Pluripotent stem cells (PSCs) are considered as a potent tool for use in regenerative medicine. Highly efficient generation of PSCs through chromatin modulators such as trichostatin A (TSA) might change their MHC molecule expression profile. The efficiency of PSC generation and their immunogenicity is major obstacles for clinical use. Hence, we aim to investigate whether the use of TSA during PSC generation affects MHC expression level. Three PSC lines were generated by iPSCs, NT-ESCs, and IVF-ESCs’ reprogramming methods from B6D2F1 mouse embryonic fibroblast cells. Established PSC lines were characterized by alkaline phosphatase assay (ALP) and immunocytochemistry. Their chromosome fidelity was checked by karyotyping. The expression level of pluripotent genes (oct4, nanog, sox2, klf4), HDACs (hdac1, hdac2, and hdac3), and immune-related genes (including Qa-1, Qa-2, H2kb, H2kd, H2db, H2db, CIITA, H2-IE-βb, H2-IE-βd) in iPSC and ESC lines were assessed by real-time PCR analysis. The presence of MHC molecules on the surface of pluripotent stem cells was also checked by flow cytometry technique. Significant increase of pluripotency markers, oct4, nanog, sox2, and klf4, was observed in 100 nM TSA-treated samples. 100 nM TSA induced significant upregulation of H2db in generated iPSCs. H2-IE-βd was remarkably downregulated in 50 and 100 nM TSA-treated iPSC lines. The expression level of other immune-related genes was not greatly affected by TSA in iPSC and NT-ESC lines. It is concluded that the use of short-term and low concentration of TSA during reprogramming in PSC generation procedure significantly increases PSC generation efficiency, but do not affect the MHC expression in established cell lines, which is in the benefit of cell transplantation in regenerative medicine.
Human dermal fibroblasts (HDF) can be reprogrammed through different strategies to generate human induced pluripotent stem cells (hiPSCs). However, most of these strategies require high-cost materials and specific equipment not readily accessible in most laboratories. Hence, liposomal and virus-based techniques can replace with polyethyleneimine (PEI)-mediated transfection to overcome these challenges. However, few researchers have addressed the PEI's ability to transfect HDFs. This study used PEI reagent to transfer oriP/EBNA1-based vector into HDFs to produce hiPSC lines. We first described conditions allowing the efficient transfection of HDFs with low cytotoxicity and without specific types of equipment and optimized several parameters relevant to the transfection procedure. We then monitored the effect of different N/P ratios on transfection efficiency and cytotoxicity using flow cytometry and fluorescent microscopy. By the results, we found that transfection efficiency was greatly affected by plasmid DNA (pDNA) concentration, PEI concentration, order of combining reagents, serum presence in polyplexes, and the duration of serum starvations. Moreover, using the optimized condition, we found that the N/P ratio of 3 achieved the highest percentage of HDFs positive for pGFP (~ 40%) with minimal cell toxicity. We finally generated hiPSCs using the optimized protocol and oriP/EBNA1-based vectors. We confirmed hiPSC formation by characterizing tests: Alkaline Phosphatase (AP) staining, immunocytochemistry (ICC) assay, real-time PCR analysis, in vitro differentiation into three germ layers, and karyotyping test. In conclusion, our results indicated that 25kD branched PEI could efficiently transfect HDFs toward generating hiPSCs via a simple, cost-effective, and optimized condition.
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