During cellular reprogramming, only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of the previously suggested reprogramming markers Fbxo15, Fgf4, and Oct4. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc, and Nanog, can activate the pluripotency circuitry.
Bibliographical noteFunding Information:
We thank Yarden Katz, Sovan Sarkar, Malkiel Cohen, Bryce Carey, and Jonathan Friedman for fruitful discussions, Patti Wisniewski and Chad Araneo for their help with cell sorting, and Stuart Levine for help with pilot Fluidigm experiments. Y.B. was supported by an NIH Kirschstein NRSA (1 F32 GM099153-01A1). D.A.F. is a Vertex Scholar and was supported by an NSF Graduate Research Fellowship and Jerome and Florence Brill Graduate Student Fellowship. A.W.C. was supported by a Croucher and Ludwig Research Fellowship. R.J. is an adviser to Stemgent and cofounder of Fate Therapeutics. This work was supported by NIH grants HD 045022 and R37CA084198 to R.J. and the NIH/NCI Physical Sciences Oncology Center at MIT (U54CA143874) and a NIH Pioneer award (1DP1OD003936) to A.v.O.