Optimization of episomal reprogramming for generation of human induced pluripotent stem cells from fibroblasts

Abstract

Generation of induced pluripotent stem cells (iPSCs) by defined factors (OCT4, SOX2, C-MYC, and KLF4) from various human primary cells has been reported. Human fibroblasts have been widely used as a cellular source in reprogramming studies over recent decades. The original method of iPSC generation uses retro- or lentivirus vectors that require integration of viral DNA into the target cells. The integration of exogenous genes encoding transcription factors (OCT4, SOX2, C-MYC, and KLF4) can be detected in iPSCs, raising concern about the risk of mutagenesis and tumor formation. Therefore, stem cell therapy would ideally require generation of integration-free iPSCs using non-integration gene delivery system such as Sendai virus, recombinant proteins, synthetic mRNA, and episomal vectors. Several groups have reported that episomal vectors are capable of reprogramming human fibroblasts into iPSCs. Although vector concentration and cell density are important in the episomal vector reprogramming method, optimization of this method for human fibroblasts has not been reported. In this study, we determined optimal conditions for generating integration-free iPSCs from human fibroblasts through the use of different concentrations of episomal vectors (OCT4/p53, SOX2/KLF4, L-MYC/LIN28A) and different plating cell density. We found that optimized vector concentration and cell density accelerate reprogramming and improve iPSC generation. Our study provides a detailed stepwise protocol for improved generation of integration-free iPSCs from human fibroblasts by transfection with episomal vectors.

Human induced pluripotent cells in personalized treatment of monogenic epilepsies

“…However, this trend is changing, especially in case of young patients, for whom the most favored cell type are PBMCs, where their collection is far less invasive [44] . The pros and cons of the different cell types for hiPSC generation are summarized in Table 1 [45][46][47][48][49][50][51][52][53][54][55][56][57][58][59] . These hiPSCs obtained via the different reprogramming protocols are the starting material to generate any given cell type in a patient-specific manner by targeted differentiation, including neurons affected by epileptic disorders.…”

Episomal Induced Pluripotent Stem Cells: Functional and Potential Therapeutic Applications

“…The results are promising in showing that direct application of EiPSCsderived cardiomyocytes is possible. The local environment and conditions under which the EiPSCs were directly Bharathan et al 36 , Weltner et al 37 , Yu et al 40 , Wong et al 77 , Bang et al 78 , Hey et al 79 , Jaffer et al 80 , Trevisan et al 81 , Fidan et al 82 [165][166][167] Amniotic fluid cells injected allowed for their direct use and differentiation according to clinical need. A diagram of the potential application for an EiPSCs-engineered cardiac cell sheet is shown in Fig.…”

“…The human cells reported to have been successfully reprogrammed into EiPSCs include fibroblasts, epithelial cells, keratinocytes, mononuclear cells from adult peripheral blood, cord blood cells, amniotic fluid stem cells, mesenchymal stromal cells, lymphoblasts, lamina propria progenitor cells from oral mucosa, and urothelial cells obtained from urine. A summary of these reported human EiPSC sources is shown in Table 1 8,20,21,32 –43,47 –52,55,56,58,60 …”

Stem cells and tooth regeneration: prospects for personalized dentistry

“…The integration of exogenous genes encoding required transcription factors for generation of iPSCs (e.g., Oct4, Sox2, C-Myc, and Klf4) raises concern about the risk of mutagenesis and tumor formation [26]. Thus, nonintegration gene delivery systems (e.g., Sendai virus, recombinant proteins, synthetic mRNA and episomal vectors) are increasingly utilized for generation of iPSCs-i.e., generation of integration-free iPSCs [27]. In this context, it is important to note that generation of integration-free iPSCs from human urine-derived cells has further raised the prospect for personalized medicine and dentistry as described below [28].…”