos. (i) Representative images of an iPSC clone (SSEA1+/dTomato-, arrow) and a non-iPSC clone (SSEA1-/dTomato +, dash circle). Scale bar: 100 μm. (j) Apoptosis analysis in Atg5 KO MEFs transduced with Ctrl or Atg5. Means ± s.e.m. from n = 3 independent experiments with 3 technical replicates are shown. (k) Effect of Atg5 rescue on reprogramming in 4F transduced OG2-Atg5 KO MEFs. (l) Atg5 rescue has no effect on reprogramming efficiency in 3F-dT transduced Atg5 KO MEFs. Data shown are the mean of 3 replicates and are from 1 of 2 (a), 3 (d,f,l) and 5 (k) representative experiments. Source data are provided in Supplementary Table 4. Uncropped images of blots are shown in Supplementary Fig. 6.

Supplementary Figure 7 Characterization of iPSC clones.

(a) Genomic PCR analysis of transgene integration of 3 representative iPSC clones generated with retroviral 4F and shBeclin1, shVps34 and Atg16l-M. Negative indicates ESCs; positive indicates Actin. (b) Phase contrast, fluorescence and immunofluorescence microscopy of representative iPSC clones. Scale bars: 100 μm. (c) Bright-field view of Atg5 KO 4F iPSC clone C1. Scale bar: 100 μm. (d) Western blotting for LC3B and ATG5-12 in Atg5 KO-4F C1. (e) qPCR analysis of the expression of endogenous pluripotent genes and the silencing of 4F transgenes in iPSCs. Data are plotted from 1 experiment with 3 technical replicates. (f) DNA methylation profile of the Oct4 and Nanog proximal promoters in the indicated iPSCs, ESCs and MEFs. (g) Karyotype analysis of these iPSCs. (h) Hematoxylin/eosin staining of the teratomas formed from the shRNA iPSCs. Scale bar: 100 μm. (i) Chimeric mice produced with the above clones and germline transmission using Atg16l-M-C2 iPSCs (F1). Source data are provided in Supplementary Table 4. Uncropped images of blots are shown in Supplementary Fig. 6.

Supplementary Figure 8 Inhibition of mTORC1 mediates mitochondrial remodelling.

(a) Western blotting for MTOR and ATK-mTORC1 signalling in early reprogramming of 2° system (+ DOX). (b) Western blotting for mTORC1 activity in MEFs and ESCs. (c) Western blotting shows the change of mTORC1 signalling pathway in p53 KO TTFs in early reprogramming. Ctrl, 4XFlag. (d) qPCR analysis for Mtor in MEFs transduced with indicated factors on day 1. (e) Western blotting for MTOR, LC3B and p62 in MEFs transduced with the indicated factors on day 1 (−/+Baf). (f) qPCR analysis of genes responsible for mitochondrial biogenesis. (g) FACS analysis shows the effect of Tsc2 shRNAs on cell size. (h) qPCR analysis to confirm the expression of the indicated genes in 4F reprogramming on day 5. Data are plotted from 1 experiment with 3 technical replicates. (i) qPCR analysis of genes responsible for mitochondrial biogenesis in MEFs transduced with 4F and the indicated genes on day 5. (j) FACS analysis for cell size in MEFs transduced with indicated genes on day 5. (k) FACS analysis cell proliferation in MEFs transduced with indicated genes on day 5. Data shown are the mean of 3 replicates and are from 1 of 2 representative experiments (d,k). Means ± s.e.m. from n = 3 (f,g,i) and 4 (j) independent experiments with 3 technical replicates are shown. Source data are provided in Supplementary Table 4. Uncropped images of blots are shown in Supplementary Fig. 6.

Supplementary Table 1 Genomic binding pattern of Atg genes by Yamanaka factors in 48 h human fibroblasts reprogramming, summarized and modified from Soufi et al., 201245. ‘+’ shaded in light green represents bound and ‘-’ represents unbound.
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Supplementary Table 2 Primers for qPCR assays in this study.
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Supplementary Table 3 Primers for cDNA cloning in this study.
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Wu, Y., Li, Y., Zhang, H. et al. Autophagy and mTORC1 regulate the stochastic phase of somatic cell reprogramming. Nat Cell Biol 17, 715–725 (2015). https://doi.org/10.1038/ncb3172

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