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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

Enhanced induction of pluripotency in human fibroblast using the 4-in-1 CoMiP vector.(A) Alkaline phosphatase (AP) staining revealed faster and superior reprogramming efficiency of the 4-in-1 CoMiP plasmid compared to the 3 Yamanaka episomal plasmids in younger and older human subjects within the first 20 days. (B) The chart summarizes the quantification of the AP-positive iPSC colonies observed in panel (A). 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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f3: Enhanced induction of pluripotency in human fibroblast using the 4-in-1 CoMiP vector.(A) Alkaline phosphatase (AP) staining revealed faster and superior reprogramming efficiency of the 4-in-1 CoMiP plasmid compared to the 3 Yamanaka episomal plasmids in younger and older human subjects within the first 20 days. (B) The chart summarizes the quantification of the AP-positive iPSC colonies observed in panel (A). 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 compared the reprogramming efficiency of our newly developed 4-in-1 CoMiP vector against the minicircle and the Yamanaka EBNA/OriP-based episomal plasmids (Fig. 3). From each reprogramming method, we established at least 5 different iPSC clones and confirmed the pluripotent phenotype of those cells via immunostaining and teratoma assays (Supplementary Fig. 5). To compare the different reprogramming methods, we transfected 1 × 106 fibroblasts from either younger (18–23 years old; n = 5) or older (50–70 years old; n = 5) human subjects with 12 μg total DNA of the different reprogramming plasmids. Afterwards, we plated 5 × 105 of the transfected cells onto a Matrigel-coated plate and changed the media every other day. After 12 days, a significant number of alkaline phosphatase (AP) positive colonies (97 ± 6, per 500,000 transfected cells) appeared in the culture transfected with the 4-in-1 CoMiP vector (Fig. 3A). By contrast, fewer number of AP-positive colonies were evident in the culture transfected with the Yamanaka EBNA/OriP episomal reprogramming plasmids after 16 days (49 ± 9, per 500,000 transfected cells) (Fig. 3A). A comparison of these two different techniques and time points shows a greater than two-fold superior reprogramming efficiency for the 4-in-1 CoMiP vector (Fig. 3B). Moreover, it appears that the higher expression rates of the 4-in-1 CoMiP transgenes led to an early establishment of the pluripotent state within the transfected cells. As shown before, the reprogramming efficiency of the minicircle technique was low for human fibroblasts and highly variable in each individual experiment, resulting in 3–10 potential iPSC clones per transfection (one million cells, below 0.005%) (Figs. 3A, 3B)6. Interestingly, we were able to reprogram human fibroblasts using the 4-in-1 CoMiP vector with a single Lipofectamine LTX-mediated transfection, albeit with significantly lower efficiency (0.002%) (Supplementary Fig. 6). By contrast, we could not derive any iPSC clones performing a single lipofection with either the minicircle or the 3 individual Yamanaka reprogramming vectors (Supplementary Fig. 6).


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)

Enhanced induction of pluripotency in human fibroblast using the 4-in-1 CoMiP vector.(A) Alkaline phosphatase (AP) staining revealed faster and superior reprogramming efficiency of the 4-in-1 CoMiP plasmid compared to the 3 Yamanaka episomal plasmids in younger and older human subjects within the first 20 days. (B) The chart summarizes the quantification of the AP-positive iPSC colonies observed in panel (A). 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

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

f3: Enhanced induction of pluripotency in human fibroblast using the 4-in-1 CoMiP vector.(A) Alkaline phosphatase (AP) staining revealed faster and superior reprogramming efficiency of the 4-in-1 CoMiP plasmid compared to the 3 Yamanaka episomal plasmids in younger and older human subjects within the first 20 days. (B) The chart summarizes the quantification of the AP-positive iPSC colonies observed in panel (A). 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 compared the reprogramming efficiency of our newly developed 4-in-1 CoMiP vector against the minicircle and the Yamanaka EBNA/OriP-based episomal plasmids (Fig. 3). From each reprogramming method, we established at least 5 different iPSC clones and confirmed the pluripotent phenotype of those cells via immunostaining and teratoma assays (Supplementary Fig. 5). To compare the different reprogramming methods, we transfected 1 × 106 fibroblasts from either younger (18–23 years old; n = 5) or older (50–70 years old; n = 5) human subjects with 12 μg total DNA of the different reprogramming plasmids. Afterwards, we plated 5 × 105 of the transfected cells onto a Matrigel-coated plate and changed the media every other day. After 12 days, a significant number of alkaline phosphatase (AP) positive colonies (97 ± 6, per 500,000 transfected cells) appeared in the culture transfected with the 4-in-1 CoMiP vector (Fig. 3A). By contrast, fewer number of AP-positive colonies were evident in the culture transfected with the Yamanaka EBNA/OriP episomal reprogramming plasmids after 16 days (49 ± 9, per 500,000 transfected cells) (Fig. 3A). A comparison of these two different techniques and time points shows a greater than two-fold superior reprogramming efficiency for the 4-in-1 CoMiP vector (Fig. 3B). Moreover, it appears that the higher expression rates of the 4-in-1 CoMiP transgenes led to an early establishment of the pluripotent state within the transfected cells. As shown before, the reprogramming efficiency of the minicircle technique was low for human fibroblasts and highly variable in each individual experiment, resulting in 3–10 potential iPSC clones per transfection (one million cells, below 0.005%) (Figs. 3A, 3B)6. Interestingly, we were able to reprogram human fibroblasts using the 4-in-1 CoMiP vector with a single Lipofectamine LTX-mediated transfection, albeit with significantly lower efficiency (0.002%) (Supplementary Fig. 6). By contrast, we could not derive any iPSC clones performing a single lipofection with either the minicircle or the 3 individual Yamanaka reprogramming vectors (Supplementary Fig. 6).

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