iPSCs
integrated into the DNA of the cell. However, both of these can be more expensive than more traditional methods while doing little to improve the other drawbacks of this method. 15
The episomal vectors method of iPSC generation
Episomal vectors are introduced into the cell by electroporation. Then they are introduced as an EBNA- 1 (Epstein-Barr nuclear antigen-1) backbone that carries six reprogramming factors. These are Oct4, Sox2, Nanog, Lin28, Klf4 and L-Myc. Once inside the cell activation of replication into iPSCs begins by binding to multiple oriP sections of EBNA-1 to the DNA within the nucleus. This only occurs once per cell division cycle. 16 Episomal vectors have been used to generate iPSCs from a range of somatic cells including fibroblasts, bone marrow mononuclear cells, PBMCs, lymphoblast B cells, and various others. 17 When the correct proportions of factors is achieved, they work to provide an optimal system for generating transgene-free and virus-free iPSCs in a feeder-free environment.
Benefits and drawbacks
Episomes are non-integrating and non-viral, plasmid-based vectors meaning that they are stable and can be conveniently thawed and re-frozen through several usages. This allows the method to be very cheap and easily applicable for reprogramming multiple cells compared to other methods. 18 Additionally, episomes generate exogenous DNA-free induced pluripotent stem cells (EF-iPSCs) due to the integration free method of reprogramming. This also means that episomal plasmids are undetectable after five cell replications. 19 This reduces the chance of insertion of unwanted genetic information being inserted into the DNA. 20
15 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 . 16 ‘ About Episomal Ipsc Reprogramming Vectors - Thermo Fisher Scientific. ’ Episomal iPSC Reprogramming Vectors. Thermo Fisher Scientific. Accessed March 1, 2023. https://assets.thermofisher.com/TFS- Assets/LSG/manuals/FAQ_Episomal_iPSC_Reprogramming_Vectors.pdf. 17 ‘ Episomal IPSC Reprogramming Vectors. ’ Thermo Fisher Scientific - US. Thermo Fisher Sceintific. Accessed March 1, 2023. https://www.thermofisher.com/order/catalog/product/A14703. 18 Wang, A.Y., & Loh, C.Y. (2019). Episomal Induced Pluripotent Stem Cells: Functional and Potential Therapeutic Applications. Cell Transplantation, 28 , 112S - 131S ; ‘ About Episomal Ipsc Reprogramming Vectors - Thermo Fisher Scientific. ’ Episomal iPSC Reprogramming Vectors. Thermo Fisher Scientific. Accessed March 1, 2023. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/FAQ_Episomal_iPSC_Reprogramming_Vectors.pdf. 19 Drozd, A.M., Walczak, M.P., Piaskowski, S., Stoczyńska -Fidelus, E., Rieske, P., & Grzela, D.P. (2015). Generation of human iPSCs from cells of fibroblastic and epithelial origin by means of the oriP/EBNA-1 episomal reprogramming system. Stem Cell Research & Therapy, 6 . 20 Lee, M., Ha, J., Son, Y.S., Ahn, H., Jung, K.B., Son, M., & Kim, J. (2019). Efficient exogenous DNA-free reprogramming with suicide gene vectors. Experimental & Molecular Medicine, 51 .
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