R-iPSCs

Picture Blog: mRNA Reprogramming for Human iPSCs without B18R!

Human induced pluripotent stem cells provide a great route towards personalized medicine and high accuracy drug screening. Allele Biotech has developed the most efficient method of making human iPSCs by using enhanced mRNAs, which have been adopted by leading pharmaceutical companies for clinical trials. The effects from medium-supplementing mRNAs are robust yet transient, and highly specific compared to both miRNAs (off-targets) and small molecules (unknown targets). To repress cellular immune response to introduced RNA molecules, viral protein B18R was previously used during mRNA reprogramming.

B18R is relatively expensive and inconvenient to use because it requires pre-aliquoting and -80C storage. The protocol has recently been dramatically improved at Allele through an NIDA-funded project. In our latest reprogramming run, all we needed to do was to include mRNA complex in the supplement during medium change for just a week without the need of adding any other type of molecules (such as B18R, miRNA, or chemicals) to help the mRNA mix, unlike all other known mRNA-reprogramming protocols. This advancement can make reprogramming human fibroblasts to footprint-free and xeno-free iPSCs a routine experiment for any lab to perform.

Human R-iPSCs were created without the need of B18R, dramatically reduced the cost and inconvenience. Shown is a newly formed iPSC colony.

mRNA reprogramming used to require B18R to repress cellular immune response from repeated exposure to RNA molecules. Allele scientists lead by Dr. Jiwu Wang further developed their technology to bypass the use of B18R or any immune repressor.

Tags: apaptosis, B18R, cellular immune response, footprint-free iPSC, iPSCs, miRNA reprogramming, mRNA iPSC, R-iPSCs, reprogramming, small-molecule compounds

Thursday, August 15th, 2013 iPSCs and other stem cells Comments Off

Picture Blog — Making mRNAs by In Vitro Transcription for Transgene Expression and R-iPSCs

R-iPS Cell FAQ 2:
What is the expected yield from the in vitro trancription (IVT) reactions?

Performed as described, you should recover around 40 ug RNA from each 40 uL IVT reaction.

R-iPS Cell FAQ 3:
How can the success of the RNA synthesis protocol be assessed?

Run 500 ng (5 uL) of the concentration-adjusted products on an E-gel to check for consistent product yield and relative product sizes, and to confirm the absence of secondary bands or smears.