Helmholtz-Institute for Biomedical Technology, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Aachen, Germany.
PLoS One. 2013 May 29;8(5):e65324. doi: 10.1371/journal.pone.0065324. Print 2013.
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.
诱导多能干细胞（iPSCs）通常是克隆衍生的。完全重编程细胞的选择通常涉及挑选形态类似于胚胎干细胞（ESCs）的单个克隆。鉴于完全重编程的细胞具有高度增殖性并且逃避细胞衰老，因此可以想象，在没有克隆选择的情况下，它们在培养物中增殖，从而超过非多能和部分重编程的细胞。在这项研究中，我们使用附加体质粒载体对人真皮成纤维细胞（HDFs）进行了重编程。与使用 HDF 或人间充质基质细胞（MSC）作为基质支持相比，使用鼠胚胎成纤维细胞（MEF）作为基质支持时，集落频率更高，大小更大。然后，我们比较了通过手动选择单个集落进行克隆衍生的 iPSCs，或从所有初始集落的批量培养中衍生的 iPSCs。经过几次传代后，它们的形态、多能性标志物的表达和基因表达谱没有显示出任何显著差异。此外，克隆衍生和批量培养的 iPSCs 在体外向三个胚层的分化潜力方面表现出相似性。因此，手动选择单个集落似乎不是生成 iPSCs 所必需的 - 这对于细胞培养程序的标准化和自动化具有重要意义。
