Reprogramming of adult cells to generate induced pluripotent stem cells (iPS cells) has opened new therapeutic opportunities; however, little is known about the possibility of in vivo reprogramming within tissues. Here we show that transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs, implying that full reprogramming can occur in vivo. Analyses of the stomach, intestine, pancreas and kidney reveal groups of dedifferentiated cells that express the pluripotency marker NANOG, indicative of in situ reprogramming. By bone marrow transplantation, we demonstrate that haematopoietic cells can also be reprogrammed in vivo. Notably, reprogrammable mice present circulating iPS cells in the blood and, at the transcriptome level, these in vivo generated iPS cells are closer to embryonic stem cells (ES cells) than standard in vitro generated iPS cells. Moreover, in vivo iPS cells efficiently contribute to the trophectoderm lineage, suggesting that they achieve a more plastic or primitive state than ES cells. Finally, intraperitoneal injection of in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic markers. We conclude that reprogramming in vivo is feasible and confers totipotency features absent in standard iPS or ES cells. These discoveries could be relevant for future applications of reprogramming in regenerative medicine.
Your institute does not have access to this article
Open Access articles citing this article.
Anti-senescence ion-delivering nanocarrier for recovering therapeutic properties of long-term-cultured human adipose-derived stem cells
Journal of Nanobiotechnology Open Access 30 October 2021
Clinical Epigenetics Open Access 06 September 2021
DMRT1-mediated reprogramming drives development of cancer resembling human germ cell tumors with features of totipotency
Nature Communications Open Access 19 August 2021
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006)
Robinton, D. A. & Daley, G. Q. The promise of induced pluripotent stem cells in research and therapy. Nature 481, 295–305 (2012)
Maherali, N. & Hochedlinger, K. Guidelines and techniques for the generation of induced pluripotent stem cells. Cell Stem Cell 3, 595–605 (2008)
Cobaleda, C., Jochum, W. & Busslinger, M. Conversion of mature B cells into T cells by dedifferentiation to uncommitted progenitors. Nature 449, 473–477 (2007)
Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J. & Melton, D. A. In vivo reprogramming of adult pancreatic exocrine cells to β-cells. Nature 455, 627–632 (2008)
Qian, L. et al. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature 485, 593–598 (2012)
Song, K. et al. Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature 485, 599–604 (2012)
Banga, A., Akinci, E., Greder, L. V., Dutton, J. R. & Slack, J. M. In vivo reprogramming of Sox9+ cells in the liver to insulin-secreting ducts. Proc. Natl Acad. Sci. USA 109, 15336–15341 (2012)
Rouaux, C. & Arlotta, P. Direct lineage reprogramming of post-mitotic callosal neurons into corticofugal neurons in vivo. Nature Cell Biol. 15, 214–221 (2013)
Torper, O. et al. Generation of induced neurons via direct conversion in vivo. Proc. Natl Acad. Sci. USA 110, 7038–7043 (2013)
Stadtfeld, M., Maherali, N., Borkent, M. & Hochedlinger, K. A reprogrammable mouse strain from gene-targeted embryonic stem cells. Nature Methods 7, 53–55 (2010)
Carey, B. W., Markoulaki, S., Beard, C., Hanna, J. & Jaenisch, R. Single-gene transgenic mouse strains for reprogramming adult somatic cells. Nature Methods 7, 56–59 (2010)
Haenebalcke, L. et al. The ROSA26-iPSC mouse: a conditional, inducible, and exchangeable resource for studying cellular (de)differentiation. Cell Rep. 3, 335–341 (2013)
Hochedlinger, K., Yamada, Y., Beard, C. & Jaenisch, R. Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell 121, 465–477 (2005)
Carey, B. W. et al. Reprogramming of murine and human somatic cells using a single polycistronic vector. Proc. Natl Acad. Sci. USA 106, 157–162 (2009)
Finch, A. J., Soucek, L., Junttila, M. R., Swigart, L. B. & Evan, G. I. Acute overexpression of Myc in intestinal epithelium recapitulates some but not all the changes elicited by Wnt/beta-catenin pathway activation. Mol. Cell. Biol. 29, 5306–5315 (2009)
Eminli, S. et al. Differentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells. Nature Genet. 41, 968–976 (2009)
Yamanaka, S. Elite and stochastic models for induced pluripotent stem cell generation. Nature 460, 49–52 (2009)
Hanna, J. et al. Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Proc. Natl Acad. Sci. USA 107, 9222–9227 (2010)
Pettitt, S. J. et al. Agouti C57BL/6N embryonic stem cells for mouse genetic resources. Nature Methods 6, 493–495 (2009)
Hughes, E. D. et al. Genetic variation in C57BL/6 ES cell lines and genetic instability in the Bruce4 C57BL/6 ES cell line. Mamm. Genome 18, 549–558 (2007)
Assou, S. et al. Transcriptome analysis during human trophectoderm specification suggests new roles of metabolic and epigenetic genes. PLoS ONE 7, e39306 (2012)
Koo, T. B. et al. Differential expression of the PEA3 subfamily of ETS transcription factors in the mouse ovary and peri-implantation uterus. Reproduction 129, 651–657 (2005)
Yao, X. Q. et al. Glycogen synthase kinase-3β regulates leucine-309 demethylation of protein phosphatase-2A via PPMT1 and PME-1. FEBS Lett. 586, 2522–2528 (2012)
Glover, C. H. et al. Meta-analysis of differentiating mouse embryonic stem cell gene expression kinetics reveals early change of a small gene set. PLOS Comput. Biol. 2, e158 (2006)
Koh, K. P. et al. Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. Cell Stem Cell 8, 200–213 (2011)
Lu, C. W. et al. Ras-MAPK signaling promotes trophectoderm formation from embryonic stem cells and mouse embryos. Nature Genet. 40, 921–926 (2008)
Ng, R. K. et al. Epigenetic restriction of embryonic cell lineage fate by methylation of Elf5. Nature Cell Biol. 10, 1280–1290 (2008)
Diéguez-Hurtado, R. et al. A Cre-reporter transgenic mouse expressing the far-red fluorescent protein Katushka. Genesis 49, 36–45 (2011)
Beddington, R. S. & Robertson, E. J. An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo. Development 105, 733–737 (1989)
Macfarlan, T. S. et al. Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature 487, 57–63 (2012)
Pfister, S., Steiner, K. A. & Tam, P. P. Gene expression pattern and progression of embryogenesis in the immediate post-implantation period of mouse development. Gene Expr. Patterns 7, 558–573 (2007)
Conley, B. J., Trounson, A. O. & Mollard, R. Human embryonic stem cells form embryoid bodies containing visceral endoderm-like derivatives. Fetal Diagn. Ther. 19, 218–223 (2004)
Gordon, E. J., Gale, N. W. & Harvey, N. L. Expression of the hyaluronan receptor LYVE-1 is not restricted to the lymphatic vasculature; LYVE-1 is also expressed on embryonic blood vessels. Dev. Dyn. 237, 1901–1909 (2008)
Thier, M. et al. Direct conversion of fibroblasts into stably expandable neural stem cells. Cell Stem Cell 10, 473–479 (2012)
Kurian, L. et al. Conversion of human fibroblasts to angioblast-like progenitor cells. Nature Methods 10, 77–83 (2013)
Halley, J. D. et al. Self-organizing circuitry and emergent computation in mouse embryonic stem cells. Stem Cell Res. 8, 324–333 (2012)
Marks, H. et al. The transcriptional and epigenomic foundations of ground state pluripotency. Cell 149, 590–604 (2012)
Domínguez, O. & López-Larrea, C. Gene walking by unpredictably primed PCR. Nucleic Acids Res. 22, 3247–3248 (1994)
Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nature Protocols 7, 562–578 (2012)
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)
Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009)
Pease, S. & Sounders, T. Advanced Protocols for Animal Transgenesis, an ISTT Manual. (Springer-Verlag, 2011)
Festing, M. F. W., Overend, P., Gaines Das, R., Cortina Borja, M. & Berdoy, M. The design of animal experiments. Reducing the use of animals in research through better experimental design (Royal Society of Medicine Press, 2002)
We are grateful to M.Torres for advice, and to K. Hochedlinger and R. Jaenisch for reagents. We also thank F. Beier, R. Serrano and N. Soberón for technical support. Work in the laboratory of M.S. is funded by the CNIO and by grants from the Spanish Ministry of Economy (MINECO, SAF), the Regional Government of Madrid (ReCaRe), the European Union (RISK-IR), the European Research Council (ERC Advanced Grant), the Botin Foundation, the Ramon Areces Foundation and the AXA Foundation. Work in the laboratory of M.M. is funded by grants from the MINECO (BFU), the Regional Government of Madrid (Cell-DD) and the ProCNIC Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors declare no competing financial interests.
Extended data figures and tables
a, Southern blot of tail tip genomic DNA digested with BamHI and hybridized with specific probes for Sox2 and Klf4. b, Mice of the indicated transgenic lines carrying the reprogramming transgene (+) or without it (−) were treated with doxycycline (1 mg ml−1) for 6 days. The mRNA levels of Oct4 were determined by qRT–PCR. Values correspond to the average and s.d. (n = 3 mice per transgenic line) and are relative to the levels of wild-type mice treated with doxycycline. c, MEFs of the indicated mouse lines were treated with doxycyline (1 µg ml−1). Colonies of iPS cells in the i4F-A and i4F-B plates were stained for alkaline phosphatase (AP) 10 days after induction. In the case of i4F-C and i4F-D, plates were stained after 15 days but no iPS cell colonies were observed. In parallel, total Oct4 mRNA levels were measured at the indicated times by qRT–PCR. Values correspond to the average and s.d. For i4F-A, i4F-B, and i4F-D, n = 3 MEF preparations; for i4F-C, n = 1. d, Comparison of the in vitro reprogramming kinetics and efficiency of MEFs from lines i4F-A and i4F-B. Reprogramming was induced with two different protocols: 1 µg ml−1 of doxycycline for 6 days, or continuous treatment with 1 µg ml−1 of doxycycline. AP+ colonies were counted at the indicated times. Values correspond to the average and s.d. (n = 3 independent MEF isolates per line). In b and c, statistical significance was evaluated by the Student's t-test (unpaired, two-tailed): *P < 0.05, **P < 0.01, ***P < 0.001.
Extended Data Figure 2 Genomic insertion sites of lentiviral transgenes i4F-A and i4F-B and their effect on the host genes.
a, Primers used for PCR to confirm insertion are shown in blue and underlined. These primers were used together with a common primer hybridizing to internal lentiviral sequences (see Methods). The 4 base pairs flanking the insertion site are duplicated upon lentiviral insertion and are underlined. A map of each gene is shown indicating with an arrow the approximate location of the lentiviral transgene. The pictures of PCR agarose gels correspond to the PCR products obtained with the flanking primer (underlined sequence in blue) and the internal lentiviral primer (not shown) (see Methods). b, The indicated tissues were used to measure the levels of Neto2 (host gene for the lentiviral transgene i4F-A) or Pparg (host gene for the lentiviral transgene i4F-B). Values correspond to the average and s.d. (n = 3 mice per condition). Statistical significance was evaluated by Student’s t-test (unpaired, two-tailed). No significant differences were observed.
Extended Data Figure 3 Histological alterations of the intestine and pancreas upon induction of i4F reprogrammable mice.
Mice were treated with doxycycline (1 mg ml−1) for 6 days. Haematoxylin and eosin (H&E) staining and inmunohistochemistry of OCT4 in the intestine (a) and pancreas (b). Similar alterations were found in both lines, i4F-A and i4F-B.
Extended Data Figure 4 i4F induction leads to the appearance of tumoral masses and in situ reprogramming events.
a, Reprogrammable mouse with multiple tumoral masses in the liver and kidneys (a representative example is shown from 15 mice analysed with teratomas). b, Incidence of other tumours in reprogrammable mice with teratomas. c, Three examples of NANOG-positive tubules in different induced reprogrammable mice.
a, Expression of pluripotency markers in the indicated cell types. Data correspond to qRT–PCR from seven independent in vivo iPS cell clones, two in vitro iPS cell clones (no. 1: in vitro reprogrammed i4F MEFs; no. 2: in vitro reprogrammed wild-type MEFs infected with lenti-OSKM), and two ES cell clones (no. 1: C57BL6.10; no. 2: G4). Values correspond to the average ± s.d. of 3 technical replicates. b, Silencing of the lentiviral cassette in in vivo iPS cell clones. Upper part, location of the PCR primers used. Lower part, lentiviral RNA levels in in vivo iPS cells (7 independent clones), in an in vitro iPS cell clone (in vitro reprogrammed i4F MEFs), in an ES cell line (C57BL6.10), and in i4F-MEFs induced with doxycycline for 3 days. Values correspond to the average ± s.d. of 3 technical replicates. c, Chimaeric E14.5 testis generated with a GFP-labelled in vivo iPS cells. Magnifications show germ cells derived from in vivo iPS cells. d, Summary of the isolation of in vivo iPS cells from the bloodstream.
Extended Data Figure 6 Transcriptomic profiles of in vivo iPS cells, in vitro iPS cells and ES cells.
a, Pearson correlation coefficients among all sequenced samples. The highest and the lowest coefficients are coloured in a blue to red gradient. b, Principal component analysis of the transcriptomes of in vivo iPS cells, in vitro iPS cells and ES cells. Data correspond to 6 clones of in vivo iPS cells, 5 clones of in vitro iPS cells, and 3 lines of ES cells (C57BL6.10, JM8.F6 and Bruce4). c, Upper part, scatter plots representing the expression of each gene in the indicated pairs of cell types. Middle part, volcano plots representing the P value of the differences in expression of each gene between the corresponding cell types. Significant P values are in blue (that is, indicating differentially expressed genes). Non-significant P values are in red (that is, indicating genes that are not differentially expressed). Lower part, Pearson coefficient correlation among samples. Data correspond to 6 clones of in vivo iPS cells, 5 clones of in vitro iPS cells, and 3 lines of ES cells (C57BL6.10, JM8.F6 and Bruce4).
a, Genes upregulated in in vivo iPS and ES cells versus in vitro iPS cells. b, Genes upregulated in in vivo iPS cells versus ES cells and in vitro iPS cells. Expression levels of the indicated genes in in vivo iPS cells (n = 6 clones), in vitro iPS cells (n = 5 clones) and ES cells (n = 3 lines C57BL6.10, JM8.F6 and Bruce4). A sample of RNA derived from a preparation of ∼170 morulas was also included in b. Values correspond to the average ± s.d. Statistical significance was evaluated relative to in vitro iPS cells (a) or relative to in vivo iPS cells (b) by the Student's t-test (unpaired, two-tailed): *P < 0.05, **P < 0.01, ***P < 0.001.
a, Induction of trophectoderm markers (Fgfr2, Eomes) in the indicated cell types after culture in TS differentiation medium (see Methods) during the indicated period of time. Other markers were used as controls: Sox1 (ectoderm), T (mesoderm) and Gata6 (endoderm). For each cell type, values are relative to the average levels at day 0. Values correspond to the average and s.d. For ES cells, n = 3 (lines C57BL6.10, JM8.F6 and Bruce4); for in vitro iPS cells, n = 5 clones; and for in vivo iPS cells, n = 5 clones. Statistical significance was determined using the Student’s t-test (unpaired, two-tailed): *P < 0.05, **P < 0.01. The lower line of asterisks refers to the comparison with in vitro iPS cells, and the upper line of asterisks to the comparison with ES cells. b, Example of a chimaeric blastocyst derived from a Katushka morula injected with GFP-labelled in vivo iPS cells. Two different confocal planes are shown containing GFP-labelled cells that have contributed to the trophectoderm and to the inner cell mass, as indicated. c, Chimaerism of GFP-labelled in vivo iPS cells in the proper embryo and placenta (E14.5). A wild-type embryo at the same stage of development is shown as a control. Fluorescence pictures were taken with the same settings.
Analysis of the expression of genes enriched in the 2C state: the retrotrasposable elements MuERV-L, Zscan4, and intracisternal A particles (IAP) showed no differences between in vivo iPS cells compared to ES cells and in vitro iPS cells. For ES cells, n = 3 (lines C57BL6.10, JM8.F6 and Bruce4); for in vitro iPS cells, n = 5 clones; and for in vivo iPS cells, n = 6 clones. Values correspond to the average and s.d. Statistical significance was determined using the Student’s t-test (unpaired, two-tailed). None of the differences was statistically significant.
Haematoxylin and eosin and immunostaining analysis of two examples of embryo-like structures generated upon in vivo iPS cells intraperitoneal injection. The following markers were used: SOX2 (ectoderm), T/BRACHYURY (mesoderm), GATA4 (endoderm), CDX2 (trophectoderm), AFP and CK8 (visceral endoderm of the yolk sac). All lateral panels are at the same magnification.
This table shows differentially expressed genes in in vivo iPS cells vs in vitro iPS cells. (XLSX 69 kb)
This table shows differentially expressed genes in in vivo iPS cells vs ES cells. (XLSX 63 kb)
This table shows differentially expressed genes in ES cells vs in vitro iPS cells. (XLSX 100 kb)
This table shows upregulated and downregulated genes in in vivo iPS cells and ES cells (vs in vitro iPS cells). (XLSX 13 kb)
This table shows upregulated and downregulated genes in in vivo iPS cells (vs in vitro iPS cells and ES cells). (XLSX 8 kb)
About this article
Cite this article
Abad, M., Mosteiro, L., Pantoja, C. et al. Reprogramming in vivo produces teratomas and iPS cells with totipotency features. Nature 502, 340–345 (2013). https://doi.org/10.1038/nature12586
Nature Reviews Molecular Cell Biology (2022)
Nature Aging (2022)
In vivo partial reprogramming alters age-associated molecular changes during physiological aging in mice
Nature Aging (2022)
BMC Genomics (2021)
Anti-senescence ion-delivering nanocarrier for recovering therapeutic properties of long-term-cultured human adipose-derived stem cells
Journal of Nanobiotechnology (2021)