Novel codon-optimized mini-intronic plasmid for efficient, inexpensive, and xeno-free induction of pluripotency

Sebastian Diecke, Jiamiao Lu, Jaecheol Lee, Vittavat Termglinchan, Nigel G. Kooreman, Paul W. Burridge, Antje D. Ebert, Jared M. Churko, Arun Sharma, Mark A. Kay, Joseph C. Wu

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Abstract

The development of human induced pluripotent stem cell (iPSC) technology has revolutionized the regenerative medicine field. This technology provides a powerful tool for disease modeling and drug screening approaches. To circumvent the risk of random integration into the host genome caused by retroviruses, non-integrating reprogramming methods have been developed. However, these techniques are relatively inefficient or expensive. The mini-intronic plasmid (MIP) is an alternative, robust transgene expression vector for reprogramming. Here we developed a single plasmid reprogramming system which carries codon-optimized (Co) sequences of the canonical reprogramming factors (Oct4, Klf4, Sox2, and c-Myc) and short hairpin RNAagainst p53 ("4-in-1 CoMiP"). Wehave derived human and mouse iPSC lines from fibroblasts by performing a single transfection. Either independently or together with an additional vector encoding for LIN28, NANOG, and GFP, we were also able to reprogram blood-derived peripheral blood mononuclear cells (PBMCs) into iPSCs. Taken together, the CoMiP system offers a new highly efficient, integration-free, easy to use, and inexpensive methodology for reprogramming. Furthermore, the CoMIP construct is color-labeled, free of any antibiotic selection cassettes, and independent of the requirement for expression of the Epstein-Barr Virus nuclear antigen (EBNA), making it particularly beneficial for future applications in regenerative medicine.

Original languageEnglish (US)
Article number8081
JournalScientific reports
Volume5
DOIs
Publication statusPublished - Jan 28 2015
Externally publishedYes

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Diecke, S., Lu, J., Lee, J., Termglinchan, V., Kooreman, N. G., Burridge, P. W., ... Wu, J. C. (2015). Novel codon-optimized mini-intronic plasmid for efficient, inexpensive, and xeno-free induction of pluripotency. Scientific reports, 5, [8081]. https://doi.org/10.1038/srep08081