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

doi:10.1038/srep08081

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^*

^*Corresponding author for this work

Pharmacology

Research output: Contribution to journal › Article › peer-review

52 Scopus citations

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 language	English (US)
Article number	8081
Journal	Scientific reports
Volume	5
DOIs	https://doi.org/10.1038/srep08081
State	Published - Jan 28 2015

Funding

This work was supported by the German Research Foundation (SD, DI 1877/1-1), Leducq Fondation, American Heart Association Established Investigator Award 14420025, the National Institutes of Health (NIH) R01 HL113006, R01 HL 123968, U01 HL099776, and R24 HL 117756 (JCW). Nicholas M. Mordwinkin helped with the endothelial cell differentiation and staining. Sang Ging Ong helped with the minicircle production.

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This output contributes to the following UN Sustainable Development Goals (SDGs)

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

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.",

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

note = "Funding Information: This work was supported by the German Research Foundation (SD, DI 1877/1-1), Leducq Fondation, American Heart Association Established Investigator Award 14420025, the National Institutes of Health (NIH) R01 HL113006, R01 HL 123968, U01 HL099776, and R24 HL 117756 (JCW). Nicholas M. Mordwinkin helped with the endothelial cell differentiation and staining. Sang Ging Ong helped with the minicircle production.",

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doi = "10.1038/srep08081",

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AU - Diecke, Sebastian

AU - Lu, Jiamiao

AU - Lee, Jaecheol

AU - Termglinchan, Vittavat

AU - Kooreman, Nigel G.

AU - Burridge, Paul W.

AU - Ebert, Antje D.

AU - Churko, Jared M.

AU - Sharma, Arun

AU - Kay, Mark A.

AU - Wu, Joseph C.

N1 - Funding Information: This work was supported by the German Research Foundation (SD, DI 1877/1-1), Leducq Fondation, American Heart Association Established Investigator Award 14420025, the National Institutes of Health (NIH) R01 HL113006, R01 HL 123968, U01 HL099776, and R24 HL 117756 (JCW). Nicholas M. Mordwinkin helped with the endothelial cell differentiation and staining. Sang Ging Ong helped with the minicircle production.

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N2 - 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.

AB - 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.

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

Abstract

Funding

ASJC Scopus subject areas

UN SDGs

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