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Lineage conversion induced by pluripotency factors involves transient passage through an iPSC stage

Nature Biotechnology volume 33, pages 761–768 (2015)Cite this article

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Abstract

Brief expression of pluripotency-associated factors such as Oct4, Klf4, Sox2 and c-Myc (OKSM), in combination with differentiation-inducing signals, has been reported to trigger transdifferentiation of fibroblasts into other cell types. Here we show that OKSM expression in mouse fibroblasts gives rise to both induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) under conditions previously shown to induce only iNSCs. Fibroblast-derived iNSC colonies silenced retroviral transgenes and reactivated silenced X chromosomes, both hallmarks of pluripotent stem cells. Moreover, lineage tracing with an Oct4-CreER labeling system demonstrated that virtually all iNSC colonies originated from cells transiently expressing Oct4, whereas ablation of Oct4+ cells prevented iNSC formation. Lastly, an alternative transdifferentiation cocktail that lacks Oct4 and was reportedly unable to support induced pluripotency yielded iPSCs and iNSCs carrying the Oct4-CreER-derived lineage label. Together, these data suggest that iNSC generation from fibroblasts using OKSM and other pluripotency-related reprogramming factors requires passage through a transient iPSC state.

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Figure 1: Generation of iPSCs and iNSCs from MEFs under neural culture conditions.
Figure 2: Oct4 lineage tracing and ablation during iNSC induction.
Figure 3: Silencing of retroviral transgenes and X chromosome reactivation during iNSC induction.
Figure 4: An alternative transdifferentiation cocktail generates iNSCs and iPSCs via an Oct4+ intermediate stage.

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Acknowledgements

We thank members of the Hochedlinger laboratory for critical evaluation and discussion of this manuscript. We thank E. Apostolou and S. Cheloufi for helpful comments and N. Maherali for suggesting the idea of X chromosome reactivation. We are also grateful to B. Payer and J. Lee for providing tail-tip fibroblasts carrying an X-linked GFP reporter and to A. Brack for sharing Rosa26-lsl-DTA mice. We are grateful to L. Prickett, M. Weglarz and K. Folz-Donahue at the MGH/HSCI flow cytometry core for their constant assistance and support. O.B.-N. is supported by a Gruss-Lipper postdoctoral fellowship from the EGL foundation. B.A.S. has been supported by a T32 (5-T32-CA-9216-33) grant and through a Postdoctoral Fellowship Award by the MGH Executive Committee on Research Fund for Medical Discovery. J.B. and I.L. are supported by Ruth L. Kirschstein F32 Post-doctoral Fellowships from the US National Institutes of Health (NIH) (1F32HD078029-01A1 to J.B. and 1F32HD079225-01A1 to I.L.). Support to K.H. is from the NIH (R01HD058013) and Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA

    Ori Bar-Nur, Cassandra Verheul, Justin Brumbaugh, Benjamin A Schwarz, Inna Lipchina, Aaron J Huebner & Konrad Hochedlinger

  2. Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA

    Ori Bar-Nur, Cassandra Verheul, Justin Brumbaugh, Benjamin A Schwarz, Inna Lipchina, Aaron J Huebner & Konrad Hochedlinger

  3. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA

    Ori Bar-Nur, Cassandra Verheul, Justin Brumbaugh, Benjamin A Schwarz, Inna Lipchina, Aaron J Huebner & Konrad Hochedlinger

  4. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA

    Ori Bar-Nur, Cassandra Verheul, Justin Brumbaugh, Benjamin A Schwarz, Inna Lipchina, Aaron J Huebner & Konrad Hochedlinger

  5. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA

    Ori Bar-Nur, Cassandra Verheul, Justin Brumbaugh, Benjamin A Schwarz, Inna Lipchina, Aaron J Huebner & Konrad Hochedlinger

  6. Center for Regenerative Medicine (CReM), Boston University School of Medicine, Boston, Massachusetts, USA

    Andreia G Sommer & Gustavo Mostoslavsky

  7. Boston Medical Center, Boston, Massachusetts, USA

    Andreia G Sommer & Gustavo Mostoslavsky

Authors
  1. Ori Bar-Nur
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  2. Cassandra Verheul
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  3. Andreia G Sommer
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  4. Justin Brumbaugh
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  5. Benjamin A Schwarz
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  9. Konrad Hochedlinger
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Contributions

O.B.-N. and K.H. conceived the experiments, interpreted results and wrote the manuscript. O.B.-N. conducted all reprogramming experiments, performed statistical analysis, bioinformatics analysis of expression data and generated figures; C.V. assisted in most reprogramming experiments and interpretation of results; A.G.S. and G.M. generated the BKSM lentiviral cassette; J.B. generated chimeric animals by blastocyst injections; B.A.S. conducted reprogramming experiments from FACS-sorted intermediates; I.L. performed allele-specific real-time PCR for an X-linked marker gene; A.J.H. confirmed specificity of the Oct4-CreER lineage tracing system in vivo.

Corresponding author

Correspondence to Konrad Hochedlinger.

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Integrated supplementary information

Supplementary Figure 1 Characterization of OKSM-iNSCs

(A) Gene expression levels of Sox1 and Sox2 in the indicated cell types by microarray gene expression analysis. (B) Dendrogram cluster analysis of global gene expression of indicated samples by expression microarrays. (C) Scatter plot analysis shows linear regression coefficient (R2) of global gene expression by microarray analysis between MEFs and OKSM-iNSCs and brain-derived NSCs and OKSM-iNSCs, respectively. (D) Functional annotation analysis of upregulated genes (2-fold or more) in OKSM-iNSCs vs. MEFs by gene expression microarray. Top categories are shown together with the number of genes and Benjamini-Hochberg-corrected p-value. (E) Gene expression levels of Nanog in the indicated cell types by microarray analysis.

Supplementary Figure 2 Molecular characterization of iPSCs generated in NSC media and propagated in ESC media

(A) Graph showing quantification of Oct4+ and Nanog+ iPSC-like colonies generated in NSC media. For each replicate, 3x104 cells were used (n=3 independent replicates; error bars, s.e.m. *P<0.05). (B) Representative immunofluorescence images showing co-localization of Oct4+ and Nanog+ in colonies emerging in NSC media. (C) Morphology of NSC media-derived iPSCs (upper panel) and alkaline phosphatase (AP) staining for an expanded iPSC clone (lower panel). (D) Representative images of immunofluorescence staining for Nanog in NSC media-derived iPSC clones #1-3. (E) Expression of pluripotency-associated surface markers in NSC media-derived iPSCs by live-staining immunofluorescence analysis. (F) Flow cytometry analysis of surface markers characteristic of nascent iPSCs (loss of Thy1, gain of Epcam) in rep-MEFs 6 days after dox administration in conventional NSC media. (G) Gene expression levels of Epcam in the indicated cell types by microarray gene expression analysis. Note that Epcam is not expressed in brain-NSCs or OKSM-iNSCs.

Supplementary Figure 3 Activity of the Oct4-CreER; R26-lsl-EYFP lineage tracing system in brain-derived NSCs

(A) Flow cytometric analysis of Oct4-CreER; R26-lsl-EYFP NSC cultures derived from the brains of five independent E13.5 embryos. Tamoxifen was administered to pregnant mice at E8.5 and brain-derived NSCs were recovered at day E13.5. EYFP expression was examined at passage 3 of in vitro culture. OKSM-iNSC clone #2 served as a positive control for EYFP expression. The PE-Cy7 channel was used to control for autofluorescence. (B) Representative immunofluorescence images show that the Oct4-CreER; R26-lsl-EYFP brain-derived NSC lines #1-5 express the NSC markers Sox1 and Sox2. (C) Flow cytometry analysis for uninduced Oct4-CreER; R26-lsl-EYFP brain-derived NSCs to confirm faithful regulation of the Oct4-CreER allele. Brain-NSCs were isolated at E13.5 and 4-OHT was added after 2 passages before performing flow cytometry analysis for EYFP. Note that no labeled cells were detected with or without 4-OHT in brain-NSCs of this genotype, indicating specificity of the Oct4-CreER allele. The PE-Cy7 channel was used to control for autofluorescence.

Supplementary Figure 4 Specificity of the Oct4-GFP and Oct4-CreER; R26-lsl-EYFP reporters

(A) Flow cytometry analysis for EYFP expression at the indicated time points of OKSM induction in Oct4-CreER; R26-lsl-EYFP MEFs. Note that no aberrant EYFP expression is detected after 2 and 4 days of OKSM induction (OKSM ind.). The PE-Cy7 channel was used to control for autofluorescence. (B) Flow cytometry analysis for Oct4-GFP reporter expression at the indicated time points of OKSM induction shows absence of GFP signal. The PE-Cy7 channel was used to control for autofluorescence. Note that more cells are EYFP+ in panel A than are GFP+ in panel B after 11 days of dox treatment and 5 days of dox withdrawal (13.6% vs. 0.14%) because differentiated cells remain labeled with the Oct4-CreER; R26-lsl-EYFP system, whereas they downregulate the Oct4-GFP reporter.

Supplementary Figure 5 Molecular characterization of iPSCs generated from Oct4-CreER; R26-lsl-EYFP MEFs in NSC media and propagated in ESC media

(A) iPSC clones remain EYFP- in the absence of 4-OHT (left panels) while they become EYFP+ in the presence of 4-OHT (right panel). (B) Representative immunofluorescence stains for Nanog and Oct4 in NSC media-derived iPSC clones generated from Oct4-CreER; R26-lsl-EYFP MEFs. (C) Flow cytometry analysis for EYFP expression in MEFs derived from chimeric embryos generated from EYFP+ iPSCs clone #6 (generated in conventional NSC media). Shown are MEF preparations established from 2 different E13.5 embryos and analyzed at passage 2. Non-chimeric EYFP- MEFs served as a negative control. The PE-Cy7 channel was used to control for autofluorescence.

Supplementary Figure 6 Characterization of iNSC subclones

(A) Representative images of iNSC sucblones #2-6 at passage 20, derived by sorting single cells from EYFP+ iNSCs expanded from a single colony. (B) Flow cytometry analysis for EYFP expression of OKSM-iNSC subclones #2-6 at passage 12. Note that all subclones are at least 98% EYFP+ at passage 12. The PE-Cy7 channel was used to control for autofluorescence. (C) Immunofluorescence images show staining for Nestin and Sox1 in OKSM-iNSC sucblones #1-6 at passage 22.

Supplementary Figure 7 Oct4 lineage tracing and ablation systems to track the origin of iNSCs

(A) Representative images of OKSM-iNSC (EYFP+) colonies generated with modified NSC media after 4-5 days of doxycycline (dox) induction, followed by 17 days of dox-independent growth. Note that images depicting neurites were taken at higher magnification than those of the corresponding EYFP+ OKSM-iNSCs. (B) Schematic of lineage ablation approach using Oct4-CreER; R26-lsl-DTA MEFs to test whether all iNSCs pass through an Oct4+ stage. (C) 4-OHT treatment triggers death of Oct4+ cells as judged by alkaline phosphatase staining (AP) of dox-independent iPSC colonies in ESC media. The presence of residual AP+ colonies in 4-OHT treated Oct4-CreER; R26-lsl-DTA cells likely represents partially reprogrammed iPSCs or iPSC colonies that failed to recombine the R26-lsl-DTA allele. (D) Quantification of AP+, dox-independent iPSC formation shown in (C). For each replicate, 3x104 cells were used (n=3 independent replicates; error bars, s.e.m.).

Supplementary Figure 8 Retroviral tdTomato expression in MEFs cultured in NSC media

(A) Flow cytometry analysis of MEFs infected with the retroviral pMXs-tdTomato vector. Shown are bulk cultures expressing tdTomato (left panel), sorted tdTomato+ MEFs (middle panel) and uninfected MEFs (right panel). The PacB channel was used to control for autofluorescence. (B) Sorted and explanted tdTomato+ MEFs retain tdTomato expression after growth in NSC media for 1 week (left panel). Uninfected MEFs are shown on the right. The PE-Cy7 channel was used to control for autofluorescence.

Supplementary Figure 9 Expression of the X-linked CMV-GFP reporter and X-linked Uba1 gene in female TTFs and derivative iNSCs

(A) Flow cytometry analysis of OKSM-iNSC clones generated either from sorted XaXiGFP (top) or XaGFPXi tail tip fibroblasts (TTFs) (bottom). Note that some iNSC clones partially lose CMV-GFP reporter expression after prolonged culture, regardless of their origin from XaXiGFP or XaGFPXi TTFs. The PE-Cy7 channel was used to control for autofluorescence. (B) Sorted XaXiGFP TTFs do not reactivate the CMV-GFP reporter located on the silent X chromosome after 2 weeks of culture in NSC media. A positive control for GFP expression is shown on the right. The APC channel was used to control for autofluorescence. (C) Allele-specific quantitative PCR analysis of the X-linked gene Uba1. Primers distinguishing between the maternal mus musculus musculus (left) and the paternal mus musculus castaneous (right) alleles of the Uba1 gene were used. Sorted GFP+ and GFP- TTFs served as controls.

Supplementary Figure 10 Effect of small molecules on the generation of iNSCs

(A) Quantification of Sox1+ iNSC colonies generated in the presence of ascorbic acid (AA) or ascorbic acid and GSK3βi (“AGi”). For each replicate, 3x104 cells were used (n=3 independent replicates; error bars, s.e.m. *P<0.05). (B) Representative immunofluorescence images showing a Sox1+ iNSC colony generated with AGi.

Supplementary Figure 11 Cloning of the BKSM polycistronic cassette.

(A) A schematic representation of the cloning strategy to generate the BKSM lentiviral vector cassette. A DNA fragment consisting of the Oct4 ORF, followed by an F2A peptide and part of the Klf4 ORF, was excised from the STEMCCA (OKSM) vector using NotI and BstZ17I and replaced by a fragment corresponding to Brn4-F2A-Klf4, generated by overlapping PCR. (B) Representative immunofluorescence images for Oct4 expression in MEFs 3 days after transduction with the tetOP-OKSM (top) and tetOP-BKSM (bottom) cassettes. Note that Oct4+ nuclear staining is detected only in cells that were infected with the tetOP-OKSM cassette. (C) Representative immunofluorescence images for Brn4 in MEFs 3 days following transduction with the tetOP-BKSM (top) or tetOP-OKSM (bottom) cassettes. Note that Brn4+ nuclear staining is detected only in cells that were infected with the tetOP-BKSM cassette.

Supplementary Figure 12 Characterization of BKSM-iNSCs

(A) Morphology of a BKSM-iNSC colony generated from Sox2-GFP MEFs (upper panel) and Sox2-GFP reporter expression (lower panel). (B) Scatter plot display of global microarray data of indicated samples, showing low linear regression coefficient (R2) of global gene expression between MEFs and BKSM-iNSCs and high R2 between BKSM-iNSCs and brain-derived NSCs. (C) Functional annotation of upregulated genes (2-fold or more) in BKSM-iNSCs vs. MEFs based on microarray data; top categories are shown together with the number of genes and Benjamini-Hochberg-corrected P-values.

Supplementary Figure 13 Characterization of BKSM-iPSCs

(A) Representative images of iPSCs generated with the BKSM cassette from Sox2-GFP MEFs (BKSM-iPSCs). (B) Expression of key pluripotency genes in BKSM-iPSCs based on microarray analysis. (C) Scatter plot analysis showing linear regression coefficient (R2) of global gene expression between MEFs and BKSM-iPSCs, and mouse ESCs and BKSM-iPSCs. (D) Teratoma sections from a BKSM-iPSC clone showing cell types representative of the three germ layers. (E) Activation of Sox2-GFP expression in the brain of a neonatal chimera generated from BKSM-iPSCs (derived from Sox2-GFP MEFs) (left panel) relative to a non-chimeric control (right panel).

Supplementary Figure 14 Reprogramming of BKSM-iNSCs into iPSCs

(A) Experimental design to generate iPSCs using a secondary system of reprogrammable BKSM-iNSCs. (B) Morphology of iPSC colony generated from BKSM-iNSCs. (C) Alkaline phosphatase (AP) staining of iPSCs derived from BKSM-iNSCs.

Supplementary Figure 15 Oct4 lineage tracing during induced cardiomyocyte (iCM) or induced neuron (iN) generation from MEFs

(A) Quantification of EYFP+ and Cardiac Troponin (cTnT)+ induced cardiomyocytes derived from Oct4-CreER; R26-lsl-EYFP MEFs upon brief OKSM expression. For each replicate, 5x105 cells were used (n=3 biological replicates; error bars, s.e.m. ** P<0.005 n.s., not significant P=0.07). (B) Representative immunofluorescence images showing co-staining for EYFP and cTnT expression in induced cardiomyocytes. (C) Schematic of the lineage tracing approach to test if iN-like cells derived from Oct4-CreER; R26-lsl-EYFP MEFs upon transduction with tetOP-Ascl1, tetOP-Brn2 and tetOP-Myt1l vectors pass through an Oct4+ stage. (D) Lack of EYFP expression in non-transduced (upper panel) or transduced (middle panel) MEFs grown in iN transdifferentiation medium for 12 days. Tuj1+ iN-like cells were detected exclusively in cultures transduced with tetOP-Ascl1, tetOP-Brn2 and tetOP-Myt1l vectors. We calculated the frequency of iN formation by counting Tuj1+ cells per DAPI+ cells in 8 random fields, indicating a reprogramming efficiency of 9%. (13 iN cells/142 DAPI+ cells) (E) Flow cytometry analysis of bulk cultures undergoing transdifferentiation. Note the lack of EYFP expression in transduced MEFs relative to positive control (EYFP+ OKSM-iNSC clone #2). The PE-Cy7 channel was used to control for autofluorescence.

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Bar-Nur, O., Verheul, C., Sommer, A. et al. Lineage conversion induced by pluripotency factors involves transient passage through an iPSC stage. Nat Biotechnol 33, 761–768 (2015). https://doi.org/10.1038/nbt.3247

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