A continuous molecular roadmap to iPSC reprogramming through progression analysis of single-cell mass cytometry.
To analyze cellular reprogramming at the single-cell level, mass cytometry was used to simultaneously measure markers of pluripotency, differentiation, cell-cycle status, and cellular signaling throughout the reprogramming process. Time-resolved progression analysis of the resulting data sets was used to construct a continuous molecular roadmap for three independent reprogramming systems. Although these systems varied substantially in Oct4, Sox2, Klf4, and c-Myc stoichiometry, they presented a common set of reprogramming landmarks. Early in the reprogramming process, Oct4(high)Klf4(high) cells transitioned to a CD73(high)CD104(high)CD54(low) partially reprogrammed state. Ki67(low) cells from this intermediate population reverted to a MEF-like phenotype, but Ki67(high) cells advanced through the M-E-T and then bifurcated into two distinct populations: an ESC-like Nanog(high)Sox2(high)CD54(high) population and a mesendoderm-like Nanog(low)Sox2(low)Lin28(high)CD24(high)PDGFR-α(high) population. The methods developed here for time-resolved, single-cell progression analysis may be used for the study of additional complex and dynamic systems, such as cancer progression and embryonic development.
Department of Microbiology and Immunology, Baxter Laboratory for Stem Cell Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Eli R
ER
Zunder
Department of Pathology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.
Ernesto
E
Lujan
Department of Microbiology and Immunology, Baxter Laboratory for Stem Cell Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Yury
Y
Goltsev
Department of Pathology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
Marius
M
Wernig
Department of Microbiology and Immunology, Baxter Laboratory for Stem Cell Biology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address: gnolan@stanford.edu.
Garry P
GP
Nolan
D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis / Q000235:genetics; D000067470:Cellular Reprogramming Techniques; D057809:HEK293 Cells; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051379:Mice; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics
D000943:Antigens, Differentiation; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D014157:Transcription Factors