Abstract
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.
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Change history
16 October 2013
Scale bar values were added to Fig. 5a.
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Acknowledgements
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.
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M.A. performed most of the experiments, contributed to experimental design, data analysis, discussion and writing; L.M. performed a substantial amount of experimental work, contributed to experimental design, data analysis, discussion and writing; C.P. contributed to experimental work, data analysis, discussion and writing; M.C. performed all the histopathological and immunohistochemical analyses; T.R. and I.O. contributed to the trophoblast stem cell and giant cell differentiation assays; O.G. analysed the RNAseq data; D. Megías supervised and helped with the confocal microscopy; O.D. performed RNAseq and determined the lentiviral genomic insertion sites; D. Martínez performed cell sorting and contributed to the bone marrow and peripheral blood analyses; M.M. supervised trophoblast differentiation assays and gave advice; S.O. generated the transgenic mice, constructed chimeras, and perfomed morula and blastocyst assays; M.S. designed and supervised the study, secured funding, analysed the data, and wrote the manuscript. All authors discussed the results and commented on the manuscript.
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Extended data figures and tables
Extended Data Figure 1 Characterization of four independent i4F transgenic mouse lines.
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.
Extended Data Figure 5 Characterization of in vivo iPS cells.
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).
Extended Data Figure 7 Validation of RNA-seq data.
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.
Extended Data Figure 8 In vivo iPS cell contribution to the trophectoderm lineage.
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.
Extended Data Figure 9 Expression levels of 2C marker genes.
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.
Extended Data Figure 10 Immunohistochemical characterization of embryo-like structures.
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.
Supplementary information
Supplementary Table 1
This table shows differentially expressed genes in in vivo iPS cells vs in vitro iPS cells. (XLSX 69 kb)
Supplementary Table 2
This table shows differentially expressed genes in in vivo iPS cells vs ES cells. (XLSX 63 kb)
Supplementary Table 3
This table shows differentially expressed genes in ES cells vs in vitro iPS cells. (XLSX 100 kb)
Supplementary Table 4
This table shows upregulated and downregulated genes in in vivo iPS cells and ES cells (vs in vitro iPS cells). (XLSX 13 kb)
Supplementary Table 5
This table shows upregulated and downregulated genes in in vivo iPS cells (vs in vitro iPS cells and ES cells). (XLSX 8 kb)
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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
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DOI: https://doi.org/10.1038/nature12586
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