Integration-Free Methods for Generating Induced Pluripotent Stem Cells

Abstract

Induced pluripotent stem (iPS) cells can be generated from mouse or human fibroblasts by exogenous expression of four factors, Oct4, Sox2, Klf4 and c-Myc, and hold great potential for transplantation therapies and regenerative medicine. However, use of retroviral vectors during iPS cell generation has limited the technique’s clinical application due to the potential risks resulting from genome integration of transgenes, including insertional mutations and altered differentiation potentials of the target cells, which may lead to pathologies such as tumorigenesis. Here we review recent progress in generating safer transgene-free or integration-free iPS cells, including the use of non-integrating vectors, excision of vectors after integration, DNA-free delivery of factors and chemical induction of pluripotency.

Efficient mRNA delivery with graphene oxide-polyethylenimine for generation of footprint-free human induced pluripotent stem cells

“…The retrovirus-or lentivirus-based iPSC production procedures show high reprogramming efficiency but have hampered therapeutic applications of iPSCs due to the concerns about insertional mutagenesis, tumorigenesis, and continued expression of potentially oncogenic proteins by the integrated transgenes [11,25]. Adenoviral or other DNA-based vectors were also developed to generate iPSCs from mouse [47] and human [48] somatic cells [17]. These vectors allow for the transient expression of exogenous genes, but this DNA-based iPSC production procedure can cause integration of the foreign DNA into the host genome at a low frequency.…”

“…Proteins have also been used to generate DNA-free iPSCs [55,56], but the difficulties with production of safe cell-penetrating reprogramming proteins as well as poor reprogramming efficiency have restricted the applicability of the protein-based technology in iPSC production [56]. Low reprogramming efficiency is also an obstacle for the general use of chemical-based technology in iPSC production [14,17,18]. Currently, the Sendai virus is popular as a non-integrating reprogramming vector [16,57].…”

“…However, utilization of viral vectors or any other DNA-based gene delivery system gives rise to serious problems with the therapeutic applications because of the risks of insertional mutagenesis, tumorigenesis, and continued expression of potentially oncogenic proteins by the integrated transgenes [10][11][12]. Generation of iPSCs free of exogenous DNA by means of RNA viruses such as Sendai virus, synthetic proteins, or chemicals has been pursued to overcome the above problems [13,14], but there are still concerns about safety and efficiency [13][14][15][16][17][18].…”

Human pluripotent stem cells: Prospects and challenges as a source of cardiomyocytes for in vitro modeling and cell-based cardiac repair

“…These early iPSCs were made with integrating viral vectors, which raised concerns about potential genetic alterations and neoplastic transformation. More recently, many groups have reported alternative approaches to produce iPSCs including the use of non-integrating viral and non-viral vectors or reprogramming via protein-, mRNA-, and small molecule-based methods [26]. Importantly, equivalent methods can be used to induce the cardiac differentiation of both human ESCs and iPSCs, and the resultant cardiomyocytes have comparable phenotypes, at least to first approximation [27].…”

Advances and Current Challenges Associated with the Use of Human Induced Pluripotent Stem Cells in Modeling Neurodegenerative Disease

“…With the expression of just 4 factors, i.e., Oct4, Sox2, Klf4, and c-Myc, a terminally differentiated cell can be reverted into a pluripotent state [Takahashi and Yamanaka, 2006], making it possible to direct cells down almost any cell lineage. This groundbreaking discovery has been improved upon and alternative strategies have been developed Shahbazi et al, 2018], but the reprogramming process from somatic cell to iPSC remains inefficient [Zhou and Zeng, 2013;Schlaeger et al, 2015]. Part of this inefficiency is due to epigenetic barriers to this process that restrict the transition from terminally differentiated to pluripotent states [Ebrahimi, 2015].…”