title: FAS-based cell depletion facilitates the selective isolation of mouse induced pluripotent stem cells.
abstract: Cellular reprogramming of somatic cells into induced pluripotent stem cells (iPSC) opens up new avenues for basic research and regenerative medicine. However, the low efficiency of the procedure remains a major limitation. To identify iPSC, many studies to date relied on the activation of pluripotency-associated transcription factors. Such strategies are either retrospective or depend on genetically modified reporter cells. We aimed at identifying naturally occurring surface proteins in a systematic approach, focusing on antibody-targeted markers to enable live-cell identification and selective isolation. We tested 170 antibodies for differential expression between mouse embryonic fibroblasts (MEF) and mouse pluripotent stem cells (PSC). Differentially expressed markers were evaluated for their ability to identify and isolate iPSC in reprogramming cultures. Epithelial cell adhesion molecule (EPCAM) and stage-specific embryonic antigen 1 (SSEA1) were upregulated early during reprogramming and enabled enrichment of OCT4 expressing cells by magnetic cell sorting. Downregulation of somatic marker FAS was equally suitable to enrich OCT4 expressing cells, which has not been described so far. Furthermore, FAS downregulation correlated with viral transgene silencing. Finally, using the marker SSEA-1 we exemplified that magnetic separation enables the establishment of bona fide iPSC and propose strategies to enrich iPSC from a variety of human source tissues.
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany; Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; REBIRTH Cluster of Excellence, Hannover, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Warlich
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; REBIRTH Cluster of Excellence, Hannover, Germany; Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Lock
authors.affiliation: Institute for Laboratory Animal Science, Hannover Medical School, Hannover, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Wedekind
authors.affiliation: REBIRTH Cluster of Excellence, Hannover, Germany; Institute for Laboratory Animal Science, Hannover Medical School, Hannover, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Glage
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Eckardt
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Bosio
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Knöbel
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D065150:Cellular Reprogramming; D000071858:Epithelial Cell Adhesion Molecule; D005786:Gene Expression Regulation; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D016256:Lewis X Antigen / Q000378:metabolism; D008280:Magnetics; D051379:Mice; D019014:fas Receptor / Q000378:metabolism*
chemical_list: D000951:Antigens, Neoplasm; D015415:Biomarkers; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; C491899:Fas protein, mouse; D016256:Lewis X Antigen; D019014:fas Receptor