Abstract
Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.
Highlights
Since Yamanaka’s breakthrough in 2006 [1], induced pluripotent stem cells have revolutionized the stem cell field and have been applied to several branches of studies. iPSCs have been generated with several integrative [2,3,4,5,6,7] and non-integrative methods [8,9,10,11], the former exploiting viral vectors that integrate into the host cell genome and stably express the transgene, and the latter including any approach that enables the transient expression of the transgene in target cells
Reprogramming experiments using three different methods, i.e., based on retroviral vectors, Sendai virus vectors and episomal vectors, were conducted in parallel starting from BJ fibroblasts at early passages, with the same batches of reagents, hoods and incubators and by the same operator
This study showed that three different reprogramming strategies adopted to obtain human iPSCs (hiPSCs) from somatic differentiated cells had comparable efficiency and generated hiPSCs with very similar gene expression profiles
Summary
Since Yamanaka’s breakthrough in 2006 [1], induced pluripotent stem cells (iPSCs) have revolutionized the stem cell field and have been applied to several branches of studies. iPSCs have been generated with several integrative [2,3,4,5,6,7] and non-integrative methods [8,9,10,11], the former exploiting viral vectors that integrate into the host cell genome and stably express the transgene, and the latter including any approach that enables the transient expression of the transgene in target cells. On the other hand, represent a very efficient and low cost way to generate virus-free iPSCs [17] They have been used to generate iPSCs from skin fibroblasts and blood cells through nucleofection techniques and they allow replication of episomes into transfected cells in order to maintain high transgene expression levels and a transient effect [9]. Requirements for clinical application of human iPSCs (hiPSCs), besides safety, include standardization and reproducibility of laboratory protocols In this regard, variability among hiPSC clones has been ascribed to the different reprogramming techniques, genetic background, batch of material used, and even laboratory personnel manipulating the cells [6,20,21,22,23]. We conclude that the different reprogramming methods are equivalent since they did not influence the hiPSC gene expression profiles, arguing that the differences previously reported [24,25] are ascribable to other factors, such as lab-to-lab technical biases and to the genetic background of the starting samples
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