title: Stem Cell Surface Marker Expression Defines Late Stages of Reprogramming to Pluripotency in Human Fibroblasts.
abstract: Our current understanding of the induction of pluripotency by defined factors indicates that this process occurs in discrete stages characterized by specific alterations in the cellular transcriptome and epigenome. However, the final phase of the reprogramming process is incompletely understood. We sought to generate tools to characterize the transition to a fully reprogramed state. We used combinations of stem cell surface markers to isolate colonies emerging after transfection of human fibroblasts with reprogramming factors and then analyzed their expression of genes associated with pluripotency and early germ lineage specification. We found that expression of a subset of these genes, including the cell-cell adhesion molecule CDH3, characterized a late stage in the reprogramming process. Combined live-cell staining with the antibody GCTM-2 and anti-CDH3 during reprogramming identified colonies of cells that showed gene expression patterns very similar to those of embryonic stem cell or established induced pluripotent stem cell lines, and gave rise to stable induced pluripotent stem cell lines at high frequency. Our findings will facilitate studies of the final stages of reprogramming of human cells to pluripotency and will provide a simple means for prospective identification of fully reprogrammed cells.
  
  
  CONCLUSIONS
  Reprogramming of differentiated cells back to an embryonic pluripotent state has wide ranging applications in understanding and treating human disease. However, how cells traverse the barriers on the journey to pluripotency still is not fully understood. This report describes tools to study the late stages of cellular reprogramming. The findings enable a more precise approach to dissecting the final phases of conversion to pluripotency, a process that is particularly poorly defined. The results of this study also provide a simple new method for the selection of fully reprogrammed cells, which could enhance the efficiency of derivation of cell lines for research and therapy.
authors.affiliation: Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
authors.forename: Jordan E
authors.initials: JE
authors.lastname: Pomeroy
authors.affiliation: Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California, Los Angeles, California, USA Department of Anatomy and Neuroscience, University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Shelley R
authors.initials: SR
authors.lastname: Hough
authors.affiliation: University of Melbourne and Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, Victoria, Australia Australian Institute of Regenerative Medicine, Monash University, Clayton, Victoria, Australia.
authors.forename: Kathryn C
authors.initials: KC
authors.lastname: Davidson
authors.affiliation: Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
authors.forename: Alex M
authors.initials: AM
authors.lastname: Quaas
authors.affiliation: Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
authors.forename: Jordan A
authors.initials: JA
authors.lastname: Rees
authors.affiliation: Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California, Los Angeles, California, USA Department of Anatomy and Neuroscience, University of Melbourne, Melbourne, Victoria, Australia The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia The Florey Neuroscience and Mental Health Institute, Parkville, Victoria, Australia mpera@unimelb.edu.au.
authors.forename: Martin F
authors.initials: MF
authors.lastname: Pera
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000378:metabolism / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D046228:Microarray Analysis; D059467:Transcriptome / Q000502:physiology*
chemical_list: D015415:Biomarkers; D008565:Membrane Proteins
keywords: CDH3; Cell surface marker; Gene expression; Induced pluripotent stem cell; Reprogramming