bulk conversion

Is “100% Conversion” No Longer a Taboo Phrase in the iPSC Field?

“By removing a single protein called Mbd3, a team at the Weizmann Institute of Science in Rehovot, Israel, was able to increase the conversion rate to almost 100%”. A staggering statement written by Monya Baker for Nature News, after her journal’s online publication by Rais et al. reported their discovery. This discovery displays that by removing the expression of nucleosome remodeling and deacetylation repressor complex member Mbd3, all cells can be reprogrammed with the OSKM Yamanaka factors. What we have described last September in Nature’s Scientific Reports (Warren et al. 2012) that by fusing Oct4 with strong transcription activation domain, we achieved essentially the same results which we call “bulk conversion”. One can imagine that the strong transcription activation domain very likely effects such strong reprogramming through some type of chromosomal remodeling indirectly.

As we later learned, people in the field believe that reprogramming is a stochastic event and it is impossible to achieve something like “bulk conversion”. We are happy to see that sea change now that a separate group also demonstrated “bulk conversion”. Recently we have shown that instead of 2 weeks or more, as required when mRNA reprogramming was first reported or commonly practiced, we could start taking up iPSCs around a week without doing any traditional transfection—just change the medium everyday as for all stem cell cultures, with mRNA complex as a supplement to be added together with bFGF.

Knocking-down Mbd3 is potentially useful, especially in situations where we have difficulty reprogramming a particular line, and it could make reprogramming more successful in more demanding conditions such as leaving out B18R as we recently established. However one needs to be cautious about the unavoidable off-target effects of siRNAs when including siRNA against Mbd3 in their RNA mix.

Rais et al. Nature (2013)

Warren et al. Scientific Reports (2012)

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Thursday, September 19th, 2013 Allele Mail Bag, iPSCs and other stem cells No Comments

Picture Blog: How Do You Like Your iPSCs, Clonal or Bulk-Conversion?

Reprogramming of differentiated cells into induced pluripotent stem cells (iPSCs) is commonly considered a stochastic process, i.e. with randomness, which offers an excuse for the commonly seen low efficiency and low constancy of making iPSCs. We have demonstrated time and again that by using potent mRNA cocktails, the majority of the fibroblasts seeded in a well can be converted into pluripotent stage in a nearly synchronized manner (Warren et al. 2012, Warren and Wang 2013, and this Allele Picture Blog series). mRNA molecules can function robustly yet transiently while avoiding the need of entering the nucleus, a bottle-neck for all DNA-based vehicles.

Other researchers are used to the idea of clonal expansion partly because isolating iPSCs from “clones” was a common step during reprogramming using viruses or other low efficiency methods, even though those clones were not necessarily from single precursor cells. This week, the Allele iPSC team developed a new way of managing our mRNA reprogramming that allowed us to achieve clonal iPSCs that appear to be a lot purer and more likely true clones compared to previous reports, without compromising any of the main benefits of our protocol, e.g. feeder-free, xeno-free, footprint-free, very fast and highly efficient. This work is currently supported by an NIDA/NIH grant to Dr. Jiwu Wang at Allele Biotech.

Allele scientists lead by Dr. Jiwu Wang have been able to consistently bulk convert

Traditional bulk-conversion by the Allele mRNA reprogramming protocol developed by Warren et al. The picture shows large patches of cells becoming stem cells almost overnight around the 9th day of adding mRNA-cocktail supplement to the media.

With clonal lineage, at bulk-conversion rate, new mRNA reprogramming

Clonal iPSC formation using a modified mRNA reprogramming protocol. The picture shows a typical clone of stem cells that originated from likely single cells.

Warren, Ni, Wang, and Guo, Scientific Reports, 2012
Warren and Wang, Current Protocols, 2013, in press

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Wednesday, September 4th, 2013 iPSCs and other stem cells No Comments