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Small molecules facilitate rapid and synchronous iPSC generation

Nature Methods volume 11, pages 1170–1176 (2014)Cite this article

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Abstract

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) upon overexpression of OCT4, KLF4, SOX2 and c-MYC (OKSM) provides a powerful system to interrogate basic mechanisms of cell fate change. However, iPSC formation with standard methods is typically protracted and inefficient, resulting in heterogeneous cell populations. We show that exposure of OKSM-expressing cells to both ascorbic acid and a GSK3-β inhibitor (AGi) facilitates more synchronous and rapid iPSC formation from several mouse cell types. AGi treatment restored the ability of refractory cell populations to yield iPSC colonies, and it attenuated the activation of developmental regulators commonly observed during the reprogramming process. Moreover, AGi supplementation gave rise to chimera-competent iPSCs after as little as 48 h of OKSM expression. Our results offer a simple modification to the reprogramming protocol, facilitating iPSC induction at unparalleled efficiencies and enabling dissection of the underlying mechanisms in more homogeneous cell populations.

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Figure 1: Ascorbic acid and GSK3-β inhibitor (AGi) act synergistically on reprogramming.
Figure 2: AGi enhances and accelerates iPSC formation across different cell types.
Figure 3: Clonal analysis of GMP reprogramming indicates synchronous reprogramming with AGi.
Figure 4: AGi rescues the reprogramming defect of refractory cells.
Figure 5: Effect of AGi on gene expression patterns in reprogramming intermediates.

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Acknowledgements

We thank members of the Hochedlinger lab for helpful suggestions and critical reading of the manuscript. We thank M. Borkent for providing study material and to A. Foudi and D. Kramer for scientific discussion. We thank L. Prickett, M. Weglarz and K. Folz-Donahue at the Massachusetts General Hospital/Harvard Stem Cell Institute flow cytometry core. O.B.-N. was supported by a Gruss-Lipper postdoctoral fellowship. Support to K.H. was from the US National Institutes of Health (NIH; R01HD058013) and the Howard Hughes Medical Institute. J.B. is grateful for support from the Tosteson Fund for Medical Discovery Post-doctoral Fellowship by the MGH Executive Committee on Research and NIH (1F32HD078029-01A1).

Author information

Author notes

  1. Ori Bar-Nur and Justin Brumbaugh: These authors contributed equally to this work.

Authors and Affiliations

  1. Massachusetts General Hospital Cancer Center, Boston, Massachusetts, USA

    Ori Bar-Nur, Justin Brumbaugh, Cassandra Verheul, Effie Apostolou, Iulian Pruteanu-Malinici, Ryan M Walsh, Sridhar Ramaswamy & Konrad Hochedlinger

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

    Ori Bar-Nur, Justin Brumbaugh, Cassandra Verheul, Effie Apostolou, Ryan M Walsh, Sridhar Ramaswamy & Konrad Hochedlinger

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

    Ori Bar-Nur, Justin Brumbaugh, Cassandra Verheul, Effie Apostolou, Ryan M Walsh & Konrad Hochedlinger

  4. Department of Stem Cell and Regenerative Biology, Cambridge, Massachusetts, USA

    Ori Bar-Nur, Justin Brumbaugh, Cassandra Verheul, Effie Apostolou, Ryan M Walsh & Konrad Hochedlinger

  5. Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA

    Iulian Pruteanu-Malinici, Sridhar Ramaswamy & Konrad Hochedlinger

  6. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Iulian Pruteanu-Malinici & Sridhar Ramaswamy

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  1. Ori Bar-Nur
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Contributions

O.B.-N., J.B. and K.H. conceived of the experiments, interpreted results and wrote the manuscript. O.B.-N. and J.B. conducted all iPSC experiments, performed statistical analyses and generated figures; C.V. assisted in experiments; E.A. produced transgenic OKSmC mice; J.B. and R.M.W. generated chimeric animals by blastocysts injections; O.B.-N., I.P.-M. and S.R. performed bioinformatics analysis of expression data.

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Correspondence to Konrad Hochedlinger.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Transgene-independent colonies stained positive for NANOG.

Representative images were obtained for colonies generated under the indicated conditions. Scale bar is 200 μm.

Supplementary Figure 2 Effect of AGi treatment on cell cycle, cell proliferation and cell death following OKSM expression.

(a) Representative FLOW data for BrdU analysis (top panels) and propidium iodine staining (lower panels) on MEFs. (b) Quantitative measurements for BrdU analysis on MEFs showing percentage of cells in each phase of the cell cycle. This analysis was performed with and without exogenous expression of c-Myc, as indicated. (c) Cell proliferation analysis for MEFs during reprogramming with or without AGi. This analysis was performed with and without exogenous expression of c-Myc, as indicated. Error bars represent standard deviation for three independent replicates. (d) Cell proliferation analysis for GMPs during reprogramming with or without AGi. Error bars represent standard deviation for three independent replicates. (e) Annexin staining showing the percentage of MEF cells undergoing apoptosis or necrosis during reprogramming with or without AGi. Error bars represent standard deviation for three biological replicates.

Supplementary Figure 3 NANOG staining of iPSC-like colonies after exposure to OKSM, OKSM+ascorbic acid (AA), OKSM+CHIR-99021 (GSK3i), and OKSM+AGi.

Cells were stained after removal of doxycycline (OKSM) for at least 7 days. Scale bar is 50 μm.

Supplementary Figure 4 Expression of select pluripotency genes in the indicated cell types.

Supplementary Figure 5 Clonal reprogramming analysis of different cell types.

(a) Intra-well heterogeneity of OCT4-GFP expression in proB cells during reprogramming. Each time point represents analysis of a 96-well plate. (b) Intra-well heterogeneity of OCT4-GFP expression in hematopoietic stem cells (HSCs) during reprogramming. Each time point represents analysis of a 96-well plate. (c) Schematic of clonal reprogramming assay in GMPs using OCT4-GFP expression after doxycycline withdrawal as readout. (d) Quantification of clonal iPSCs generated after administering doxycycline with or without AGi for the indicated times. Doxycycline was withdrawn for at least 3 days prior to analysis to ensure that iPSC colonies were stable. Box and whisker plots were used in which the band in the middle of the box represents the median; the top and bottom of the box represent the first and third quartiles. The whiskers represent the lowest and highest datum within 1.5-fold of the inter-quartile ranges.

Supplementary Figure 6 AGi overcomes the refractory phenotype observed during iPSC formation.

Thy1+ cells were sorted and treated for an additional 7 days with the indicated supplements. Alkaline phosphatase staining and the corresponding quantitation are shown for three independent experiments after 4 days of doxycycline withdrawal. The number of sorted Thy1+ cells plated for each experiment is indicated.

Supplementary Figure 7 AGi-dependent gene expression changes after OKSM induction.

(a) Unsupervised clustering of replicate samples for iPSCs, MEFs, and reprogramming MEFs expressing OKSM in the absence or presence of AGi after 48 hours of factor expression. (b) Examples of signaling genes that are rapidly downregulated by at least 1.5-fold following AGi treatment at the indicated time points.

Supplementary Figure 8 qRT-PCR for key transient genes at early time points of reprogramming following AGi treatment.

iPSCs are shown for reference. Relative expression is calculated as 2-(δδCt) relative to GAPDH. Error bars represent the standard deviation for three independent experiments.

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Bar-Nur, O., Brumbaugh, J., Verheul, C. et al. Small molecules facilitate rapid and synchronous iPSC generation. Nat Methods 11, 1170–1176 (2014). https://doi.org/10.1038/nmeth.3142

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