Picture Blog: A Short Path from Human mRNA-iPSCs to Neurons in Record Speed

http://blog.allelebiotech.com/2014/02/picture-blog-a-short-path-from-human-mrna-ipscs-to-neurons-in-record-speed/

 AlleleBlog:

Traditional differentiation protocols use embryoid body (EB) formation as the first step of lineage restriction to mimic early human embryogenesis, which is then followed by manual selection of neuroepithelial precursors. This procedure is tedious and often inconsistent. We have developed a novel neural differentiation scheme that directs human iPSCs (created with the Allele 6F mRNA reprogramming kit) that progressed, as attached culture, to neural precursor cells (NPCs) in just 4-6 days, half the time it typically takes by other methods. From NPCs it takes about another 5-6 days for neural rosettes to form (see figures below); upon passage, cells in neural rosettes differentiate into neurons in 24 hours.

The neural progenitors at the rosettes stage can be stocked and expanded, before differentiated into different types of neurons. We are working on specifically and efficiently different these neural progenitor cells into dopaminergic, glutamatergic, GABAergic, and other types of sub-types of neurons with Allele’s technologies (Questions? email the Allele Stem Cell Group at iPSatAllelebiotech.com).

Neural rosettes formed efficiently in wells without going through EB.

Human iPSC-derived neurons are created in a short regimen developed at Allele Biotech

mRNA Delivery And the Next Wave of Regenerative Medicine

AlleleBlog: Published online by Nature Biotechnology, researchers from Ken Chien’s lab at Harvard and other coauthors showed that modified mRNA of VEGF-A injected intramyocardially resulted in the expansion and directed differentiation of endogenous heart progenitors. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients by directing epicardial progenitor cells toward cardiovascular cell types. This publication appears to be the first example of using mRNA as a delivery platform for cell fate-related therapy. AstraZeneca recently invested $240 million on mRNA-related delivery via Moderna, a company with roots within the Harvard stem cell group.

The drastically increased efficacy of using the mRNA platform was accredited to the pulse-like kinetics of mRNA expression profile. It was explained by the fact that native paracrine signals are often transient and precisely regulated in time and space, therefore the pulse-like expression profile of modRNA might be well suited to delivering paracrine-factor signals. Transfected mRNA molecules do not need to penetrate the nuclear membrane, which greatly enhances the efficiency of protein expression on a per transfected molecule over DNA. mRNAs turn over in a much faster pace than plasmid-mediated transgene expression. This is beneficial to many cell fate decisions as exemplified by this recent publication.

Allele Biotech’s reprogramming technologies, licensed by some of the leading stem cell therapy companies, are built around the mRNA platform. We chose mRNA as our core technology to not only change cell fate, but also direct differentiation. We know this platform is the future for cell fate manipulation because we have seen how robustly mRNA expression made the day-and-night difference in gene expression when compared to plasmid DNA (episomal or not), retrovirus, lentivirus, baculo virus, or even transfected proteins. We could convert human fibroblasts into iPSCs, in bulk, in as short as one week with no more effort than changing mRNA complex-containing medium.

Another recent development in iPSC research is in situ reprogramming. Abad et al. generated mice carrying a Tet-inducible cassette of the four cell-reprogramming factors. They then added feed doxycycline to the animals. After several weeks, teratomas appeared in various tissues, indicating that in situ reprogramming had occurred. The iPSCs created this way did not appear to have much advantage over in vitro produced iPSCs other than they are totipotent (helpful if you are studying placenta). Nevertheless, the concept of changing cell fate in situ as dramatically as complete reprogramming is an important leap of faith. As for the next big step, it is easy to see that mRNAs are well suited for in situ reprogramming, as well as transdifferentiation, and more complex gene delivery than the above mentioned VEGF-A alone in heart treatment.

References:
Zangi et al. Nature Biotechnology, http://www.nature.com/nbt/journal/vaop/ncurrent/abs/nbt.2682.html
Abad et al. Nature, http://www.nature.com/nature/journal/vaop/ncurrent/abs/nature12586.html

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.