Abstract
A systematic evaluation of three different methods for generating induced pluripotent stem (iPS) cells was performed using the same set of parental cells in our quest to develop a feeder independent and xeno-free method for somatic cell reprogramming that could be transferred into a GMP environment. When using the BJ fibroblast cell line, the highest reprogramming efficiency (1.89% of starting cells) was observed with the mRNA based method which was almost 20 fold higher than that observed with the retrovirus (0.2%) and episomal plasmid (0.10%) methods. Standard characterisation tests did not reveal any differences in an array of pluripotency markers between the iPS lines derived using the various methods. However, when the same methods were used to reprogram three different primary fibroblasts lines, two derived from patients with rapid onset parkinsonism dystonia and one from an elderly healthy volunteer, we consistently observed higher reprogramming efficiencies with the episomal plasmid method, which was 4 fold higher when compared to the retroviral method and over 50 fold higher than the mRNA method. Additionally, with the plasmid reprogramming protocol, recombinant vitronectin and synthemax® could be used together with commercially available, fully defined, xeno-free essential 8 medium without significantly impacting the reprogramming efficiency. To demonstrate the robustness of this protocol, we reprogrammed a further 2 primary patient cell lines, one with retinosa pigmentosa and the other with Parkinsons disease. We believe that we have optimised a simple and reproducible method which could be used as a starting point for developing GMP protocols, a prerequisite for generating clinically relevant patient specific iPS cells.
Highlights
Mature somatic cells can be reprogrammed to a pluripotent state through ectopic expression of key transcription factors, in a process known as induced pluripotency
Several retrovirus induced pluripotent stem (iPS) cell lines (BJ-RV-iPS) were established and one line used as a standard for further characterisation
While the morphology of iPS colonies grown in vitronectin and synthemax® is distinct, our results show that these two matrices have the capacity to support both plasmid reprogramming and the growth of pluripotent stem cells which is consistent with previous findings [20]
Summary
Mature somatic cells can be reprogrammed to a pluripotent state through ectopic expression of key transcription factors, in a process known as induced pluripotency. While many patient specific iPS cell lines have already been derived, most have been generated using genome integrating methods which raises concerns of insertional mutagenesis and continued expression of potentially oncogenic proteins by the integrated transgenes [1]. These concerns are important when considering clinical translation. Subsequent reports have shown that the replacement of SV40 large T antigen, Nanog and c-Myc, with a shp and L-Myc can improve reprogramming efficiencies over 10 fold [1] These studies demonstrate that reprogramming using episomal plasmids is a viable approach for generating integration free iPS cells. The RNA based method is not associated with chromosomal integration, which is an important safety attribute
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