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

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.

SUBMITTER: Goh PA

PROVIDER: S-EPMC3841145 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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

Goh Pollyanna A PA Caxaria Sara S Casper Catharina C Rosales Cecilia C Warner Thomas T TT Coffey Pete J PJ Nathwani Amit C AC

PloS one 20131126 11

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 ...[more]

PMID: 24303062

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Project description:As flattening filter-free (FFF) photon beams become readily available for treat-ment delivery in techniques such as SBRT, thorough investigation of skin dose from FFF photon beams is necessary under clinically relevant conditions. Using a parallel-plate PTW Markus chamber placed in a custom water-equivalent phantom, surface-dose measurements were taken at 2 × 2, 3 × 3, 4 × 4, 6 × 6, 8 × 8, 10 × 10, 20 × 20, and 30 × 30 cm2 field sizes, at 80, 90, and 100 cm source-to-surface distances (SSDs), and with fields defined by jaws and multileaf collimator (MLC) using multiple beam energies (6X, 6XFFF, 10X, and 10XFFF). The same set of measurements was repeated with the chamber at a reference depth of 10cm. Each surface measurement was normalized by its corresponding reference depth measurement for analysis. The FFF surface doses at 100 cm SSD were higher than flattened surface doses by 45% at 2 × 2 cm2 to 13% at 20 × 20 cm2 for 6MV energy. These surface dose differences varied to a greater degree as energy increased, ranging from +63% at 2 × 2 cm2 to -2% at 20 × 20 cm2 for 10 MV. At small field sizes, higher energy increased FFF surface dose relative to flattened surface dose; while at larger field sizes, relative FFF surface dose was higher for lower energies. At both energies investigated, decreasing SSD caused a decrease in the ratios of FFF-to-flattened surface dose. Variability with SSD of FFF-to-flattened surface dose differences increased with field size and ranged from 0% to 6%. The field size at which FFF and flattened beams gave the same skin dose increased with decreasing beam energy. Surface dose was higher with MLC fields compared to jaw fields under most conditions, with the difference reaching its maximum at a field size between 4 × 4 cm2 and 6 × 6 cm2 for a given energy and SSD. This study conveyed the magnitude of surface dose in a clinically meaning-ful manner by reporting results normalized to 10 cm depth dose instead of depth of dose maximum.

Dataset Information

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

Publications

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

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