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Reprogramming fibroblasts toward cardiomyocytes, neural stem cells and hepatocytes by cell activation and signaling-directed lineage conversion

Nature Protocols volume 10, pages 959–973 (2015)Cite this article

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Abstract

Induction of tissue-specific cell types via a conventional transdifferentiation strategy typically uses overexpression of the corresponding lineage-specific transcription factors. Alternatively, somatic cells can be temporarily activated via a common set of reprogramming factors into a transition state, which can then be directed into various cell types via soluble lineage-specific signals, without establishing a pluripotent state. Here, we provide protocols for the generation of cardiomyocytes, neural stem cells and hepatocytes from fibroblasts with such a cell activation (CA) and signaling-directed (SD; CASD) strategy. In these protocols, beating cardiomyocytes can be induced from mouse fibroblasts in 2–5 weeks; expandable neural stem cells and definitive endoderm progenitors can be obtained from human fibroblasts as early as 2.5 weeks; and human definitive endoderm progenitors can be differentiated into functional hepatocytes in 2 weeks. Through further developments, the CASD strategy can serve as a unique avenue for generating diverse functional cell types for biomedical research and therapeutic applications.

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Figure 1: The CASD reprogramming strategy and potential applications.
Figure 2: Small molecules in conjunction with Oct4 induce the conversion of mouse fibroblasts into cardiac cells.
Figure 3: Reprogramming human fibroblasts into neural stem cells.
Figure 4: Reprogramming human fibroblasts into hepatocytes.

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Acknowledgements

We thank all members of Ding laboratory for their valuable suggestion and discussion, particularly X. Wang for her contribution on episomal Nucleofection, and I. Namro Redwan, P. Liu and M. Xie for proofreading of the manuscript. We also thank C. Brennecka for editing the manuscript. S.D. is supported by funding from the California Institute for Regenerative Medicine (CIRM), the US National Institutes of Health (NIH) and the Gladstone Institutes. S.Z. is a CIRM training scholar.

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Author notes

  1. Saiyong Zhu and Haixia Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA

    Saiyong Zhu, Haixia Wang & Sheng Ding

  2. Roddenberry Center for Stem Cell Biology and Medicine at the Gladstone Institutes, San Francisco, California, USA

    Saiyong Zhu, Haixia Wang & Sheng Ding

  3. Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USA

    Sheng Ding

Authors
  1. Saiyong Zhu
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Contributions

S.Z., H.W. and S.D. conceived the study and the experimental design and wrote the manuscript. S.Z. performed the NSC and hepatocyte reprogramming experiments. H.W. performed the cardiomyocyte reprogramming experiments. All authors edited and approved the final manuscript.

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Correspondence to Sheng Ding.

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The authors declare no competing financial interests.

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Zhu, S., Wang, H. & Ding, S. Reprogramming fibroblasts toward cardiomyocytes, neural stem cells and hepatocytes by cell activation and signaling-directed lineage conversion. Nat Protoc 10, 959–973 (2015). https://doi.org/10.1038/nprot.2015.059

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