title: To clone or not to clone? Induced pluripotent stem cells can be generated in bulk culture.
abstract: Induced pluripotent stem cells (iPSCs) are usually clonally derived. The selection of fully reprogrammed cells generally involves picking of individual colonies with morphology similar to embryonic stem cells (ESCs). Given that fully reprogrammed cells are highly proliferative and escape from cellular senescence, it is conceivable that they outgrow non-pluripotent and partially reprogrammed cells during culture expansion without the need of clonal selection. In this study, we have reprogrammed human dermal fibroblasts (HDFs) with episomal plasmid vectors. Colony frequency was higher and size was larger when using murine embryonic fibroblasts (MEFs) as stromal support instead of HDFs or human mesenchymal stromal cells (MSCs). We have then compared iPSCs which were either clonally derived by manual selection of a single colony, or derived from bulk-cultures of all initial colonies. After few passages their morphology, expression of pluripotency markers, and gene expression profiles did not reveal any significant differences. Furthermore, clonally-derived and bulk-cultured iPSCs revealed similar in vitro differentiation potential towards the three germ layers. Therefore, manual selection of individual colonies does not appear to be necessary for the generation of iPSCs - this is of relevance for standardization and automation of cell culture procedures.
authors.affiliation: Helmholtz-Institute for Biomedical Technology, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Aachen, Germany.
authors.forename: Charlotte A
authors.initials: CA
authors.lastname: Willmann
authors.forename: Hatim
authors.initials: H
authors.lastname: Hemeda
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Pieper
authors.forename: Michael
authors.initials: M
authors.lastname: Lenz
authors.forename: Jie
authors.initials: J
authors.lastname: Qin
authors.forename: Sylvia
authors.initials: S
authors.lastname: Joussen
authors.forename: Stephanie
authors.initials: S
authors.lastname: Sontag
authors.forename: Paul
authors.initials: P
authors.lastname: Wanek
authors.forename: Bernd
authors.initials: B
authors.lastname: Denecke
authors.forename: Herdit M
authors.initials: HM
authors.lastname: Schüler
authors.forename: Martin
authors.initials: M
authors.lastname: Zenke
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wagner
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000235:genetics; D049109:Cell Proliferation; D002478:Cells, Cultured; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D007621:Karyotyping; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D020411:Oligonucleotide Array Sequence Analysis; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction