title: CD44 is a negative cell surface marker for pluripotent stem cell identification during human fibroblast reprogramming.
abstract: Induced pluripotent stem cells (iPSCs) are promising tools for disease research and cell therapy. One of the critical steps in establishing iPSC lines is the early identification of fully reprogrammed colonies among unreprogrammed fibroblasts and partially reprogrammed intermediates. Currently, colony morphology and pluripotent stem cell surface markers are used to identify iPSC colonies. Through additional clonal characterization, we show that these tools fail to distinguish partially reprogrammed intermediates from fully reprogrammed iPSCs. Thus, they can lead to the selection of suboptimal clones for expansion. A subsequent global transcriptome analysis revealed that the cell adhesion protein CD44 is a marker that differentiates between partially and fully reprogrammed cells. Immunohistochemistry and flow cytometry confirmed that CD44 is highly expressed in the human parental fibroblasts used for the reprogramming experiments. It is gradually lost throughout the reprogramming process and is absent in fully established iPSCs. When used in conjunction with pluripotent cell markers, CD44 staining results in the clear identification of fully reprogrammed cells. This combination of positive and negative surface markers allows for easier and more accurate iPSC detection and selection, thus reducing the effort spent on suboptimal iPSC clones.
authors.affiliation: Cell Biology & Stem Cell Sciences, Life Technologies, Carlsbad, California, United States of America.
authors.forename: Rene H
authors.initials: RH
authors.lastname: Quintanilla
authors.affiliation: Cell Biology & Stem Cell Sciences, Life Technologies, Carlsbad, California, United States of America.
authors.forename: Joanna S T
authors.initials: JS
authors.lastname: Asprer
authors.affiliation: Bioinformatics Institute, Agency for Science Technology & Research (AStar), Singapore, Republic of Singapore.
authors.forename: Candida
authors.initials: C
authors.lastname: Vaz
authors.affiliation: Bioinformatics Institute, Agency for Science Technology & Research (AStar), Singapore, Republic of Singapore ; Institute for Medical Biology, Agency for Science Technology & Research (A*Star), Singapore, Republic of Singapore.
authors.forename: Vivek
authors.initials: V
authors.lastname: Tanavde
authors.affiliation: Cell Biology & Stem Cell Sciences, Life Technologies, Carlsbad, California, United States of America.
authors.forename: Uma
authors.initials: U
authors.lastname: Lakshmipathy
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D065150:Cellular Reprogramming; D002999:Clone Cells; D061252:Feeder Cells / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D016130:Immunophenotyping; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D059467:Transcriptome
chemical_list: D015415:Biomarkers; C497483:CD44 protein, human; D018960:Hyaluronan Receptors