Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA

Luigi Warren, Philip D. Manos, Tim Ahfeldt, Yuin-Han Loh, Hu Li, Frank Lau, Wataru Ebina, Pankaj K. Mandal, Zachary D. Smith, Alexander Meissner, George Q. Daley, Andrew S. Brack, James J. Collins, Chad Cowan, Thorsten M. Schlaeger, Derrick J. Rossi

All figures and captions are from the above authors.

Introduction

Induced pluripotent stem cells (iPSCs) allow researchers to examine the processes of development and differentiation, and can provide a source of autologous cells for the treatment of diseases. Current approaches to induced pluripotency, which include enforced gene expression via retroviral or excisable vectors and serial protein transduction, result in low efficiencies. Furthermore, the modification of cellular genome leads to risks of recombination or mutation, and recombinant proteins for transduction are difficult to generate and isolate. This paper examines an alternative to the current methods by inducing pluripotency through the administration of synthetic mRNA modified to overcome innate antiviral responses.

Summary

To develop modified RNAs, the researchers complexed RNA with a cationic vehicle to facilitate cellular uptake by endocytosis. Immunogenic responses were evident with dose-dependent toxicity after transfecting murine embryonic fibroblasts and human epidermal keratinocytes with synthetic RNA coding for GFP. The synthetic RNA was treated with phosphate to deactivate protein kinase R, a global repressor of protein translation. Modified nucleoside bases were shown to improve cell viability by reducing interferon signaling (Figure 1A-D). Furthermore, media supplementation with B18R, a virus decoy receptor for type I interferons, increased cell viability. Due to RNA and protein degradation, expression is transient and repetitive transfections are required to maintain high levels of protein expression over an extended period of time. After transfecting modified RNA with myogenic transcription factor (MYOD) into murine C3H10T1/2 cells, myotubes emerged that stained positive for myogenic factors (Figure 1I).

The authors repeatedly transfected modified RNA with Yamanka factors OCT4, KLF4, SOX2, and c-MYC into human ESC-derived fibroblasts. Cells were cultured in low oxygen conditions with a KMOS stoichiometry to promote conversion to iPSCs. Human ESC-like colonies emerged toward the second week of transfection and were mechnically picked after 20 days, leading to 14 prospective iPSC lines (Figure 2C). The authors also transfected somatic derived cells with modified RNA encoding LIN28. Daily transfection led to numerous hESC like colonies in all cell lines which expressed pluripotency markers OCT4, NANOG, TRA-1-60, TRA-1-81, SSEA3, and SSEA4 (Figure 2D).

To determine the effectiveness of RNA reprogramming, cells were evaluated by RT-PCR. All cells had robust expression of OCT4, SOX2, NANOG, and hTERT (Figure 3A). RNA-induced pluripotent stem cells (RiPSCs) had molecular signatures very similar to those of human ESC and highly divergent from parental fibroblasts (Figure 3C). Unsupervised hierarchical clustering analysis revealed that RiPSCs clustered more closely to hESC than did virally derived iPSCs (Figure 3D). In embryoid bodies generated from five RiPSC derivations, cardiomyocytes were observed in the majority. RiPSCs were capable of neuron and endodermal cell differentiation (Figure 4B) along with in vivo teratoma formation (Figure 4C).

The efficiency of reprogramming with RNA was measured according to the expression of pluripotency markers TRA-1-60 and TRA-1-81. In one particular experiment, RNA induced conversion efficiency was found to be two times higher than virus-based derivations. Efficiency was markedly improved when a low-oxygen condition was combined with a five-factor cocktail with RNA encoding LIN28. The authors tested a protocol which directly compared transfection of either KMOS-modified RNAs or KMOS retroviruses in dH1f fibroblasts. Whereas RNA transfected cultures were overgrown with ESC-like colonies by the 16^th day, colonies did appear on retroviral cultures until day 24. Furthermore, iPSC derivation efficiency was found to be 36 fold higher with modified RNA (Figures 5E, F). The authors successfully transfected RiPSCs with modified RNA encoding MYOD resulting in differentiation to myogenic lineage (Figure 6).

Commentary:

The research presented in this paper appears to be a significant step forward for the field of induced pluripotency. The authors were able to effectively demonstrate that RNA technology is highly efficient in comparison with current reprogramming techniques while eliminating the risk of genomic modification and mutation. They followed a thorough protocol for ensuring pluripotency of the cells by testing for various markers, gene expression, and developmental potential. While the authors tested methods of reducing immune response to RNA, they did not mention which was the most effective. The upregulation of interferon genes which was still evident after RNA modification needs to be addressed in future studies to determine long-term safety. The authors could have strengthened the paper by testing the differentiation of RiPSCs into various cell types. Overall, the work done in this paper has strong support for the use of RiPSCs in the future, but more studies need to be conducted to determine its application and safety.