Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq.
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.
Department of Bioengineering, Stanford University, Stanford, California 94305, USA.|Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig 04103, Germany.
Barbara
B
Treutlein
Department of Bioengineering, Stanford University, Stanford, California 94305, USA.|Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.|Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, USA.
Qian Yi
QY
Lee
Department of Developmental Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
J Gray
JG
Camp
Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.|Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, USA.
Moritz
M
Mall
Department of Bioengineering, Stanford University, Stanford, California 94305, USA.
Winston
W
Koh
Department of Biology, Stanford University, Stanford, California 94305, USA.
Seyed Ali Mohammad
SA
Shariati
Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.
Sopheak
S
Sim
Department of Bioengineering, Stanford University, Stanford, California 94305, USA.
Norma F
NF
Neff
Department of Biology, Stanford University, Stanford, California 94305, USA.
Jan M
JM
Skotheim
Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.|Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, USA.
Marius
M
Wernig
Department of Bioengineering, Stanford University, Stanford, California 94305, USA.|Howard Hughes Medical Institute, Stanford, California 94305, USA.
Stephen R
SR
Quake
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics; D019070:Cell Lineage / Q000235:genetics; D054338:Cell Transdifferentiation / Q000235:genetics; D065150:Cellular Reprogramming / Q000235:genetics*; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism*; D020869:Gene Expression Profiling; D020868:Gene Silencing; D018398:Homeodomain Proteins / Q000378:metabolism; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D050809:POU Domain Factors / Q000378:metabolism; D017423:Sequence Analysis, RNA; D059010:Single-Cell Analysis; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D059467:Transcriptome / Q000235:genetics; D019076:Transgenes / Q000235:genetics
D051792:Basic Helix-Loop-Helix Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D050809:POU Domain Factors; D014157:Transcription Factors; C499317:Ascl1 protein, mouse; C561019:Myt1l protein, mouse; C085076:transcription factor Brn-2