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

Diecke S, Lu J, Lee J, Termglinchan V, Kooreman NG, Burridge PW, Ebert AD, Churko JM, Sharma A, Kay MA, Wu JC - Sci Rep (2015)

Bottom Line: We have 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute for Stem Cell Biology and Regenerative Medicine; Stanford University School of Medicine, Stanford, California 94305, USA [2] Stanford Cardiovascular Institute; Stanford University School of Medicine, Stanford, California 94305, USA [3] Department of Medicine, Division of Cardiology; Stanford University School of Medicine, Stanford, California 94305, USA [4] Max Delbrück Center, Robert-Rössle Strasse 10, 13125 Berlin, Germany [5] Berlin Institute of Health, Kapelle-Ufer 2, 10117 Berlin, Germany.

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 RNA against p53 ("4-in-1 CoMiP"). We have 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.

No MeSH data available.


Related in: MedlinePlus

CoMiP-derived iPSCs show a similar gene expression as seen in the hESC line H7.(A–B) 4-in-1 CoMiP-derived iPSCs generated by either electroporation or lipofection showed a similar gene expression and promoter methylation patterns as those observed in the standard human ESC line H7. The 4-in-1 CoMiP-derived iPSCs were negative for the expression of cardiac specific markers such as TNNT2 and MYH6, which were used as a negative control. Statistical significance was analyzed using the student's t-test and expressed as a P-value. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.
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f4: CoMiP-derived iPSCs show a similar gene expression as seen in the hESC line H7.(A–B) 4-in-1 CoMiP-derived iPSCs generated by either electroporation or lipofection showed a similar gene expression and promoter methylation patterns as those observed in the standard human ESC line H7. The 4-in-1 CoMiP-derived iPSCs were negative for the expression of cardiac specific markers such as TNNT2 and MYH6, which were used as a negative control. Statistical significance was analyzed using the student's t-test and expressed as a P-value. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Mentions: We next established multiple 4-in-1 CoMiP-derived iPSC lines using either lipofection or electroporation and confirmed their pluripotency. iPSCs demonstrate the expression of the key pluripotency genes OCT4, NANOG, SOX2, and TRA-1-81, whereas cardiac genes were not expressed compared to differentiated iPSC-derived cardiomyocytes (Fig. 4A). Furthermore chromatin immunoprecipitation (ChiP) followed by qRT-PCR demonstrated that the newly established iPSCs had similar epigenetic pattern as the human ESC line H7 (Fig. 4B). Promoters of pluripotency genes (OCT4, SOX2, NANOG, and REX1) exhibited histone methylation patterns associated with active gene transcription (H3K4me3), whereas promoter regions of lineage specific genes (NKX2.5) exhibited repressive methylation patterns (H4K27me3). All 4-in-1 CoMiP-derived iPSCs showed pluripotent immunostaining and normal karyotype, and were able to differentiate in vivo into all three germ layers (Fig. 5A–C). Using a small molecule-based monolayer differentiation protocol, we were also able to differentiate the 4-in-1 CoMiP-derived iPSCs effectively in vitro into cardiomyocytes, endothelial cells, and neurons (Fig. 5D)2324. A specific PCR assay using primers targeting a 2A-linked junction region between the codon optimized OCT4 and KLF4 regions, and a separate Southern blot analysis using a portion of the tdTomato gene as a probe, confirmed that 4-in-1 CoMiP-derived iPSCs are mainly integration-free (Supplementary Fig. 8). However, some of the CoMiP-derived iPSCs showed traces of plasmid integration or episomal persistence until passage 15 (data not shown). The rate of genomic integration of the 4-in-1 CoMiP plasmid was comparable to the integration rate reported for other reprogramming plasmids25. Interestingly, we noticed that the PCR-based screening method for integration is not sufficient. With this method, it is not possible to distinguish among residual episomal persistence, partial plasmid integration, and complete plasmid integration. Therefore, we recommend using the PCR primer as a pre-screening method followed by verification by Southern blot. This combined approach enabled us to identify some iPSCs that showed no sign of integration using PCR primer but were positive for integration in a Southern blot experiment (Supplementary Figs. 8A and 8B).


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

Diecke S, Lu J, Lee J, Termglinchan V, Kooreman NG, Burridge PW, Ebert AD, Churko JM, Sharma A, Kay MA, Wu JC - Sci Rep (2015)

CoMiP-derived iPSCs show a similar gene expression as seen in the hESC line H7.(A–B) 4-in-1 CoMiP-derived iPSCs generated by either electroporation or lipofection showed a similar gene expression and promoter methylation patterns as those observed in the standard human ESC line H7. The 4-in-1 CoMiP-derived iPSCs were negative for the expression of cardiac specific markers such as TNNT2 and MYH6, which were used as a negative control. Statistical significance was analyzed using the student's t-test and expressed as a P-value. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4308704&req=5

f4: CoMiP-derived iPSCs show a similar gene expression as seen in the hESC line H7.(A–B) 4-in-1 CoMiP-derived iPSCs generated by either electroporation or lipofection showed a similar gene expression and promoter methylation patterns as those observed in the standard human ESC line H7. The 4-in-1 CoMiP-derived iPSCs were negative for the expression of cardiac specific markers such as TNNT2 and MYH6, which were used as a negative control. Statistical significance was analyzed using the student's t-test and expressed as a P-value. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.
Mentions: We next established multiple 4-in-1 CoMiP-derived iPSC lines using either lipofection or electroporation and confirmed their pluripotency. iPSCs demonstrate the expression of the key pluripotency genes OCT4, NANOG, SOX2, and TRA-1-81, whereas cardiac genes were not expressed compared to differentiated iPSC-derived cardiomyocytes (Fig. 4A). Furthermore chromatin immunoprecipitation (ChiP) followed by qRT-PCR demonstrated that the newly established iPSCs had similar epigenetic pattern as the human ESC line H7 (Fig. 4B). Promoters of pluripotency genes (OCT4, SOX2, NANOG, and REX1) exhibited histone methylation patterns associated with active gene transcription (H3K4me3), whereas promoter regions of lineage specific genes (NKX2.5) exhibited repressive methylation patterns (H4K27me3). All 4-in-1 CoMiP-derived iPSCs showed pluripotent immunostaining and normal karyotype, and were able to differentiate in vivo into all three germ layers (Fig. 5A–C). Using a small molecule-based monolayer differentiation protocol, we were also able to differentiate the 4-in-1 CoMiP-derived iPSCs effectively in vitro into cardiomyocytes, endothelial cells, and neurons (Fig. 5D)2324. A specific PCR assay using primers targeting a 2A-linked junction region between the codon optimized OCT4 and KLF4 regions, and a separate Southern blot analysis using a portion of the tdTomato gene as a probe, confirmed that 4-in-1 CoMiP-derived iPSCs are mainly integration-free (Supplementary Fig. 8). However, some of the CoMiP-derived iPSCs showed traces of plasmid integration or episomal persistence until passage 15 (data not shown). The rate of genomic integration of the 4-in-1 CoMiP plasmid was comparable to the integration rate reported for other reprogramming plasmids25. Interestingly, we noticed that the PCR-based screening method for integration is not sufficient. With this method, it is not possible to distinguish among residual episomal persistence, partial plasmid integration, and complete plasmid integration. Therefore, we recommend using the PCR primer as a pre-screening method followed by verification by Southern blot. This combined approach enabled us to identify some iPSCs that showed no sign of integration using PCR primer but were positive for integration in a Southern blot experiment (Supplementary Figs. 8A and 8B).

Bottom Line: We have 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute for Stem Cell Biology and Regenerative Medicine; Stanford University School of Medicine, Stanford, California 94305, USA [2] Stanford Cardiovascular Institute; Stanford University School of Medicine, Stanford, California 94305, USA [3] Department of Medicine, Division of Cardiology; Stanford University School of Medicine, Stanford, California 94305, USA [4] Max Delbrück Center, Robert-Rössle Strasse 10, 13125 Berlin, Germany [5] Berlin Institute of Health, Kapelle-Ufer 2, 10117 Berlin, Germany.

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 RNA against p53 ("4-in-1 CoMiP"). We have 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.

No MeSH data available.


Related in: MedlinePlus