Abstract
Here we show that conventional reprogramming towards pluripotency through overexpression of Oct4, Sox2, Klf4 and c-Myc can be shortcut and directed towards cardiogenesis in a fast and efficient manner. With as little as 4 days of transgenic expression of these factors, mouse embryonic fibroblasts (MEFs) can be directly reprogrammed to spontaneously contracting patches of differentiated cardiomyocytes over a period of 11–12 days. Several lines of evidence suggest that a pluripotent intermediate is not involved. Our method represents a unique strategy that allows a transient, plastic developmental state established early in reprogramming to effectively function as a cellular transdifferentiation platform, the use of which could extend beyond cardiogenesis. Our study has potentially wide-ranging implications for induced pluripotent stem cell (iPSC)-factor-based reprogramming and broadens the existing paradigm.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Change history
11 February 2011
In the version of this article initially published online, the commerically available cardiomyocytes Cor.At were incorrectly referred to as CorAT in Figure 4. Similarly, in the methods Axiogenesis was not correctly referenced as the manufacturers of Cor.At cells. This error has been corrected in both the HTML and PDF versions of the article.
References
Ieda, M. et al. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell 142, 375–386 (2010).
Schenke-Layland, K. et al. Reprogrammed mouse fibroblasts differentiate into cells of the cardiovascular and hematopoietic lineages. Stem Cells 26, 1537–1546 (2008).
Hanna, J. et al. Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 462, 595–601 (2009).
Stadtfeld, M., Maherali, N., Breault, D. T. & Hochedlinger, K. Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell 2, 230–240 (2008).
Cai, C. L. et al. T-box genes coordinate regional rates of proliferation and regional specification during cardiogenesis. Development 132, 2475–2487 (2005).
Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).
Snyder, A., Fraser, S.T. & Baron, M. H. Bone morphogenetic proteins in vertebrate hematopoietic development. J. Cell Biochem. 93, 224–232 (2004).
Martin-Puig, S., Wang, Z. & Chien, K. R. Lives of a heart cell: tracing the origins of cardiac progenitors. Cell Stem Cell 2, 320–331 (2008).
Snyder, M., Huang, X. Y. & Zhang, J. J. Stat3 directly controls the expression of Tbx5, Nkx2.5, and GATA4 and is essential for cardiomyocyte differentiation of P19CL6 cells. J. Biol. Chem. 285, 23639–23646 (2010).
Cohen, E. D., Tian, Y. & Morrisey, E. E. Wnt signaling: an essential regulator of cardiovascular differentiation, morphogenesis and progenitor self-renewal. Development 135, 789–798 (2008).
Klaus, A. & Birchmeier, W. Developmental signaling in myocardial progenitor cells: a comprehensive view of Bmp- and Wnt/β-catenin signaling. Pediatr. Cardiol. 30, 609–616 (2009).
Qyang, Y. et al. The renewal and differentiation of Isl1+ cardiovascular progenitors are controlled by a Wnt/β-catenin pathway. Cell Stem Cell 1, 165–179 (2007).
Kuzmenkin, A. et al. Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro. FASEB J. 23, 4168–4180 (2009).
Shah, A. P. et al. Genetic background affects function and intracellular calcium regulation of mouse hearts. Cardiovasc. Res. 87, 683–693 (2010).
Stieber, J. et al. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc. Natl Acad. Sci. USA 100, 15235–15240 (2003).
Markoulaki, S. et al. Transgenic mice with defined combinations of drug-inducible reprogramming factors. Nat. Biotechnol. 27, 169–171 (2009).
Blelloch, R., Venere, M., Yen, J. & Ramalho-Santos, M. Generation of induced pluripotent stem cells in the absence of drug selection. Cell Stem Cell 1, 245–247 (2007).
Nakagawa, M. et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat. Biotechnol. 26, 101–106 (2008).
Wernig, M., Meissner, A., Cassady, J. P. & Jaenisch, R. c-Myc is dispensable for direct reprogramming of mouse fibroblasts. Cell Stem Cell 2, 10–12 (2008).
Okada, M., Oka, M. & Yoneda, Y. Effective culture conditions for the induction of pluripotent stem cells. Biochim. Biophys. Acta 1800, 956–963 (2010).
Wernig, M. et al. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nat. Biotechnol. 26, 916–924 (2008).
Hanna, J. et al. Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell 133, 250–264 (2008).
Silva, J. et al. Nanog is the gateway to the pluripotent ground state. Cell 138, 722–737 (2009).
Brambrink, T. et al. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2, 151–159 (2008).
Stadtfeld, M., Maherali, N., Borkent, M. & Hochedlinger, K. A reprogrammable mouse strain from gene-targeted embryonic stem cells. Nat. Methods 7, 53–55 (2010).
Anjos-Afonso, F. & Bonnet, D. Nonhematopoietic/endothelial SSEA-1+ cells define the most primitive progenitors in the adult murine bone marrow mesenchymal compartment. Blood 109, 1298–1306 (2007).
Koso, H. et al. SSEA-1 marks regionally restricted immature subpopulations of embryonic retinal progenitor cells that are regulated by the Wnt signaling pathway. Dev. Biol. 292, 265–276 (2006).
Christen, B., Robles, V., Raya, M., Paramonov, I. & Belmonte, J. C. Regeneration and reprogramming compared. BMC Biol. 8, 5 (2010).
Meshorer, E. et al. Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells. Dev. Cell 10, 105–116 (2006).
Hochedlinger, K. & Plath, K. Epigenetic reprogramming and induced pluripotency. Development 136, 509–523 (2009).
Artyomov, M. N., Meissner, A. & Chakraborty, A. K. A model for genetic and epigenetic regulatory networks identifies rare pathways for transcription factor induced pluripotency. PLoS Comput. Biol. 6, e1000785 (2010).
Woltjen, K. et al. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458, 766–770 (2009).
Zhou, H. et al. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4, 381–384 (2009).
David, R. et al. MesP1 drives vertebrate cardiovascular differentiation through Dkk-1-mediated blockade of Wnt-signalling. Nat. Cell Biol. 10, 338–345 (2008).
Takahashi, K., Okita, K., Nakagawa, M. & Yamanaka, S. Induction of pluripotent stem cells from fibroblast cultures. Nat. Protoc. 2, 3081–3089 (2007).
Ouyang, K., Wu, C. & Cheng, H. Ca(2+)-induced Ca(2+) release in sensory neurons: low gain amplification confers intrinsic stability. J. Biol. Chem. 280, 15898–15902 (2005).
Acknowledgements
We thank C. Desponts, X. Yuan, S. Zhu, R. Ambasudhan, R. Abujarour and W. Li for discussions, technical assistance, and critical reading of the manuscript. We also thank H. Schöler for providing TTFs, and D. Watry for assistance with FACS analyses. S.D. is supported by funding from NICHD, NHLBI and NIMH/NIH, California Institute for Regenerative Medicine, Prostate Cancer Foundation, Fate Therapeutics, Esther B. O'Keeffe Foundation and the Scripps Research Institute. J.A.E. is a Lowe Family Foundation fellow.
Author information
Authors and Affiliations
Contributions
J.A.E. and S.D. conceived the project and wrote the manuscript. J.A.E. designed and carried out experiments. S.H., J.K., H.Z., K.O., G.W. and J.C. provided materials and assisted with experiments.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Information
Supplementary Information (PDF 241 kb)
Supplementary Movie 1
Supplementary Information (MOV 3091 kb)
Supplementary Movie 2
Supplementary Information (MOV 17149 kb)
Supplementary Movie 3
Supplementary Information (MOV 11795 kb)
Supplementary Movie 4
Supplementary Information (MOV 3380 kb)
Supplementary Movie 5
Supplementary Information (MOV 2189 kb)
Supplementary Movie 6
Supplementary Information (MOV 3850 kb)
Rights and permissions
About this article
Cite this article
Efe, J., Hilcove, S., Kim, J. et al. Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat Cell Biol 13, 215–222 (2011). https://doi.org/10.1038/ncb2164
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ncb2164
This article is cited by
-
Cell-fate conversion of intestinal cells in adult Drosophila midgut by depleting a single transcription factor
Nature Communications (2024)
-
Cell-mediated exon skipping normalizes dystrophin expression and muscle function in a new mouse model of Duchenne Muscular Dystrophy
EMBO Molecular Medicine (2024)
-
Cardiomyocyte precursors generated by direct reprogramming and molecular beacon selection attenuate ventricular remodeling after experimental myocardial infarction
Stem Cell Research & Therapy (2023)
-
Harnessing stem cell and lineage reprogramming technology to treat cardiac fibrosis
Cell Regeneration (2023)
-
Direct cardiac reprogramming: basics and future challenges
Molecular Biology Reports (2023)