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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

Human peripheral blood mononuclear cells (PBMCs) were successfully reprogrammed using the 4-in-1 CoMiP vector in combination with a Yamanaka vector co-expressing human c-Myc, LIN28, and NANOG.A single transfection of 2 × 106 PBMCs and subsequent cultivation in blood media and chemical defined media was sufficient to generate multiple iPSC colonies. Representative brightfield and fluorescent pictures exemplified the expected outcome of normal PBMC reprogramming experiment. A robust transfection efficiency (tdTomato expression in PBMCs observed 2 days after the electroporation) and optimal culture condition supporting the initial proliferation of the PBMCs are crucial for a successful experimental outcome.
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f6: Human peripheral blood mononuclear cells (PBMCs) were successfully reprogrammed using the 4-in-1 CoMiP vector in combination with a Yamanaka vector co-expressing human c-Myc, LIN28, and NANOG.A single transfection of 2 × 106 PBMCs and subsequent cultivation in blood media and chemical defined media was sufficient to generate multiple iPSC colonies. Representative brightfield and fluorescent pictures exemplified the expected outcome of normal PBMC reprogramming experiment. A robust transfection efficiency (tdTomato expression in PBMCs observed 2 days after the electroporation) and optimal culture condition supporting the initial proliferation of the PBMCs are crucial for a successful experimental outcome.

Mentions: For purposes of clinical application, it is easier to obtain cells for reprogramming via phlebotomy than skin biopsy. For this reason, we next attempted to reprogram freshly isolated peripheral blood mononuclear cells (PBMCs) using the 4-in-1 CoMiP plasmid. Previous studies already described the successful reprogramming of PBMCs with different episomal vectors expressing the canonical reprogramming factors OCT4, KLF4, SOX2, and c-MYC, either together with NANOG and LIN28 or in different combinations82627. Therefore, we transfected 2 × 106 cells with either the 4-in-1 CoMiP plasmid independently or with an additional plasmid expressing LIN28, NANOG, and GFP (CoMiP LNG). As expected, the reprogramming efficiency of the 4-in-1 CoMiP plasmid alone was relatively low (5–7 colonies, 0.00025%), but was up to 3-fold higher when we transfected both plasmids simultaneously (10–17 colonies, 0.00085%). The subsequently isolated iPSC colonies were positive for the expression of the pluripotency markers OCT4, NANOG, and TRA-1-81 (Fig. 6) and demonstrated a normal karyotype (Fig. 5B). Furthermore, we confirmed the pluripotent phenotype of these cells using either the teratoma or the Scorecard assay. The teratoma assay demonstrated the potential of PBMC-derived iPSCs to differentiate in vivo into all 3 germ layers (Supplementary Fig. 9A). The Scorecard assay is a real-time PCR-based method comparing pluripotency and differentiation data sets derived with multiple hESC cell3. Using this gene expression panel, we also confirmed the pluripotency of our PBMC-derived iPSCs. Furthermore, upon spontaneous differentiation we observed a massive down-regulation of pluripotency-related marker genes and in parallel a strong up-regulation of genes specific for the 3 germ layers (Supplementary Fig. 10A). Based on a previously published robust data set, the Scorecard assay could predict the differentiation propensity of the newly derived iPSCs compared to standardized hESCs3. According to those results, our PBMC-derived iPSC clones 1 and 2 have a comparable endoderm and an increased mesoderm and ectodermal differentiation propensity (Supplementary Fig. 10B).


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)

Human peripheral blood mononuclear cells (PBMCs) were successfully reprogrammed using the 4-in-1 CoMiP vector in combination with a Yamanaka vector co-expressing human c-Myc, LIN28, and NANOG.A single transfection of 2 × 106 PBMCs and subsequent cultivation in blood media and chemical defined media was sufficient to generate multiple iPSC colonies. Representative brightfield and fluorescent pictures exemplified the expected outcome of normal PBMC reprogramming experiment. A robust transfection efficiency (tdTomato expression in PBMCs observed 2 days after the electroporation) and optimal culture condition supporting the initial proliferation of the PBMCs are crucial for a successful experimental outcome.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Human peripheral blood mononuclear cells (PBMCs) were successfully reprogrammed using the 4-in-1 CoMiP vector in combination with a Yamanaka vector co-expressing human c-Myc, LIN28, and NANOG.A single transfection of 2 × 106 PBMCs and subsequent cultivation in blood media and chemical defined media was sufficient to generate multiple iPSC colonies. Representative brightfield and fluorescent pictures exemplified the expected outcome of normal PBMC reprogramming experiment. A robust transfection efficiency (tdTomato expression in PBMCs observed 2 days after the electroporation) and optimal culture condition supporting the initial proliferation of the PBMCs are crucial for a successful experimental outcome.
Mentions: For purposes of clinical application, it is easier to obtain cells for reprogramming via phlebotomy than skin biopsy. For this reason, we next attempted to reprogram freshly isolated peripheral blood mononuclear cells (PBMCs) using the 4-in-1 CoMiP plasmid. Previous studies already described the successful reprogramming of PBMCs with different episomal vectors expressing the canonical reprogramming factors OCT4, KLF4, SOX2, and c-MYC, either together with NANOG and LIN28 or in different combinations82627. Therefore, we transfected 2 × 106 cells with either the 4-in-1 CoMiP plasmid independently or with an additional plasmid expressing LIN28, NANOG, and GFP (CoMiP LNG). As expected, the reprogramming efficiency of the 4-in-1 CoMiP plasmid alone was relatively low (5–7 colonies, 0.00025%), but was up to 3-fold higher when we transfected both plasmids simultaneously (10–17 colonies, 0.00085%). The subsequently isolated iPSC colonies were positive for the expression of the pluripotency markers OCT4, NANOG, and TRA-1-81 (Fig. 6) and demonstrated a normal karyotype (Fig. 5B). Furthermore, we confirmed the pluripotent phenotype of these cells using either the teratoma or the Scorecard assay. The teratoma assay demonstrated the potential of PBMC-derived iPSCs to differentiate in vivo into all 3 germ layers (Supplementary Fig. 9A). The Scorecard assay is a real-time PCR-based method comparing pluripotency and differentiation data sets derived with multiple hESC cell3. Using this gene expression panel, we also confirmed the pluripotency of our PBMC-derived iPSCs. Furthermore, upon spontaneous differentiation we observed a massive down-regulation of pluripotency-related marker genes and in parallel a strong up-regulation of genes specific for the 3 germ layers (Supplementary Fig. 10A). Based on a previously published robust data set, the Scorecard assay could predict the differentiation propensity of the newly derived iPSCs compared to standardized hESCs3. According to those results, our PBMC-derived iPSC clones 1 and 2 have a comparable endoderm and an increased mesoderm and ectodermal differentiation propensity (Supplementary Fig. 10B).

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