Abstract
IntroductionThe prospect of therapeutic applications of the induced pluripotent stem cells (iPSCs) is based on their ability to generate virtually any cell type present in human body. Generation of iPSCs from somatic cells has opened up new possibilities to investigate stem cell biology, to better understand pathophysiology of human diseases, and to design new therapy approaches in the field of regenerative medicine. In this study, we focus on the ability of the episomal system, a non-viral and integration-free reprogramming method to derive iPSCs from somatic cells of various origin.MethodsCells originating from neonatal and adult tissue, renal epithelium, and amniotic fluid were reprogrammed by using origin of replication/Epstein-Barr virus nuclear antigen-1 (oriP/EBNA-1)-based episomal vectors carrying defined factors. The iPSC colony formation was evaluated by using immunocytochemistry and alkaline phosphatase assay and by investigating gene expression profiles. The trilineage formation potential of generated pluripotent cells was assessed by embryoid body-mediated differentiation. The impact of additionally introduced factors on episome-based reprogramming was also investigated.ResultsReprogramming efficiencies were significantly higher for the epithelial cells compared with fibroblasts. The presence of additional factor miR 302/367 in episomal system enhanced reprogramming efficiencies in fibroblasts and epithelial cells, whereas the downregulation of Mbd3 expression increased iPSC colony-forming efficiency in fibroblasts solely.ConclusionsIn this study, we performed a side-by-side comparison of iPSC colony-forming efficiencies in fibroblasts and epithelial cells transiently transfected with episomal plasmids and demonstrated that iPSC generation efficiency was highest when donor samples were derived from epithelial cells. We determined that reprogramming efficiency of episomal system could be further improved. Considering results obtained in the course of this study, we believe that episomal reprogramming provides a simple, reproducible, and efficient tool for generating clinically relevant pluripotent cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0112-3) contains supplementary material, which is available to authorized users.
Highlights
The prospect of therapeutic applications of the induced pluripotent stem cells is based on their ability to generate virtually any cell type present in human body
Such a tendency was not observed for induced pluripotent stem cell (iPSC) generated from renal epithelial cells, which originated from mesodermal tissue
Our results demonstrated that the incorporation of short hairpin RNA (shRNA) against methyl-binding protein 3 (Mbd3) protein into the episomal system improved the efficiency by 47 % for fibroblasts but that it showed no effect for epithelial cells
Summary
The prospect of therapeutic applications of the induced pluripotent stem cells (iPSCs) is based on their ability to generate virtually any cell type present in human body. The reprogramming factors were introduced by retroviral transduction that caused the genomic integration of delivered transgenes This method is simple and efficient, the concern of clinical application of iPSCs established in such a manner involves the risk of insertional mutagenesis and oncogenic potential of some factors, especially Klf and c-Myc. To comprise high efficiency and safety of integrative vectors, excisable systems have been developed. With application of Cre recombinase, it is possible to excise floxed reprogramming genes after the generation of iPSCs [7, 8] Another approach involves the use of transposons, which have been shown to be efficient to the abovementioned viruses regarding long-term transgene expression [9, 10]. None of the genomeintegrating vectors can be regarded as completely safe, because of DNA footprint left after transposon or Cre/ loxP-based viral excision or because of possible homologous recombination events between closely positioned identical sequences that could lead to DNA deletion and genomic rearrangements
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