Table 2.
Advantages and disadvantages of srRNA- or mRNA-based reprogramming of fibroblasts to obtain footprint-free iPSCs.
| srRNA reprogramming | mRNA reprogramming | |
|---|---|---|
| Advantages of srRNA | ||
| RNA generation | No modified nucleotides∗ | Modified nucleotides∗ |
| Identical RNA molecules | Multiple mRNAs | |
| RNA transfection | Once (1 μg) | Daily (1.2 μg) for 14-20 days |
| Transfection efficiency | GFP reporter on the same srRNA construct | Additional transfection with GFP mRNA for monitoring |
| First iPSCs emerged after | 12 days | 14 days |
| Reprogramming efficiency | Very high efficiency after positive selection | High efficiency (0.8%) |
| Reprogramming costs of RNA∗∗ | ~2.5€ (1 μg srRNA once) | ~60€ (~2.5€/1 μg mRNA for 20 days) |
| Transgene-free iPSCs | Yes (total elimination of srRNA was demonstrated after p3) | Yes (no integration of mRNA into the host genome) |
| Disadvantages of srRNA | ||
| RNA modification | Posttranscriptional enzymatic 5′-capping and 3′-polyadenylation | Cotranscriptional 5′-capping and 3′-polyadenylation |
| Immune system activation counteraction | Interferon inhibitor B18R required∗∗∗ | Interferon inhibitor B18R required∗∗∗ |
| Transgene expression | Check for residual srRNA expression (VEE virus-derived RNA construct) | Natural degradation of mRNA in cells after 2-3 days |
∗Modified nucleotides (e.g., 5mCTP, Pseudo-UTP, and N1-methylpseudo-UTP) can improve the translation of proteins but are also expensive to purchase. ∗∗Costs for the synthesis and purification of RNA (with commercially available kits, without plasmid generation) needed for one reprogramming experiment. ∗∗∗B18R-containing medium (BcM) can vary from batch to batch; therefore, the functionality of B18R has to be assessed before use, for example, by the determination of the positive transfection of fibroblasts with GFP mRNA with 25% BcM compared to the transfection without B18R. To date, there are no commercially available antibodies against B18R for specific analysis of the B18R content. For a constant quality, B18R can also be purchased as a recombinant protein, but this is much more expensive than the use of BcM.