Generation and characterization of induced pluripotent stem cells from human embryonic kidney cells via codon-optimized mini-intronic plasmids

Objective: Induced pluripotent stem cells (iPSCs) have a great potential to be used in many fields such as regenerative medicine and in vitro disease modeling. Strategies such as overexpression of the reprogramming factors, use of small molecules to increase efficiency, and use of various different vector systems are utilized for improvement of the efficiency and safety of iPSC generation methodologies. Codon-optimized mini intronic plasmids (CoMiPs) serve as effective, cost-efficient, and safe non-integrating vectors. We aimed to generate and characterize iPSCs from HEK293T human embryonic kidney cells via a combined strategy involving CoMiP plasmids and sodium butyrate. Methods: "MIP 247 CoMiP 4in1 with shRNA p53" plasmids that code for the OSKM factors (Oct-4, Sox2, Klf4 and c-Myc) and short hairpin RNA against p53, and "MIP 339 CoMiP coLin28 2A Nanog IRES eGFP" plasmids that code for Lin28 and Nanog were used for reprogramming of HEK293T cells. Efficiencies of the electroporation- and lipofectamine-mediated co-transfections were evaluated via fluorescent markers encoded by the plasmid vectors. Morphological criteria, immunocytochemical/immunofluorescence analyses for pluripotency markers, and potential for formation of embryoid bodies were utilized for characterization of the iPSC colonies. Results: iPSCs were generated from HEK293T cells via an efficient approach comprising CoMiP mini intronic plasmids and sodium butyrate. The obtained colonies stained positive for the pluripotency markers alkaline phosphatase, Tra-1-81, Tra-1-60, Oct4, Klf4, c-Myc and Nanog, and successfully formed embryoid bodies (EBs); thus were characterized as iPSC clusters. Conclusion: The reprogramming approach used in our study may be utilized for reprogramming of various different cells as an efficient, cost-effective, and safe methodology. The iPSCs obtained has the potential to be used in disease modeling and for various therapeutic purposes, following differentiation. Keywords: induced pluripotent stem cells, CoMiP plasmids, electroporation, lipofection, sodium butyrate