Reprogramming in vivo produces teratomas and iPS cells with totipotency features

Journal name:
Nature
Volume:
502,
Pages:
340–345
Date published:
DOI:
doi:10.1038/nature12586
Received
Accepted
Published online
Corrected online

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.

At a glance

Figures

  1. Generation of teratomas upon in vivo induction of the four factors Oct4, Sox2, Klf4 and c-Myc.
    Figure 1: Generation of teratomas upon in vivo induction of the four factors Oct4, Sox2, Klf4 and c-Myc.

    a, Reprogrammable mouse generation. b, Reprogrammable mouse with multiple teratomas (arrowheads). c, Teratomas in the intestine of a reprogrammable mouse. d, Histological section of a teratoma with mesoderm (mes), endoderm (end) and ectoderm (ect). Asterisk indicates giant trophoblast cells and haemorrhages. e, Survival of reprogrammable mice after the indicated doxycycline treatments. Time refers to the initiation of the treatments. f, Incidence of teratomas. Data corresponds to their time of death or week 30. g, Localization of teratomas in mice with teratomas.

  2. Many cell types are reprogrammed in vivo.
    Figure 2: Many cell types are reprogrammed in vivo.

    a, Incidence of teratomas in wild-type mice transplanted with reprogrammable bone marrow (BM). Time refers to the initiation of the treatment. b, Same as a in reprogrammable mice transplanted with wild-type BM. Ticks indicate censored mice dead without teratomas due to pulmonary oedemas secondary to irradiation (a) or systemic i4F induction (b). c, Double immunohistochemistry of NANOG (dark brown) and cytokeratin 19 (CK19, magenta) in the stomach of whole-body reprogrammable mice. d, Same staining as c in the large intestine. e, Same staining as c in the pancreas. All scale bars correspond to 100µm.

  3. Isolation and characterization of in vivo iPS cells.
    Figure 3: Isolation and characterization of in vivo iPS cells.

    a, In vivo iPS cell colony 10days after blood plating. b, Expansion of in vivo iPS cells. c, Immunofluorescence of in vivo iPS cell colony. d, Immunoblot of in vivo iPS cells, in vitro iPS cells (no. 1 from i4F MEFs; no. 2 from MEFs infected with lenti-OSKM) and C57BL/6.10 ES cells. e, Subcutaneous teratoma from in vivo iPS cells injection. Asterisk indicates giant trophoblast cells and haemorrhages. f, In vivo iPS cells derived chimaera. g, Frequency of in vivo iPS cells isolation. h, Unsupervised hierarchical clustering of in vivo iPS cells, in vitro iPS cells and ES cells (6, 5 and 3 clones respectively). i, Venn diagram of differentially expressed genes. j, qPCR analysis of differentially expressed genes in the same clones as in h. Average±s.d. and unpaired two-tailed Student’s t-test are shown. *P<0.05; **P<0.01; ***P<0.001.

  4. In vivo iPS cells efficiently contribute to the trophectoderm.
    Figure 4: In vivo iPS cells efficiently contribute to the trophectoderm.

    a, Teratomas with trophoblast giant cells. b, Trophoblast stem cell differentiation of the indicated cells. c, Cdx2 expression in in vivo iPS cells, in vitro iPS cells and ES cells (6, 5 and 3 clones, respectively) during TS differentiation, relative to day 0. Average±s.d. and unpaired, two-tailed Student’s t-test are shown: *P<0.05, **P<0.01. d, Immunofluorescence of in vivo iPS cells derived trophoblast stem cells. e, Giant cells differentiated from trophoblast stem cells and in vivo iPS cells derived trophoblast stem cells. f, Chimaeric blastocysts from GFP in vivo iPS cells and GFP-ES cells. Arrowheads mark GFP+ trophectoderm cells. g, Frequency of blastocysts with GFP+ trophectoderm cells from in vivo iPS cells (n = 2 clones) and ES cells (JM8.F6). Fisher’s exact test: **P<0.01. h, GFP in vivo iPS cells chimaeric embryo and placenta. i, Immunostaining against GFP.

  5. In vivo reprogramming and in vivo iPS cells generate embryo-like structures.
    Figure 5: In vivo reprogramming and in vivo iPS cells generate embryo-like structures.

    a, Cysts in the abdominal cavity of a reprogrammable mouse. b, Frequency of embryo-like structures after intraperitoneal injection of in vivo iPS cells (3 clones), in vitro iPS cells (2 clones) and ES cells (JM8.F6). Fisher’s exact test: *P<0.05. c, Cyst generated by intraperitoneal injection. Left panels, germ layer markers: SOX2 (ectoderm), T/BRACHYURY (mesoderm) and GATA4 (endoderm). Right panels, extraembryonic markers: CDX2 (trophectoderm), and AFP and CK8, both specific for visceral endoderm of the yolk sac. d, Cyst generated by intraperitoneal injection presenting TER-119+ nucleated erythrocytes and LYVE-1+ endothelial cells in structures resembling yolk sac blood islands.

  6. Characterization of four independent i4F transgenic mouse lines.
    Extended Data Fig. 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 (1mgml−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 (1µgml−1). Colonies of iPS cells in the i4F-A and i4F-B plates were stained for alkaline phosphatase (AP) 10days after induction. In the case of i4F-C and i4F-D, plates were stained after 15days 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: 1µgml−1 of doxycycline for 6days, or continuous treatment with 1µgml−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.

  7. Genomic insertion sites of lentiviral transgenes i4F-A and i4F-B and their effect on the host genes.
    Extended Data Fig. 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.

  8. Histological alterations of the intestine and pancreas upon induction of i4F reprogrammable mice.
    Extended Data Fig. 3: Histological alterations of the intestine and pancreas upon induction of i4F reprogrammable mice.

    Mice were treated with doxycycline (1mgml−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.

  9. i4F induction leads to the appearance of tumoral masses and in situ reprogramming events.
    Extended Data Fig. 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.

  10. Characterization of in vivo iPS cells.
    Extended Data Fig. 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 3days. 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.

  11. Transcriptomic profiles of in vivo iPS cells, in vitro iPS cells and ES cells.
    Extended Data Fig. 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).

  12. Validation of RNA-seq data.
    Extended Data Fig. 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.

  13. In vivo iPS cell contribution to the trophectoderm lineage.
    Extended Data Fig. 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.

  14. Expression levels of 2C marker genes.
    Extended Data Fig. 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.

  15. Immunohistochemical characterization of embryo-like structures.
    Extended Data Fig. 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.

Accession codes

Referenced accessions

Gene Expression Omnibus

Change history

Corrected online 16 October 2013
Scale bar values were added to Fig. 5a.

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Author information

Affiliations

  1. Tumour Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid E-28029, Spain

    • María Abad,
    • Lluc Mosteiro,
    • Cristina Pantoja &
    • Manuel Serrano
  2. Histopathology Unit, Spanish National Cancer Research Centre (CNIO), Madrid E-28029, Spain

    • Marta Cañamero
  3. Cardiovascular Development and Repair Department, Spanish National Cardiovascular Research Centre (CNIC), Madrid E-28029, Spain

    • Teresa Rayon,
    • Inmaculada Ors &
    • Miguel Manzanares
  4. Bioinformatics Unit, Spanish National Cancer Research Centre (CNIO), Madrid E-28029, Spain

    • Osvaldo Graña
  5. Confocal Microscopy Unit, Spanish National Cancer Research Centre (CNIO), Madrid E-28029, Spain

    • Diego Megías
  6. Genomics Unit, Spanish National Cancer Research Centre (CNIO), Madrid E-28029, Spain

    • Orlando Domínguez
  7. Flow Cytometry Unit, Spanish National Cancer Research Centre (CNIO), Madrid E-28029, Spain

    • Dolores Martínez
  8. Transgenic Mice Unit, Spanish National Cancer Research Centre (CNIO), Madrid E-28029, Spain

    • Sagrario Ortega

Contributions

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

The primary RNA-seq data has been deposited in the GEO repository under accession number GSE48364.

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Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Characterization of four independent i4F transgenic mouse lines. (328 KB)

    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 (1mgml−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 (1µgml−1). Colonies of iPS cells in the i4F-A and i4F-B plates were stained for alkaline phosphatase (AP) 10days after induction. In the case of i4F-C and i4F-D, plates were stained after 15days 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: 1µgml−1 of doxycycline for 6days, or continuous treatment with 1µgml−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.

  2. Extended Data Figure 2: Genomic insertion sites of lentiviral transgenes i4F-A and i4F-B and their effect on the host genes. (310 KB)

    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.

  3. Extended Data Figure 3: Histological alterations of the intestine and pancreas upon induction of i4F reprogrammable mice. (1,141 KB)

    Mice were treated with doxycycline (1mgml−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.

  4. Extended Data Figure 4: i4F induction leads to the appearance of tumoral masses and in situ reprogramming events. (485 KB)

    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.

  5. Extended Data Figure 5: Characterization of in vivo iPS cells. (462 KB)

    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 3days. 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.

  6. Extended Data Figure 6: Transcriptomic profiles of in vivo iPS cells, in vitro iPS cells and ES cells. (442 KB)

    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).

  7. Extended Data Figure 7: Validation of RNA-seq data. (122 KB)

    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.

  8. Extended Data Figure 8: In vivo iPS cell contribution to the trophectoderm lineage. (452 KB)

    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.

  9. Extended Data Figure 9: Expression levels of 2C marker genes. (70 KB)

    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.

  10. Extended Data Figure 10: Immunohistochemical characterization of embryo-like structures. (704 KB)

    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

Excel files

  1. Supplementary Table 1 (69 KB)

    This table shows differentially expressed genes in in vivo iPS cells vs in vitro iPS cells.

  2. Supplementary Table 2 (63 KB)

    This table shows differentially expressed genes in in vivo iPS cells vs ES cells.

  3. Supplementary Table 3 (100 KB)

    This table shows differentially expressed genes in ES cells vs in vitro iPS cells.

  4. Supplementary Table 4 (13 KB)

    This table shows upregulated and downregulated genes in in vivo iPS cells and ES cells (vs in vitro iPS cells).

  5. Supplementary Table 5 (8.2 KB)

    This table shows upregulated and downregulated genes in in vivo iPS cells (vs in vitro iPS cells and ES cells).

Additional data