Generation of mouse-induced pluripotent stem cells by transient expression of a single nonviral polycistronic vector

Abstract

Induced pluripotent stem (iPS) cells have generated keen interest due to their potential use in regenerative medicine. They have been obtained from various cell types of both mice and humans by exogenous delivery of different combinations of Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28. The delivery of these transcription factors has mostly entailed the use of integrating viral vectors (retroviruses or lentiviruses), carrying the risk of both insertional mutagenesis and oncogenesis due to misexpression of these exogenous factors. Therefore, obtaining iPS cells that do not carry integrated transgene sequences is an important prerequisite for their eventual therapeutic use. Here we report the generation of iPS cell lines from mouse embryonic fibroblasts with no evidence of integration of the reprogramming vector in their genome, achieved by nucleofection of a polycistronic construct coexpressing Oct4, Sox2, Klf4, and c-Myc.iPS cells ͉ nonintegrative technique ͉ reprogramming ͉ pluripotency T he reprogramming of somatic cells to induced pluripotent stem (iPS) cells by delivery of exogenous factors was first achieved in mouse embryonic fibroblasts (MEFs) by retroviral transduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) (1). This result was confirmed and expanded in a series of reports which established that iPS cells are similar to ES cells in morphology, expression of pluripotency markers, and ability to differentiate into the 3 germ layers in vitro and in vivo, including the ability to produce germline-competent chimeras (2-4). The technique was quickly reproduced with human fibroblasts (5-7), providing a source of patient-specific pluripotent cells with potential for regenerative medicine. Reprogramming has been achieved in a range of cell types, including embryonic and adult fibroblasts, keratinocytes, stomach cells, liver cells, pancreatic ␤ cells, lymphocytes, and neural progenitor cells (5-13). Moreover, reprogramming can be attained using different combinations of exogenous factors: human fibroblasts have been reprogrammed with Oct4, Sox2, Nanog, and Lin28 (14), and c-Myc is not strictly required for reprogramming, although it enhances its efficiency and speed (15, 16). Certain cell types have been reprogrammed with only 2 factors, like neural progenitors with Oct4 and c-Myc or Oct4 and Klf4 (10), and both mouse and human fibroblasts with Oct4 and either Sox2 or Klf4 in combination with small molecules (17,18).Although different reprogramming protocols have been reported, the delivery of the original OSKM transcription factor set remains the most commonly used method. Reprogramming requires the delivery of all of the factors to the cell and their adequate expression for a period of Ϸ8-12 days for fibroblasts and keratinocytes (13,19,20). The retroviral and (both constitutive and inducible) lentiviral (14,19,21,22) vectors commonly used meet these requirements, but their permanent integration into the genome limits their use for eventual therapeutic applications because of the risk of bo...

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).…”

Direct reprogramming of somatic cells: an update

“…Therefore, iPSCs must be screened to select free cells for further applications (Gonzalez et al, 2009). To date, the safest technique of iPSC production is induction of pluripotency via mRNA (Warren et al, 2012;Yoshioka et al, 2013) or protein (Kim et al, 2009;Lee et al, 2012).…”

Cyclin A1 Is Essential for Setting the Pluripotent State and Reducing Tumorigenicity of Induced Pluripotent Stem Cells

“…The generation of iPS cells using MEFs from black 6 mice has been described before [26]. For reprogramming experiments, about 50,000 or 100,000 cells were seeded per well of a 6-well plate and infected with retroviral supernatants of a FLAGtagged polycistronic retroviral vector Oct3/4, Sox2, Klf4, and C-Myc (OSKM) in the presence of 1 mg/mL polybrene.…”

“…Cultures were maintained at 37°C, 5% CO 2 , with medium changes every other day. iPS cells were picked, expanded, and characterized for morphology, gene expression, cell cycle, pluripotency, and teratomas [26].…”