What is the best reprogramming method?

by Alexey Bersenev on December 2, 2014 · 0 comments

in embryonic/iPS

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Since the first report of iPS generation technique by Yamanaka in 2006, there were plenty of other methods described in the literature. So-called “non-integrating techniques” are more attractive in terms of clinical translation. A new study, published yesterday in Nature Biotechnology, provides a great guideline for choosing the most suitable (to you) non-integrating reprogramming method. If you need to set iPS cell generation technique in the lab and have no idea what method to choose – this paper for you!

The authors compared 3 non-integrating techniques – Sendai-viral (SeV), episomal (Epi) and mRNA, using two integrating (retro- and lentiviral vectors) methods as control. They used the following criteria: reproducibility, lab adoption rate, lab rejection rate, aneuploidy rate, reprogramming efficiency, workload and some other. The beauty of the study is in a survey, conducted among multiple labs, using variety of techniques to reprogram human cells. I’m going to highlight some interesting data from the study below.

Reprogramming efficiency: mRNA = 2.7% (the best), SeV = 0.077%, Epi = 0.013%, Lenti/Retro = 0.27%.
Success rate (measured as % of samples “for which at least three hiPSC colonies emerged”): mRNA = 27% (the worst), Epi = 93%, SeV = 94%, Lenti = 100%.
Workload time (from seeding of target somatic cells to picking iPS colonies): SeV = 3.5h prep – 26 days, Epi = 4h – 20 days, mRNA = 8h – 14 days.
Aneuploidy rate: Retro = 13.5% (the worst), mRNA = 2.3% (the best), Epi = 11.5%, SeV = 4.6%, Lenti = 4.5%.
There were no differences in: preexisting genomic abberations rate, pluripotent markers expression, differentiation propensity, epigenetics.

Some results of the survey:
Number of labs tried on fibroblasts: mRNA = 22, SeV = 35, Epi = 21; on blood cells: mRNA = 4, SeV = 19, Epi = 11.
Success rate (reproducibility): mRNA = 59%, SeV = 97%, Epi = 100%, Lenti = 95%, Retro = 97%.
% of labs adopted the method (means usage frequencies of ≥60%): on fibroblasts: mRNA = 46%, SeV = 62%, Epi = 33%, Lenti = 19%, Retro = 9%; on blood cells: mRNA = N/A, SeV = 61%, Epi = 44%, Lenti = 40%, Retro = 0%.

The authors discuss advantages and disadvantages of each method. They seem to be in favor of Epi- method for clinical-grade reprogramming:

The shortcomings of SeV reprogramming include the dependence on one commercial vendor…
we share this enthusiasm and highly recommend SeV reprogramming to laboratories that do not focus on generating clinical-grade hiPSCs.

The main advantages of the RNA method are the speed of colony emergence, high efficiency, a complete absence of integration, a very low aneuploidy rate and a low donor cell requirement (typically 50,000 cells, but as few as 1,000 human fibroblasts can be reprogrammed; data not shown).
The conventional RNA method can therefore be useful for easy-to-reprogram fibroblast samples, but needs to be further optimized to overcome the reprogramming resistance and excessive cell death observed with many patient samples.

Epi reprogramming seems particularly well-suited for clinical translation because it is integration-free, works reliably with patient fibroblasts and blood cells, and is based on a very simple reagent (plasmid DNA) that can easily be generated using current good manufacturing practice (cGMP)-compatible processes. SeV, on the other hand, is currently not available commercially as a cGMP-grade reagent for reprogramming, although Sendai virus has been used as a vehicle in human vaccination studies.

This study is highly recommended to everyone, who is working with iPS cells!

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