Generation of iPSCs by Nonintegrative RNA-Based Reprogramming Techniques: Benefits of Self-Replicating RNA versus Synthetic mRNA

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.