A nonviral minicircle vector for deriving human iPS cells

Abstract

Due to the risk of insertional mutagenesis, viral transduction has been increasingly replaced by nonviral methods to generate induced pluripotent stem (iPS) cells. One technique that has not yet been explored is the use of "minicircle" DNA, a novel compact vector that is free of bacterial DNA and capable of persistent high level expression in cells. Here, we report the use of a single minicircle vector to generate transgene-free iPS cells from adult human cells. Keywords minicircle DNA; reprogramming; iPS cells; viral-free; human adipose stem cellsNon-viral methods for generating iPS cells using adenovirus 1 , plasmids 2 , or excision of reprogramming factors using Cre/LoxP 3,4 or piggyBAC transposition 5 have been reported, but in general are restricted to mouse, suffer from low reprogramming efficiencies (<0.003%), and may leave behind residual vector sequences. Recently, successful reprogramming of human neonatal foreskin fibroblasts was reported using episomal vectors derived from the EpsteinBarr virus6. However, this technique required three individual plasmids carrying a total of seven factors, including the oncogene SV40, and has not been shown to successfully reprogram cells from adult donors, a more clinically-relevant target population. Further, expression of the EBNA1 protein, as was required for this technique, may increase immune cell recognition of transfected cells7, thus potentially limiting clinical application if the transgene is not completely removed. Protein-based iPS cell generation in mouse8 and human9 fetal and neonatal cells has also been published, but required either chemical treatment (valproic acid) 8 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript methods require only minimal molecular biology background, and so remain a more attractive option for a wider population of researchers interested in cellular reprogramming.Compared to plasmids, minicircle DNA benefits from higher transfection efficiencies and longer ectopic expression due to its lower activation of exogenous silencing mechanisms10 , 11 , and thus may represent an ideal mechanism for generating iPS cells. We constructed a plasmid (P2PhiC31-LGNSO) that contained a single cassette of four reprogramming factors (Oct4, Sox2, Lin28, Nanog) plus a green fluorescent protein (GFP) reporter gene, each separated by selfcleavage peptide 2A sequences 12, 13 ( Supplementary Fig. 1a,b). We next took advantage of the PhiC31-based intramolecular recombination system that allows the plasmid backbone to be excluded and degraded in bacteria, and the minicircle to be purified and isolated as described 10,11 ( Supplementary Fig. 1c). Expression of individual protein factors was validated in 293FT cells ( Supplementary Fig. 2). To determine the reprogramming ability of the minicircle vector, we chose to induce pluripotency in human adipose stem cells (hASCs). hASCs have a number of advantages over other somatic cell types such as fibroblasts since they can be isolated in large quantities (100 ml of human adipo...

Mechanism and methods to induce pluripotency

“…To meet this need, several nonintegration systems and removable systems have been developed. These include episomal vectors (Yu et al, 2009), the piggyBac transposon system (Kaji et al, 2009;Woltjen et al, 2009;Yusa et al, 2009), and minicircle vectors (Jia et al, 2010).…”

“…Minicircle vectors are supercoiled DNA molecules that have higher transfection efficiency and longer ectopic expression window than plasmid DNA (Chen et al, 2003;Chen et al, 2005) due to reduced activation of the silencing mechanisms against foreign DNA. However, they cannot self-replicate and thus the expression time is not as long as for episomal DNA (Jia et al, 2010). The non-integration DNAs will be diluted upon cell division and lost with time, so that the resulting iPSCs were free of any foreign DNA contamination (Yu et al, 2009;Jia et al, 2010).…”

“…However, they cannot self-replicate and thus the expression time is not as long as for episomal DNA (Jia et al, 2010). The non-integration DNAs will be diluted upon cell division and lost with time, so that the resulting iPSCs were free of any foreign DNA contamination (Yu et al, 2009;Jia et al, 2010). However, the reprogramming efficiency of these non-integration systems was low compared to the viral approach.…”

Concise Review: Induced Pluripotent Stem Cells Versus Embryonic Stem Cells: Close Enough or Yet Too Far Apart?

“…But how close are they really to the gold standard ESC, and related to ithow ''safe'' are they for future clinical use? Avoiding the integration of reprogramming factors into the genome during iPSC generation is already achieved using different methodologies [5][6][7][8][9]. This is a first important step toward safer cells.…”

Non-viral iPSCs: a safe way for therapy?

“…Various new approaches have been employed to generate genetically unmodified or non-integrative human iPSCs: (1) non-integrative vectors, including episomal vectors, adenoviral vectors, and sendai viral vectors (Yu et al, 2009;Zhou and Freed, 2009;Jia et al, 2010;Ban et al, 2011;Chou et al, 2011;Hiratsuka et al, 2011;Okita et al, 2011); (2) excisable integrating vectors, such as Cre-recombinase excisable viruses, piggyBac transposon (Kaji et al, 2009;Soldner et al, 2009;Woltjen et al, 2009;Yusa et al, 2009;Sommer et al, 2010); (3) DNA-free materials, such as pluripotency-associated recombinant proteins, RNA, and microRNA (Kim et al, 2009;Warren et al, 2010;Miyoshi et al, 2011); (4) small molecules that can facilitate reprogramming (Feng et al, 2009;Li and Ding, 2010;Efe and Ding, 2011). Here we will briefly summarize recent literatures on episomal vectors-or small molecules-based technologies for generation of iPSCs ( Fig.…”