Polycistronic Lentiviral Vector for “Hit and Run” Reprogramming of Adult Skin Fibroblasts to Induced Pluripotent Stem Cells

Abstract

We report the derivation of induced pluripotent stem (iPS) cells from adult skin fibroblasts using a single, polycistronic lentiviral vector encoding the reprogramming factors Oct4, Sox2, and Klf4. Porcine teschovirus-1 2A sequences that trigger ribosome skipping were inserted between human cDNAs for these factors, and the polycistron was subcloned downstream of the elongation factor 1 alpha promoter in a self-inactivating (SIN) lentiviral vector containing a loxP site in the truncated 3 0 long terminal repeat (LTR). Adult skin fibroblasts from a humanized mouse model of sickle cell disease were transduced with this single lentiviral vector, and iPS cell colonies were picked within 30 days. These cells expressed endogenous Oct4, Sox2, Nanog, alkaline phosphatase, stage-specific embryonic antigen-1, and other markers of pluripotency. The iPS cells produced teratomas containing tissue derived from all three germ layers after injection into immunocompromised mice and formed high-level chimeras after injection into murine blastocysts. iPS cell lines with as few as three lentiviral insertions were obtained. Expression of Cre recombinase in these iPS cells resulted in deletion of the lentiviral vector, and sequencing of insertion sites demonstrated that remnant 291-bp SIN LTRs containing a single loxP site did not interrupt coding sequences, promoters, or known regulatory elements. These results suggest that a single, polycistronic ''hit and run'' vector can safely and effectively reprogram adult dermal fibroblasts into iPS cells.

Overcoming barriers to the clinical utilization of iPSCs: reprogramming efficiency, safety and quality

“…There are several drawbacks for the genome integration system for example their infectivity is limited to dividing cells, thus restricting the range of cell types that can be reprogrammed; silencing occurs gradually during the course of iPSC induction, resulting in a lowered efficiency of conversion compared to nonsilencing viral methods and iPSCs made with retroviruses often maintain viral gene ex- Yamanaka, 2006 Blelloch et al, 2007;Yu et al, 2007;Brambrink et al, 2008Brambrink et al, 2008Stadtfeld et al, 2008b Free of genetic modification No genetic modification, still requires at least one factor to be transduced Okita et al, 2008Chang et al, 2009Soldner et al, 2009Stadtfeld et al, 2008cFusaki et al, 2009;Gonzalez et al, 2009Kaji et al, 2009Woltjen et al, 2009Bosnali and Edenhofer, 2008Huangfu et al, 2008aShi et al, 2008b;Ichida et al, 2009;Lyssiotis et al, 2009 pression thus limiting their utility (Maherali and Hochedlinger, 2008). Although iPSCs can be generated by constitutive lentiviruses, their poor silencing within pluripotent cells make them less suitable for direct reprogramming attempts (Blelloch et al, 2007;Yu et al, 2007;Brambrink et al, 2008).…”

“…Other groups have exploited lentiviral vectors flanked by loxP sites as factor delivery vehicles, such that Cre-mediated recombination in the resultant iPSC lines could excise the integrated transgenes (although it should be noted that the loxP sites themselves remain in the genome) (Chang et al, 2009;Soldner et al, 2009). Another recent approach has involved piggyBac transposition, wherein the reprogramming factors are delivered in a piggyBac transposon that can be subsequently induced to integrate or excise from the genome upon transient expression of a transposase enzyme (Kaji et al, 2009;Woltjen et al, 2009).…”

Advancements in reprogramming strategies for the generation of induced pluripotent stem cells

“…Unfortunately, Cremediated excisions of transgene can lead to genomic instability and genome rearrangements. To overcome these potential drawbacks, a single polycistronic vector, containing the four reprogramming factors connected with 2A peptide linkers, was developed [66,67]. The multiproteins vector does not need multiple integrations in genome, therefore minimizing overall genome modification [68].…”

Vectorology and Factor Delivery in Induced Pluripotent Stem Cell Reprogramming

“…To achieve these goals, many distinct technologies are employed in current reprogramming protocols. These include nonintegrating adenoviral vectors [7], excisable PiggyBac (PB) transposon [8], excision of transgenes with the Cre-Lox system upon completion of reprogramming [9,10], repeated transfection with conventional plasmids [11], minicircle DNA [12], Epstein-Barr virus-based replicating episomal plasmids [4][5][6], protein transduction [13], mRNA transfection [14], negative-sense RNA vectors (Sendai viral vector) [15], positive-sense RNA vector/replicons [16], and the use of polycistrons mediated by 2A peptide [9,11], and/or Internal Ribosome Entry Site (IRES) [4]. This review summarizes information relative to vector designs and factor delivery systems used in current reprogramming protocols.…”