A systematic evaluation of integration free reprogramming methods for deriving clinically relevant patient specific induced pluripotent stem (iPS) cells

PLoS One. 2013 Nov 26;8(11):e81622. doi: 10.1371/journal.pone.0081622. eCollection 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Culture Techniques
  • Cell Line
  • Fibroblasts / metabolism
  • Gene Expression
  • Humans
  • Induced Pluripotent Stem Cells / metabolism*
  • Mice
  • Plasmids / genetics
  • Transduction, Genetic
  • Transfection
  • Transgenes

Grants and funding

This work was supported by the National Institute of Health Research (NIHR) UK Programme Grant (RP-PG-0310-1001) and grants from the National Health Service Blood and Transplant (NHSBT). Catharina Casper was supported by a grant from the Brain Research Trust. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.