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 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.

Figure 1. Side-by-side comparison of 3 different reprogramming techniques.

The reprogramming vectors include (A) 4-in-1 CoMiP, (B) 4-in-1 Minicircle, and (C) 3 Yamanaka episomal plasmids. The highest transfection, expression efficiencies, and survival rate were observed with the 4-in-1 CoMiP reprogramming plasmid (A). Lower but moderate transfection efficiency and cell survival rates were observed by using either minicircle (B) or episomal (C) reprogramming plasmids.

Figure 2. Anticipated workflow and result using the 4-in-1 CoMiP reprogramming vector.

(A) Representative brightfield and fluorescent images demonstrating the pluripotent phenotype of the 4-in-1 CoMiP-derived human iPSCs. (B) Detailed timeline shows the media requirements and chemical treatments used for the reprogramming of human fibroblasts, as well as the time frame in which the first iPSCs are ready for further expansion through individual picking.

Figure 3. 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.

Figure 4. 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.

Figure 5. Confirmation of pluripotency of the 4-in-1 CoMiP-derived iPSCs.

(A) Immunofluorescence staining on the pluripotency markers NANOG, OCT4, TRA-1-60, and TRA-1-81. DAPI and brightfield images are also shown. (B) 4-in-1 CoMiP-derived iPSCs showed a normal karyotype and (C) from formed in vivo teratomas consisting of ectoderm, mesoderm, and endoderm lineages on H&E staining. (D) Directed in vitro differentiation into cardiomyocytes, endothelial cells, and neuronal cell using specific monolayer differentiation protocols further confirmed the pluripotent nature of the 4-in-1 CoMiP-derived iPSCs.

Figure 6. 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.