Abstract
Induced pluripotent stem cells (iPSCs) provide a unique opportunity for generation of patient-specific cells for use in translational purposes. We aimed to compare iPSCs generated by different reprogramming methods regarding their reprogramming efficiency, pluripotency capacity, and the possibility to use high-throughput PCR-based methods for detection of human pathogenic viruses. iPSCs from skin fibroblasts (FB), peripheral blood mononuclear cells (PBMCs), or mesenchymal stem cells (MSCs) were generated by using three different reprogramming systems including chromosomal integrating and nonintegrating methods. Reprogramming efficiencies were in accordance with the literature, indicating that the parental cell type and the reprogramming method play a major role for the reprogramming efficiencies (FB: STEMCCA: 1.30 ± 0.18, Sendai virus: 1.37 ± 0.01, and episomal plasmids: 0.04 ± 0.02; PBMCs: Sendai virus: 0.002 ± 0.001, episomal plasmids: 0) but result in the same characteristics of pluripotency. We found the highest reprogramming efficiencies for MSC with 3.32 ± 1.2 by using episomal plasmids. Since GMP standard working procedures and screening units need virus contamination-free cell lines, we studied HIV-1 contamination in the generated iPSCs. We used the high-throughput cobas® 6800/8800 system, which is normally used for detection of HIV-1 in plasma of patients, and found that footprint-free reprogramming methods as episomal plasmids and Sendai virus are useful for the described virus detection method. This fast, cost-effective, robust, and reliable assay demonstrates the feasibility to use high-throughput PCR-based methods for detection of human pathogenic viruses in ps-iPSC lines that were generated with nongenome integrating reprogramming methods.
Highlights
The ability to generate induced pluripotent stem cells from somatic cells of patients offers a great opportunity to model human diseases and creates a powerful tool for drug screening and the development of new grafts for transplantation.Until now, numerous methods have been developed to generate iPSCs
Our data of reprogramming FB and peripheral blood mononuclear cells (PBMCs) showed that success rates for FB are highest when the Sendai virus or the STEMCCA virus system was used (100%) compared to plasmids (89%) (Supplemental Table 3)
It should be noted that the reprogramming efficiency was higher in fibroblasts compared to PBMCs as the parental cell source by using Sendai virus (PBMCs: 0 002 ± 0 001) (Figure 1(a))
Summary
The ability to generate induced pluripotent stem cells (iPSCs) from somatic cells of patients offers a great opportunity to model human diseases and creates a powerful tool for drug screening and the development of new grafts for transplantation.Until now, numerous methods have been developed to generate iPSCs. To minimize vector sequences that were integrated into the reprogrammed iPSC genome, a humanized version of a single cassette reprogramming vector became popular offering the possibility of Cre-Lox-mediated transgene excision. For this vector termed STEMCCA, reprogramming efficiencies of 0.1-1.5% are reported [3]. Sendai virus was shown to be able to reprogram neonatal and adult human fibroblasts in about 25 days with a high efficiency of 1% [4] Another integration-free and nonviral method for reprogramming is the use of episomal plasmids expressing transiently the reprogramming factors. Using the OriP/EBNA-based plasmids in combination with p53 suppression and nontransforming L-Myc, the reprogramming efficiency can be increased to 0.1% [5,6,7]
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