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Nature. 2016 Jun 16;534(7607):391-5. doi: 10.1038/nature18323. Epub 2016 Jun 8.

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

Author information

1
Department of Bioengineering, Stanford University, Stanford, California 94305, USA.
2
Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig 04103, Germany.
3
Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.
4
Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, USA.
5
Department of Developmental Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
6
Department of Biology, Stanford University, Stanford, California 94305, USA.
7
Howard Hughes Medical Institute, Stanford, California 94305, USA.

Abstract

Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states. However, the intermediate stages through which individual cells progress during reprogramming are largely undefined. Here we use single-cell RNA sequencing 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.

PMID:
27281220
PMCID:
PMC4928860
DOI:
10.1038/nature18323
[Indexed for MEDLINE]
Free PMC Article

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