Promotion of direct reprogramming by transformation-deficient Myc

Masato Nakagawa

Nanako Takizawa

Megumi Narita

Tomoko Ichisaka

Shinya Yamanaka

Construction of Plasmids.

The pMXs-based retroviral vectors for mouse Myc family genes have been described previously (6). The coding regions of human L-Myc and N-Myc were amplified by RT-PCR with the primers listed in Table S1. N-terminus deleted c-Myc mutants (cdN1, 14–439 aa; cdN2, 42–439 aa) were amplified by the PCR primers listed in Table S2. These PCR products were subcloned into pENTR-D-TOPO (Invitrogen) and then recombined with pMXs-gw via the LR reaction (Invitrogen). For the construction of Myc point mutants, site-directed mutagenesis was performed using PrimeSTAR HS DNA Polymerase (TaKaRa) with the primers listed in Table S3, according to the manufacturer's instructions.

Generation of iPSCs.

Induction of mouse iPSCs was performed as described previously (1, 3, 6) with some modifications. In brief, MEFs containing the Nanog-GFP-IRES-Puro^{reporter were seeded in six-well plates at 1.0 × 10}^{cells/well. The next day (day 0), the cells were infected with retorvirsuses containing three or four factors. On day 3, the cells were replated onto mitomycin C–treated SNL feeder cells (}48). The transduced cells were cultivated with ES medium containing leukemia inhibitory factor (49). Selection with puromycin (1.5 μg/mL) was started on day 21. Between 25 and 30 d after transduction, the number of colonies was manually counted under a microscope and recorded. Some colonies were then selected for expansion. The induction of human iPSCs was performed as described previously (6, 50). Adult human dermal fibroblasts (aHDFs) from the facial dermis of a 36-y-old Caucasian female were purchased from Cell Applications.

RNA Isolation and Reverse-Transcription.

The purifications of total RNA and RT-PCR were performed as described previously (1, 3, 6, 50). The expression of L-Myc was detected with a primer set, as listed in Table S4.

Transformation Assay in NIH 3T3 Cells.

NIH 3T3 cells were plated in 24-well plates at 2.5 × 10^{cells/well. The next day, the cells were infected with WT or mutant Myc. Two days after infection, the transfomation activity was determined based on the morphological changes detected.}

DNA Microarray Analyses.

A DNA microarray analysis was performed as described previously (50). First, aHDFs were retrovirally infected with WT or mutant Myc. Then at 48 hours after infection, total RNA was extracted from the cells and used for microarray experiments ({"type":"entrez-geo","attrs":{"text":"GSE22654","term_id":"22654"}}GSE22654). Data were analyzed using the GeneSpring GX 11 software package (Agilent). The genes activated or suppressed by Myc proteins were identified and categorized as described in Results. According to the expression levels of these selected genes, hierarchical clustering of the log2 expression ratios was performed for five cancer cells, two normal cells (aHDFs and lung fibroblasts), human iPSCs (average of three clones: 201B2, 201B7, and 253G1), and human ESCs (average of four clones: H1, H9, KhES1, and KhES3). The microarray data for cancer cells and lung fibroblasts were obtained from GEO DataSets (adenocarcinomas, {"type":"entrez-geo","attrs":{"text":"GSE13213","term_id":"13213"}}GSE13213; bladder cancer, {"type":"entrez-geo","attrs":{"text":"GSE19716","term_id":"19716"}}GSE19716; glioblastoma, {"type":"entrez-geo","attrs":{"text":"GSE10878","term_id":"10878"}}GSE10878; nasopharyngeal carcinoma, {"type":"entrez-geo","attrs":{"text":"GSE15191","term_id":"15191"}}GSE15191; stromal tumor, {"type":"entrez-geo","attrs":{"text":"GSE17018","term_id":"17018"}}GSE17018; lung fibroblasts, {"type":"entrez-geo","attrs":{"text":"GSE15359","term_id":"15359"}}GSE15359).

Statistical Analyses.

Data are presented as average ± SD. All statistical analyses were performed with one-way repeated-measures ANOVA and the Bonferroni post hoc test, using KaleidaGraph 4 (HULINKS).

Supplementary Material

Supporting Information:

Click here to view.

^{Center for iPS Cell Research and Application and}

^{Institute for Integrated Cell–Material Sciences, Kyoto University, Kyoto 606-8507, Japan;}

^{Yamanaka iPS Cell Special Project, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan; and}

^{Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158}

^{To whom correspondence may be addressed. E-mail:}pj.ca.u-otoyk.aric@awagakan or pj.ca.u-otoyk.aric@akanamay.

Communicated by Yuet Wai Kan, University of California San Francisco School of Medicine, San Francisco, CA, June 30, 2010 (received for review June 24, 2010)

Author contributions: M. Nakagawa and S.Y. designed research; M. Nakagawa, N.T., M. Narita, and T.I. performed research; M. Nakagawa contributed new reagents/analytic tools; M. Nakagawa analyzed data; and M. Nakagawa and S.Y. wrote the paper.

Communicated by Yuet Wai Kan, University of California San Francisco School of Medicine, San Francisco, CA, June 30, 2010 (received for review June 24, 2010)

Abstract

Induced pluripotent stem cells (iPSCs) are generated from mouse and human fibroblasts by the introduction of three transcription factors: Oct3/4, Sox2, and Klf4. The proto-oncogene product c-Myc markedly promotes iPSC generation, but also increases tumor formation in iPSC-derived chimeric mice. We report that the promotion of iPSC generation by Myc is independent of its transformation property. We found that another Myc family member, L-Myc, as well as c-Myc mutants (W136E and dN2), all of which have little transformation activity, promoted human iPSC generation more efficiently and specifically compared with WT c-Myc. In mice, L-Myc promoted germline transmission, but not tumor formation, in the iPSC-derived chimeric mice. These data demonstrate that different functional moieties of the Myc proto-oncogene products are involved in the transformation and promotion of directed reprogramming.

Keywords: induced pluripotent stem cell, embryonic stem cell, regenerative medicine, proto-oncogene

Abstract

Induced pluripotent stem cells (iPSCs) were first generated from mouse fibroblasts by the retroviral introduction of four transcription factors: Oct3/4, Sox2, Klf4, and c-Myc (1). Mouse iPSCs are indistinguishable from embryonic stem cells (ESCs) in morphology, proliferation and gene expression. Furthermore, mouse iPSCs give rise to chimeric mice that are competent for germline transmission (2 –4). However, both the chimeras and progenies derived from mouse iPSC have an increased incidence of tumor formation, due primarily to reactivation of the c-Myc retrovirus (3). We and others successfully created mouse iPSCs without the c-Myc retrovirus by modifying the induction protocol (5, 6). Chimeric mice derived from these c-Myc–minus iPSCs did not demonstrate an increased incidence of tumor formation (6). The efficiency of iPSC generation is significantly lower without the c-Myc retrovirus, however. Indeed, c-Myc is used in most of the reported methods to generate iPSCs without viral integration (7 –15). Thus, c-Myc functions as a “double-edged sword,” promoting both iPSC generation and tumorigenicity.

In addition to the overexpression of c-Myc, we and others have shown that suppression of the tumor-suppressor gene p53 also significantly enhances iPSC generation (16 –19). The downstream targets of p53, including p21 and Arf/Ink4, also are involved in the suppression of iPSC generation. The fact that the two most common pathways associated with human cancers—activation of c-Myc and suppression of p53—both substantially enhance iPSC generation raises the possibility that the molecular mechanisms underlying iPSC generation and tumorigenicity largely overlap.

The Myc proto-oncogene family consists of three members: c-Myc, N-Myc, and L-Myc (20 –23). All three members dimerize with Max and binding to DNA (24). N-Myc is similar to c-Myc in terms of length, domain structures, and frequent association with human cancers (25). In contrast, the L-Myc protein has shorter amino acid sequences than the other two members in the N-terminal region, along with significantly lower transformation activity in cultured cells (21, 26 –29). Consistent with this property, only a small number of human cancers have been associated with the aberrant expression of L-Myc. In the present study, we analyzed the effect of L-Myc in promoting iPSC generation. Despite its weak transformation activity, L-Myc was found to have a stronger and more specific activity in promoting iPSC generation. In addtion, the mutations that significantly deteriorate the transformation activity of c-Myc more effectively and specifically promote human iPSC generation. These findings demonstrate that the promotion of nuclear-reprogramming and transformation activity are independent properties of the Myc family proteins.

Click here to view.

Acknowledgments

We thank Drs. Takashi Aoi, Yoshinori Yoshida, Keisuke Okita, and Kazutoshi Takahashi and other members of Yamanaka research group for scientific comments and valuable discussions; Mika Ohuchi for assistance in the animal experiments; Tokiko Ohkame and Yukari Matsukawa for karyotype analyses; Dr. Toshio Kitamura (The Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo) for the retroviral expression system; and Dr. Peter W. Andrews (University of Sheffield, Sheffield, UK) for anti-SSEA-3, Tra-1-60, and Tra-1-81 antibodies. We also thank Rie Kato, Eri Nishikawa, Yuko Ohtsu, Sayaka Takeshima, and Haruka Hasaba for their valuable administrative support. This study was supported in part by a grant from the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, a grant from the Leading Project of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), a grant from Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program) of the Japan Society for the Promotion of Science (JSPS), and Grants-in-Aid for Scientific Research of JSPS and MEXT.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1009374107/-/DCSupplemental.

Footnotes

References

1. Takahashi K, Yamanaka SInduction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–676.[PubMed][Google Scholar]
2. Wernig M, et al In vitro reprogramming of fibroblasts into a pluripotent ES-cell–like state. Nature. 2007;448:318–324.[PubMed][Google Scholar]
3. Okita K, Ichisaka T, Yamanaka SGeneration of germline-competent induced pluripotent stem cells. Nature. 2007;448:313–317.[PubMed][Google Scholar]
4. Maherali N, et al Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell. 2007;1:55–70.[PubMed][Google Scholar]
5. Wernig M, Meissner A, Cassady JP, Jaenisch Rc-Myc is dispensable for direct reprogramming of mouse fibroblasts. Cell Stem Cell. 2008;2:10–12.[PubMed][Google Scholar]
6. Nakagawa M, et al Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol. 2008;26:101–106.[PubMed][Google Scholar]
7. Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka SGeneration of mouse induced pluripotent stem cells without viral vectors. Science. 2008;322:949–953.[PubMed][Google Scholar]
8. Yusa K, Rad R, Takeda J, Bradley AGeneration of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon. Nat Methods. 2009;6:363–369.[Google Scholar]
9. Woltjen K, et al piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature. 2009;458:766–770.[Google Scholar]
10. Kim DH, et al Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell. 2009;4:472–476.[Google Scholar]
11. Yu J, et al Human induced pluripotent stem cells free of vector and transgene sequences. Science. 2009;324:797–801.[Google Scholar]
12. Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa MEfficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B. 2009;85:348–362.[PubMed][Google Scholar]
13. Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger KInduced pluripotent stem cells generated without viral integration. Science. 2008;322:945–949.[Google Scholar]
14. Zhou W, Freed CRAdenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells. 2009;27:2667–2674.[PubMed][Google Scholar]
15. Zhou H, et al Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell. 2009;4:381–384.[PubMed][Google Scholar]
16. Marión RM, et al A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature. 2009;460:1149–1153.[Google Scholar]
17. Kawamura T, et al Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature. 2009;460:1140–1144.[Google Scholar]
18. Hong H, et al Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature. 2009;460:1132–1135.[Google Scholar]
19. Zhao Y, et al Two supporting factors greatly improve the efficiency of human iPSC generation. Cell Stem Cell. 2008;3:475–479.[PubMed][Google Scholar]
20. Cole MDThe myc oncogene: Its role in transformation and differentiation. Annu Rev Genet. 1986;20:361–384.[PubMed][Google Scholar]
21. Birrer MJ, et al L-myc cooperates with ras to transform primary rat embryo fibroblasts. Mol Cell Biol. 1988;8:2668–2673.[Google Scholar]
22. Schwab M, Varmus HE, Bishop JMHuman N-myc gene contributes to neoplastic transformation of mammalian cells in culture. Nature. 1985;316:160–162.[PubMed][Google Scholar]
23. Yancopoulos GD, et al N-myc can cooperate with ras to transform normal cells in culture. Proc Natl Acad Sci USA. 1985;82:5455–5459.[Google Scholar]
24. Blackwell TK, et al Binding of myc proteins to canonical and noncanonical DNA sequences. Mol Cell Biol. 1993;13:5216–5224.[Google Scholar]
25. Malynn BA, et al N-myc can functionally replace c-myc in murine development, cellular growth, and differentiation. Genes Dev. 2000;14:1390–1399.[Google Scholar]
26. Oster SK, Mao DY, Kennedy J, Penn LZFunctional analysis of the N-terminal domain of the Myc oncoprotein. Oncogene. 2003;22:1998–2010.[PubMed][Google Scholar]
27. Hatton KS, et al Expression and activity of L-Myc in normal mouse development. Mol Cell Biol. 1996;16:1794–1804.[Google Scholar]
28. Barrett J, Birrer MJ, Kato GJ, Dosaka-Akita H, Dang CVActivation domains of L-Myc and c-Myc determine their transforming potencies in rat embryo cells. Mol Cell Biol. 1992;12:3130–3137.[Google Scholar]
29. Cole MD, Cowling VHTranscription-independent functions of MYC: Regulation of translation and DNA replication. Nat Rev Mol Cell Biol. 2008;9:810–815.[Google Scholar]
30. Aoi T, et al Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science. 2008;321:699–702.[PubMed][Google Scholar]
31. Brough DE, Hofmann TJ, Ellwood KB, Townley RA, Cole MDAn essential domain of the c-myc protein interacts with a nuclear factor that is also required for E1A-mediated transformation. Mol Cell Biol. 1995;15:1536–1544.[Google Scholar]
32. Herold S, et al Negative regulation of the mammalian UV response by Myc through association with Miz-1. Mol Cell. 2002;10:509–521.[PubMed][Google Scholar]
33. Blackwood EM, Eisenman RNMax: A helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science. 1991;251:1211–1217.[PubMed][Google Scholar]
34. Sridharan R, et al Role of the murine reprogramming factors in the induction of pluripotency. Cell. 2009;136:364–377.[Google Scholar]
35. Dang CV, et al The c-Myc target gene network. Semin Cancer Biol. 2006;16:253–264.[PubMed][Google Scholar]
36. Nandan MO, Yang VWThe role of Krüppel-like factors in the reprogramming of somatic cells to induced pluripotent stem cells. Histol Histopathol. 2009;24:1343–1355.[Google Scholar]
37. Rowland BD, Bernards R, Peeper DSThe KLF4 tumour suppressor is a transcriptional repressor of p53 that acts as a context-dependent oncogene. Nat Cell Biol. 2005;7:1074–1082.[PubMed][Google Scholar]
38. de Jong J, Looijenga LHStem cell marker OCT3/4 in tumor biology and germ cell tumor diagnostics: History and future. Crit Rev Oncog. 2006;12:171–203.[PubMed][Google Scholar]
39. Liu A, et al Diagnostic utility of novel stem cell markers SALL4, OCT4, NANOG, SOX2, UTF1, and TCL1 in primary mediastinal germ cell tumors. Am J Surg Pathol. 2010;34:697–706.[PubMed][Google Scholar]
40. Maddison P, Thorpe A, Silcocks P, Robertson JF, Chapman CJAutoimmunity to SOX2, clinical phenotype and survival in patients with small-cell lung cancer. Lung Cancer. 2010[PubMed][Google Scholar]
41. Tung CL, et al SOX2 modulates alternative splicing in transitional cell carcinoma. Biochem Biophys Res Commun. 2010;393:420–425.[PubMed][Google Scholar]
42. Peng S, Maihle NJ, Huang YPluripotency factors Lin28 and Oct4 identify a sub-population of stem cell–like cells in ovarian cancer. Oncogene. 2010;29:2153–2159.[PubMed][Google Scholar]
43. Vousden KH, Prives CBlinded by the light: The growing complexity of p53. Cell. 2009;137:413–431.[PubMed][Google Scholar]
44. Hemann MT, et al Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants. Nature. 2005;436:807–811.[Google Scholar]
45. Chen Z, et al Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature. 2005;436:725–730.[Google Scholar]
46. Beauséjour CM, et al Reversal of human cellular senescence: Roles of the p53 and p16 pathways. EMBO J. 2003;22:4212–4222.[Google Scholar]
47. Ferbeyre G, et al Oncogenic ras and p53 cooperate to induce cellular senescence. Mol Cell Biol. 2002;22:3497–3508.[Google Scholar]
48. McMahon AP, Bradley AThe Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell. 1990;62:1073–1085.[PubMed][Google Scholar]
49. Meiner VL, et al Disruption of the acyl-CoA:cholesterol acyltransferase gene in mice: Evidence suggesting multiple cholesterol esterification enzymes in mammals. Proc Natl Acad Sci USA. 1996;93:14041–14046.[Google Scholar]
50. Takahashi K, et al Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861–872.[PubMed][Google Scholar]