Letter | Published:

Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq

Nature volume 534, pages 391395 (16 June 2016) | Download Citation


Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states1,2,3. However, the intermediate stages through which individual cells progress during reprogramming are largely undefined. Here we use single-cell RNA sequencing4,5,6,7 at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts to induced neuronal cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts, suggesting that the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.

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Gene Expression Omnibus

Data deposits

The single-cell RNA-seq data were deposited on NCBI GEO with the accession number GSE67310.


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The authors would like to acknowledge B. Passarelli and B. Vernot for discussions regarding bioinformatic pipelines, P. Lovelace for support with FACS and other Quake and Wernig laboratory members for discussions and support. This work was supported by NIH grant RC4NS073015-01 (M.W., S.Q.R., B.T.), the Stinehart-Reed Foundation, the Ellison Medical Foundation, the New York Stem Cell Foundation, CIRM grant RB5-07466 (all to M.W.), a National Science Scholarship from the Agency for Science, Technology and Research (Q.Y.L.), NIH grant GM092925 (S.A.M.S., J.S.), the German Research Foundation (M.M.) and a PhRMA foundation Informatics fellowship (J.G.C.). S.R.Q. is an investigator of the Howard Hughes Medical Institute. M.W. is a New York Stem Cell Foundation (NYSCF) Robertson Investigator and a Tashia and John Morgridge Faculty Scholar at the Child Health Research Institute at Stanford.

Author information

Author notes

    • Barbara Treutlein
    •  & Qian Yi Lee

    These authors contributed equally to this work.

    • Marius Wernig
    •  & Stephen R. Quake

    These authors jointly supervised this work.


  1. Department of Bioengineering, Stanford University, Stanford, California 94305, USA

    • Barbara Treutlein
    • , Qian Yi Lee
    • , Winston Koh
    • , Norma F. Neff
    •  & Stephen R. Quake
  2. Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig 04103, Germany

    • Barbara Treutlein
  3. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA

    • Qian Yi Lee
    • , Moritz Mall
    • , Sopheak Sim
    •  & Marius Wernig
  4. Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Qian Yi Lee
    • , Moritz Mall
    •  & Marius Wernig
  5. Department of Developmental Biology, Stanford University School of Medicine, Stanford, California 94305, USA

    • J. Gray Camp
  6. Department of Biology, Stanford University, Stanford, California 94305, USA

    • Seyed Ali Mohammad Shariati
    •  & Jan M. Skotheim
  7. Howard Hughes Medical Institute, Stanford, California 94305, USA

    • Stephen R. Quake
  8. Department of Applied Physics, Stanford University, Stanford, California 94305, USA


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B.T., Q.Y.L., M.W. and S.R.Q. conceived the study and designed the experiments. Q.Y.L. performed direct reprogramming, qRT–PCR and western blot experiments; B.T., Q.Y.L., and S.S. performed single-cell RNA-seq experiments; N.F.N. assisted with single-cell RNA-seq experiments and sequenced the libraries; Q.Y.L., S.A.M.S. and M.M. performed time-lapse imaging experiments. B.T., J.G.C. and W.K. analysed single-cell RNA-seq data, Q.Y.L. analysed qRT–PCR and time-lapse imaging data, J.M.S., M.W. and S.R.Q. provided intellectual guidance in the interpretation of the data. B.T., Q.Y.L., J.G.C., M.W., and S.R.Q. wrote the paper.

Competing interests

S.R.Q. is a founder and consultant for Fluidigm Corporation.

Corresponding authors

Correspondence to Marius Wernig or Stephen R. Quake.

Reviewer Information Nature thanks F. Tang and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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