iPSCs
The cytoplasmic RNA delivery method of iPSC generation
The delivery of RNA to the cytoplasm is an effective way of generating induced pluripotent stem cells (iPSCs). This process involves the direct and transient introduction of pluripotency marker genes into somatic cells. 9 A very promising approach is the use of a cytoplasmic RNA vector introduced into the cell via the Sendai virus. This novel method has been shown to successfully remove SeVdp vectors from reprogrammed iPSC colonies, leading to the generation of iPSCs. Cytoplasmic RNA viruses have the advantage of enabling gene expression independent of the host's transcriptional machinery, as they do not require nuclear entry and have no DNA intermediate phase in their life cycle. 10
Benefits and drawbacks
The key advantage of using RNA to reprogram cells is that it is simple and efficient. It has a high efficiency of reprogramming when compared to other methods. 11 The ability to generate patient- specific iPSCs offers an invaluable reservoir of pluripotent cells, which could be genetically engineered and differentiated into specific cell types for disease modelling, drug discovery, and regenerative medicine. iPSCs in combination with microarray and RNA sequencing technology can be used to generate phenotype-genotype maps of complex diseases by comparing the transcriptomes of diseased cells with those of healthy cells. 12 Cytoplasmic RNA delivery can shorten the time between the biopsy and the generation of clinically relevant iPSCs. The synergistic activity of synthetic modified mRNAs encoding reprogramming factors and miRNA-367/302s delivered as mature miRNA mimics greatly enhances the reprogramming efficiency of human primary fibroblasts into iPSCs. 13 One of the major drawbacks of cytoplasmic RNA delivery to generate iPSCs is that it usually has very poor and inconsistent effectiveness and may not convert all the desired cells into iPSCs. It may also represent an oncogenic risk owing to higher expression levels of Myc affecting genomic stability. 14 This puts a limit on the applications it can be used for outside of research because has a greater risk of causing cancer of the cell type it is mimicking within an organism. This can be avoided using srRNA and synthetic mRNA as they both act to reduce the chance of unwanted genetic material being 9 Steinle, H., Weber, M., Behring, A., Mau-Holzmann, U., Schlensak, C., Wendel, H.P., & Avci-Adali, M. (2019). Generation of iPSCs by Nonintegrative RNA-Based Reprogramming Techniques: Benefits of Self-Replicating RNA versus Synthetic mRNA. Stem Cells International, 2019 . 10 ‘ A Sendai Virus-Based Cytoplasmic RNA Vector as a Novel Platform. ’ Accessed February 27, 2023. https://www.cell.com/molecular-therapy-family/methods/fulltext/S2329-0501(19)30120-2. 11 Steinle, H., Weber, M., Behring, A., Mau-Holzmann, U., Schlensak, C., Wendel, H.P., & Avci-Adali, M. (2019). Generation of iPSCs by Nonintegrative RNA-Based Reprogramming Techniques: Benefits of Self-Replicating RNA versus Synthetic mRNA. Stem Cells International, 2019 . 12 Kogut, I.D., McCarthy, S.M., Pavlova, M., Astling, D., Chen, X., Jakimenko, A., Jones, K.L., Getahun, A., Cambier, J.C., Pasmooij, A.M., Jonkman, M.F., Roop, D.R., & Bilousova, G. (2018). High-efficiency RNA-based reprogramming of human primary fibroblasts. Nature Communications, 9 . 13 Al Abbar, A., Ngai, S.C., Nograles, N., Alhaji, S.Y., & Abdullah, S. (2020). Induced Pluripotent Stem Cells: Reprogramming Platforms and Applications in Cell Replacement Therapy. BioResearch Open Access, 9 , 121 - 136. 14 Gonzãlez, F., Boué, S., & Belmonte, J.C. (2011). Methods for making induced pluripotent stem cells: reprogramming à la carte. Nature Reviews Genetics, 12 , 231-242.
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