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.
Highlights
Induced pluripotent stem cells open fascinating perspectives for drug discovery, cell therapy and basic research [1]
After seven days the cells were reseeded in parallel on three different feeder layers: irradiated murine embryonic fibroblasts (MEFs), irradiated human dermal fibroblasts (HDFs), and irradiated human bone marrow derived mesenchymal stromal cells (MSCs; Figure 1A)
Colonies were further analyzed by immunofluorescent staining for the pluripotency markers POU5F1 (OCT4), TRA-1-60 and SSEA3 demonstrating that MEFs, HDFs, and MSCs support formation of typical embryonic stem cells (ESCs)-like colonies (Figure 1C,D)
Summary
Induced pluripotent stem cells open fascinating perspectives for drug discovery, cell therapy and basic research [1]. Various methods have been described, including transfection with episomal plasmid vectors which enable the generation of integration-free iPSCs [2,3,4]. Such integration-free iPSCs are of relevance for regenerative medicine since they diminish the risk of insertion-associated genetic aberrations [5,6]. Initial colonies arise three to four weeks after induction and they typically reveal a heterogeneous morphology: pluripotent cells have relatively large nuclei and grow in flat colonies with an embryonic stem cell (ESC)-like morphology and with a sharp rim, whereas other colonies lack a sharp border and consist of larger and rather granular cells [7,8]. It is commonly accepted that this heterogeneity of initial clones reflects either successful or partial reprogramming into iPSCs [9,10]
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